Long noncoding RNAs in ubiquitination, protein degradation, and human diseases

Abstract

Protein stability and turnover is critical in normal cellular and physiological process and their misregulation may contribute to accumulation of unwanted proteins causing cellular malfunction, neurodegeneration, mitochondrial malfunction, and disrupted metabolism. Signalling mechanism associated with protein degradation is complex and is extensively studied. Many protein and enzyme machineries have been implicated in regulation of protein degradation. Despite these insights, our understanding of protein degradation mechanisms remains limited. Emerging studies suggest that long non-coding RNAs (lncRNAs) play critical roles in various cellular and physiological processes including metabolism, cellular homeostasis, and protein turnover. LncRNAs, being large nucleic acids (>200 nt long) can interact with various proteins and other nucleic acids and modulate protein structure and function leading to regulation of cell signalling processes. LncRNAs are widely distributed across cell types and may exhibit tissue specific expression. They are detected in body fluids including blood and urine. Their expressions are also altered in various human diseases including cancer, neurological disorders, immune disorder, and others. LncRNAs are being recognized as novel biomarkers and therapeutic targets. This review article focuses on the emerging role of noncoding RNAs (ncRNAs), particularly long noncoding RNAs (lncRNAs), in the regulation of protein polyubiquitination and proteasomal degradation.

Introduction

The human genome consists of over 3 billion base pairs, encoding approximately 20,000 protein-coding genes which are transcribed, translated, and processed to produce tens of thousands of proteins.^1-3 These proteins contribute towards cellular and physiological processes including enzymatic reactions, structural support, signalling, and transport.^{1, 4-12} Proteins fold into specific three-dimensional structures and may be post translationally modified and these ultimately influence interactions with other biomolecules including proteins and nucleic acids and hence determine their functions. Each protein has limited half-life inside cells and must be degraded after appropriate functions are completed. ^{13, 14} Protein turnover is a central biological process by which cells maintain the cellular homeostasis and quality control.^13-16 The proper functioning of protein quality control mechanisms is essential for cellular homeostasis, as defects in these processes can lead to the accumulation of misfolded or aggregated proteins, which are associated with various diseases, including neurodegenerative disorders, cancer, and metabolic disorders (Figure 1, Table 1). ^17-21

Figure 1. Overview of protein degradation pathways and implications in human diseases.

This figure illustrates the three primary pathways for protein degradation within cells: (a) the ubiquitin-proteasome system, (b) autophagy, and (c) molecular chaperones. Each pathway plays a crucial role in identifying and degrading misfolded or defective proteins, preventing their aggregation. The ubiquitin-proteasome system tags proteins for degradation with ubiquitin molecules, directing them to the proteasome. Autophagy involves the encapsulation of cellular debris and proteins in autophagosomes, which then fuse with lysosomes for degradation. Molecular chaperones identify and refold misfolded proteins; if unsuccessful, they target these proteins for degradation. The failure of these systems can lead to the accumulation of misfolded proteins, contributing to the development of various diseases, including proteionopathies, atherosclerosis, fibrosis, heart diseases, metabolic and immune disorders, and cancer.

Table 1:

List of Diseases Associated with Ubiquitination and Proteasomal Degradation

Disease	Target Gene	Mechanism	REF
Liposarcoma	Mdm2	Mdm2, an E3 ubiquitin ligase, targets tumor suppressor p53. When Mdm2 is amplified, it leads to more p53 being marked for destruction, which in turn impairs p53's ability to inhibit tumour growth.	315-318
Cervical Carcinoma	p53	Human papillomavirus (HPV) causes various benign and malignant growths. HPV types, especially types 16 and 18 causes cancer from their E6 and E7 proteins. The E6 protein boosts the activity of a human protein called E6-associated protein, which, is an E3 ubiquitin ligase targeting the p53 protein. By degrading p53, HPV can grow uncontrollably in cells and contribute to their transformation into cancer cells.	1, 13, 16, 23
Familial adenomatous polyposis	APC	APC protein acts as an enhancer for the breakdown of β-catenin. Consequently, when APC function is compromised, it results in the buildup of β-catenin, which continuously signals for growth. This buildup of β-catenin in the nucleus is commonly utilized as an additional diagnostic tool in desmoid fibromatosis and various similar tumor forms.	1, 13, 16, 23
Parkinson Disease (PD)	α-synuclein	Major component of Lewy body, a cytoplasmic aggregate found in substantia nigra. Lewy body is associated with ubiquitin like components. It can be confirmed that the abundance of this protein can be used as a diagnostic marker of PD.	18, 19, 24, 25
Alzheimer’s Disease	APP, PSEN1	A list of E3 ubiquitin ligases is linked to AD; for example, Parkin decreases Aβ42 levels, but the levels of Parkin are reduced in AD brains. Aβ oligomers can bind and inhibit the 20S proteasome, blocking protein degradation. In addition, alterations in the UPS and autophagy-related mechanisms are detected under AD conditions. BRCA1 (breast cancer type 1 susceptibility protein) may affect the degradation of the components of the γ-secretase complex in neurons from EOAD. An overactivation and cytosolic distribution of the BRCA1 seems to be associated with neuronal death in AD by mechanisms that imply an increase in total protein ubiquitination, alterations in the degradation of PSEN1, and consequently, an effect on APP processing with the generation of toxic amyloids.	18, 19, 31
Cystic Fibrosis	CFTR	The ER is crucial for ensuring proper protein folding; misfolded proteins are degraded via ER-associated degradation. The most prevalent cystic fibrosis mutation deletes a phenylalanine residue at position 508 (ΔF508), leading to the misfolding and degradation of the CFTR protein in the ER. Remarkably, the ΔF508 mutant retains some functionality as a chloride ion channel. Lowering the temperature, for instance, can reduce misfolding and allow it to reach the plasma membrane with diminished efficiency.	1, 13, 15, 16, 20, 52, 53
Gaucher’s Disease	GBA	Improper breakdown and recycling of cellular components. Glucocerebrosidases are part of the lysosome. When these enzymes are deficient or malfunctioning, the lysosomes cannot effectively degrade their substrates, leading to the accumulation of these substances in the cells. The inability to degrade glucocerebroside effectively in Gaucher's disease is a specific example of a broader category of disorders known as lysosomal storage diseases.	1, 13, 15, 16, 20, 52, 53
Von Hippel-Lindau (VHL) disease	VHL	VHL disease is caused by mutations in the VHL gene, which produces the VHL protein, key in regulating cellular responses to oxygen and protein degradation. Normally, VHL targets the hypoxia-inducible factor (HIF) for degradation. However, when VHL is dysfunctional due to mutation, HIF accumulates, even in normal oxygen conditions, leading to overactivation of genes involved in cell proliferation, angiogenesis, and metabolism. This results in tumor and cyst formation characteristic of VHL disease.	300-302
Amyotrophic lateral sclerosis (ALS)	SOD1, TDP-43, and C9orf72	There are disruptions in these protein degradation pathways, which include the ubiquitin-proteasome system and autophagy-lysosome pathway. In ALS, there's often an accumulation of ubiquitinated proteins in motor neurons, indicating a failure in this system to adequately dispose of these proteins. Disruptions in autophagy have also been observed, leading to the buildup of protein aggregates and dysfunctional organelles.	1, 13, 15, 16, 20, 52, 53
Acute promyelocytic leukemia (APML)	PML-RARα	Maintenance of APML is dependent on RARα. Increased levels of this fusion protein helps in the retention of the oncogenic potential of the leukemic cells. Proteasomal degradation of this protein leads to the loss of the activities of oncogenic cells.	1, 13, 15, 16, 20, 52, 53
Familial hyperkalemic hypertension syndrome	KLHL3	The variant ubiquitinates and promotes the degradation of WNK1/4 kinases, which are responsible for the phosphorylation of SPAK and ORS1, two kinases involved in the regulation of electroneutral cation-coupled chloride co-transporters, such as the sodium-chloride symporter and the Na-K-Cl cotransporter. In the absence of KLHL3, or as a result of the variants that interfere with substrate interaction, WNK1/4 kinases cannot be degraded and are upregulated in affected tissues. Remarkably, variants in WNK1 and WNK4 genes have also been found in pseudohypoaldosteronism type II patients and disrupt interaction with CRL3KLHL3 without affecting the kinase activity	1, 13, 15, 16, 20, 52, 53
Giant axonal neuropathy	Gigaxonin (GAN), or KLHL16	Substrate is microtubule-associated protein 1B (MAP1B). Accumulation leads to motor protein dysfunction	18, 19, 23
Retinitis Pigmentosa (RP)	KLHL7	Terminal uridylyl transferase 1 (TUT1) binds to and is ubiquitinated by the KLHL7 complex leading to degenerative rod cone dystrophy	18, 19, 23
Epidermolysis Bullosa (EB)	KLHL24	KLHL24 lacking short N-terminal region causes auto-ubiquitination. The complex binds and promotes ubiquitination and degradation of the keratin KRT14	1, 13, 15, 16, 20, 52, 53

Proteasomal dysfunction has been implicated in various neurodegenerative diseases characterized by the accumulation of misfolded or aggregated proteins in the brain. In Alzheimer's Disease (AD), the ubiquitin-proteasome system (UPS) plays a role in the degradation of amyloid precursor protein (APP) and tau protein, which are associated with amyloid plaques and neurofibrillary tangles in AD, respectively.^{17-19, 22, 23} Impaired proteasomal degradation may contribute to the accumulation of these toxic proteins, promoting AD progression. Parkinson's Disease (PD) is characterized by the loss of dopaminergic neurons and the presence of Lewy bodies, which contain aggregated α-synuclein.^24-30 Impaired proteasomal degradation has been implicated in the accumulation of α-synuclein and the formation of Lewy bodies.^24-30 Huntington's Disease (HD) is caused by an expanded polyglutamine (polyQ) tract in the huntingtin protein, which leads to protein aggregation and neuronal dysfunction.^31-50 Proteasomal impairment has been observed in HD models, suggesting a role for UPS dysfunction in disease progression.^31-50 Alongside its role in development of neurological disorders, UPS regulates the stability of many proteins involved in cell cycle progression, apoptosis, and DNA repair. Dysregulation of proteasomal degradation can contribute to the development and progression of cancer. Overexpression or stabilization of oncoproteins, such as c-Myc and Cyclin D1, can result from impaired proteasomal degradation, promoting uncontrolled cell proliferation.^{20, 21, 51-54} Loss of tumor suppressor function due to impaired proteasomal degradation can also lead to genomic instability and cancer development. For example, the tumor suppressor p53 is targeted for degradation by the E3 ubiquitin ligase MDM2, in coordination with HERC2 (E3 ubiquitin ligase) and MARCH7, a RING domain-containing ubiquitin E3 ligase.^{55, 56} Disruption of this process can contribute to tumorigenesis. Dysregulation of the UPS has also been implicated in the pathogenesis of various inflammatory and autoimmune diseases. Impaired proteasomal degradation has been associated with the accumulation of apoptotic cell debris, which may contribute to the production of autoantibodies in Systemic lupus erythematosus (SLE).^57-59 Proteasomal dysfunction has also been implicated in the activation of the NF-κB pathway, which plays a critical role in promoting inflammation in Rheumatoid arthritis (RA).^60-64

Despite extensive research, there is very limited understanding of the protein-degradation mechanism and their turnover. Deciphering the intricate mechanisms involved in protein quality control is crucial for developing therapeutic strategies targeting protein misfolding and aggregation-related diseases. Emerging studies suggest that, along with various protein-based factors, noncoding RNAs (ncRNAs) play critical roles in regulation of protein ubiquitination and protein degradation. In this review article, we discussed and summarised the recent advances on the function of noncoding RNAs especially long noncoding RNAs in regulation of protein polyubiquitination, proteasomal degradation, and associated diseases.

Protein degradation and quality control pathways

There are several mechanisms for protein quality control: a) The ubiquitin-proteasome system (UPS) is the primary pathway responsible for the targeted degradation and clearance of damaged proteins; ^{13, 14, 16, 18} b) Molecular chaperones are a class of proteins that assist in protein folding and prevent misfolding and aggregation;^65-67 c) Autophagy is a cellular process involved in the degradation and recycling of intracellular components, including proteins;^{68, 69} d) The Endoplasmic Reticulum-Associated Degradation (ERAD) pathway targets misfolded or unassembled proteins in the endoplasmic reticulum (ER) for degradation;^{70, 71} e) The heat shock response is a cellular stress response that is activated in response to protein misfolding caused by heat or other stresses and it involves the upregulation of molecular chaperones, such as heat shock proteins (HSPs), to assist in protein refolding and prevent protein aggregation.^{72, 73}

The Ubiquitin proteasome pathway is one of the most widely studied pathways known for its roles in protein degradation. Ubiquitination is a post-translational modification in which ubiquitin, a 76 amino acid long small protein, is covalently attached to target proteins and polyubiquitinated proteins are targeted towards proteasomal degradation.^{14, 21, 74-82} The ubiquitination process is mediated by a cascade of enzymes, including E1 ubiquitin-activating enzymes, E2 ubiquitin-conjugating enzymes, and E3 ubiquitin ligases (Figure 2). ^{13, 14, 16, 51} The ubiquitination process starts with the activation of ubiquitin by E1 enzyme. During this phase, an ATP-dependent high-energy thioester bond is formed between the C-terminal glycine of ubiquitin and a cysteine residue in the E1 enzyme. Following activation, the activated ubiquitin is transferred from E1 to an E2 enzyme via trans-thioesterification reaction. E2 then interacts with E3 ubiquitin ligases, which are responsible for recognizing the specific protein substrate to be ubiquitinated. E3 ligases facilitate the transfer of ubiquitin from the E2 enzyme to a lysine residue on the substrate protein.^{1, 74, 75, 82-85} Polyubiquitinated proteins are targeted to proteasome leading to their degradation. The proteasome is a multi-subunit protease complex responsible for the degradation of ubiquitinated proteins. ^{13, 51, 86-91} The 26S proteasome, the primary proteasome complex in eukaryotes, consists of a 20S core particle and two 19S regulatory particles. The 20S core particle contains proteolytic active sites, whereas the 19S regulatory particles recognize and unfold ubiquitinated substrates, facilitating their entry into the 20S core for degradation.

Figure 2. Schematic Representation of the Ubiquitination and Proteasomal Degradation Pathway.

This figure illustrates the multi-step process of ubiquitination and subsequent proteasomal degradation, a critical regulatory mechanism for protein turnover in cells. The process begins with the activation of ubiquitin (Ub), a small regulatory protein, by an E1 ubiquitin-activating enzyme, which forms a high-energy thioester bond with Ub. This activated Ub is then transferred to an E2 ubiquitin-conjugating enzyme. The specificity of the ubiquitination process is determined in the next step, where an E3 ubiquitin ligase facilitates the transfer of Ub from the E2 enzyme to a lysine residue on the target protein. This E3 ligase also recognizes specific protein substrates for ubiquitination, ensuring precise control over protein degradation. Multiple ubiquitin molecules can be attached, forming a polyubiquitin chain that signals for proteasomal recognition. The polyubiquitinated protein is then recognized and bound by the 26S proteasome, a large proteolytic complex. The protein is unfolded and translocated into the proteasomal core, where it is degraded into small peptides. The ubiquitin molecules are released and recycled for further use in the ubiquitination cycle. Key components highlighted in the figure include the E1, E2, and E3 enzymes involved in the ubiquitination cascade, the polyubiquitinated target protein, and the 26S proteasome.

Polyubiquitination is a well-known mark for protein degradation. However, monoubiquitination of proteins is also common and associated with regulation of cell signalling, gene expression, protein function, and localization, without causing degradation.^{13, 77, 78, 81, 92-94} The ubiquitination process is characterized by a diversity of E3 ligases, providing specificity and regulation. Additionally, proteins can be deubiquitinated by deubiquitinating enzymes (DUBs), which remove ubiquitin chains or individual ubiquitin molecules, reversing the ubiquitination process.^95-101 Protein degradation by the UPS is attributed to several different factors including non-coding genome. Recent research demonstrated that along with protein coding genes, the non-coding RNAs plays crucial roles in polyubiquitination and protein degradation and thus, has opened new avenue for the understanding of UPS mechanisms and their implications in human diseases.

Noncoding RNAs

Non-coding RNAs (ncRNAs) represent a significant portion of the RNA world, transcending the classical view that RNA's sole function is to serve as an intermediary between DNA and proteins. Emerging research has unveiled a plethora of ncRNA species, each characterized by unique structural attributes and functional roles within cellular and molecular processes. These RNA molecules do not encode proteins but are pivotal in regulating gene expression at the transcriptional, post-transcriptional, and epigenetic levels, thereby orchestrating a wide array of biological functions. ncRNA could have wide ranges in sizes: small (<50 nt long), medium (50-200 nt) and long (>200 nt) noncoding RNAs.^{1, 102-108} (Figure 3A) The discovery of MicroRNAs (miRNAs) and Small Interfering RNAs (siRNAs) highlighted the intricacies of gene regulation at the post-transcriptional level. MiRNAs, approximately 22 nucleotides in length, are small ncRNAs and they are shown to exhibit wide ranges of functions including modulation of gene expression, by binding to complementary sequences in target mRNAs, leading to their degradation or repression of translation.^109-112 SiRNAs, about 20-25 nucleotides long, are central to the RNA interference (RNAi) pathway, targeting specific mRNAs for cleavage and degradation, thus silencing gene expression. This pathway plays a vital role in defense against viral infection and the regulation of endogenous gene expression.^{16, 113-118} Piwi-interacting RNAs (piRNAs), 26-31 nucleotides in length, collaborate with Piwi proteins to safeguard the genome integrity in germ cells from transposable elements and other genomic parasites. By guiding Piwi proteins to target sequences, piRNAs facilitate the silencing of transposons, playing a crucial role in maintaining genomic stability and fertility.^{1, 119-122}

Figure 3. Classification of non-coding RNAs and long non-coding RNAs.

(A) Based on their size, non-coding RNAs are classified into small non-coding RNAs (< 100 nucleotide in length), medium non-coding RNAs (50 - 200 nucleotide in length) and long non-coding RNAs (> 200 nucleotide in length). Examples of small/medium and lncRNAs are given. (B) LncRNAs may also be categorized based on their unique mechanisms of action and functional domains within cellular processes. Based on their mechanisms of action and their roles within the cell, enhancer lncRNAs (elncRNAs) are sourced from enhancer regions and work to boost gene expression. Decoy lncRNAs are a class of lncRNAs that regulate gene expression by acting as molecular sponges or decoys. Guide lncRNAs represent a functional class of lncRNAs that modulate gene expression by directing the localization and activity of chromatin-modifying complexes to specific genomic regions. Scaffold lncRNAs are a category of lncRNAs that organize and stabilize the assembly of multiple protein complexes, facilitating their simultaneous interaction and functional synergy.

On the other hand, medium ncRNAs include transfer RNA (tRNA), ribosomal RNA (rRNA), small nuclear RNAs (snRNAs) and small nucleolar RNAs (snoRNAs).¹²³ Transfer RNA (tRNA) molecules, typically 76 to 90 nucleotides in length, adopt a characteristic cloverleaf structure in their secondary form and an L-shaped three-dimensional configuration. These molecules are central to decoding the genetic information in mRNA, as they deliver specific amino acids to the ribosome during protein synthesis, ensuring the correct assembly and translation of proteins according to the genetic code.^{14, 124-129}

Medium ncRNAs are categorized according to their regulatory functions, namely small Nuclear RNAs (snRNAs) and Small Nucleolar RNAs (snoRNAs), both of which are pivotal in the post-transcriptional modifications of other RNAs. SnRNAs are integral components of the spliceosome complex, orchestrating the precise excision of introns from pre-mRNA and the subsequent ligation of exons, a critical step in the maturation of mRNA. SnoRNAs, residing mainly within the nucleolus, guide the chemical modification of rRNAs, tRNAs, and snRNAs, playing a crucial role in the modification of nucleotides through methylation and pseudouridylation, thereby influencing the stability and function of these RNAs.^{1, 106-108, 123, 130-132}

Contrarily, long non-coding RNAs (lncRNAs), exceeding 200 nucleotides in length, embody a diverse class of ncRNAs with a myriad of functions, including chromatin remodeling, transcriptional regulation, and post-transcriptional processing. These molecules can interact with DNA, RNA, and proteins, acting as guides, scaffolds, decoys, or enhancers, thereby influencing gene expression patterns in a spatial and temporal manner (Figure 3B).^{102-108, 133-151} Prominent lncRNAs such as XIST, instrumental in X-chromosome inactivation, and HOTAIR, which regulates gene silencing and chromatin dynamics, exemplify their diverse functional repertoire.^{1, 152-158} Additionally, ribosomal RNA (rRNA), a cornerstone of the cellular machinery, underpins protein synthesis in all living cells, belong to the class of lncRNAs.^{1, 159-164} Comprising the bulk of RNA within the cell, rRNAs assemble with ribosomal proteins to form the structural and catalytic core of ribosomes, facilitating the translation of mRNA sequences into polypeptides. Their structure, varying significantly in size across different organisms, is intricate, with complex secondary and tertiary conformations that are essential for their function.

Emerging classes of ncRNAs, such as Circular RNAs (circRNAs) and Telomerase RNA Component (TERC), further expand the functional repertoire of ncRNAs. CircRNAs, characterized by their covalently closed loop structures, can act as miRNA sponges, thus modulating miRNA activity and affecting gene expression indirectly.^{1, 165-167} TERC, an essential component of the telomerase complex, provides a template for telomere extension, playing a critical role in chromosome stability and cellular lifespan.^168-170 The elucidation of ncRNAs' structures and functions has revolutionized our understanding of genetic regulation, revealing a complex network of RNA-mediated control mechanisms that contribute to the dynamic regulation of gene expression, cellular differentiation, and organismal development. As research continues to uncover the vast and intricate world of ncRNAs, their significance in health and disease becomes increasingly apparent, offering new avenues for therapeutic intervention and the potential for novel diagnostic markers.

Similar to other RNAs, ncRNAs could fold into different secondary structures and may interacts with various proteins and other nucleic acids, modulate protein structure function and enzymatic activities and hence eventually influence cellular and physiological functions. Recent studies demonstrated that ncRNAs influences protein poly-ubiquitination and protein degradation under different cellular and physiological environments.

Classification of LncRNAs

LncRNAs are a diverse class of transcripts that are widely expressed in various tissues and play crucial roles in regulating various cellular and physiological functions.^{3, 9, 11, 12, 135, 138, 149, 152-158, 171-176} Being large polynucleotide sequences, they are able to fold and adopt a variety of three-dimensional structures, interact with various proteins and other nucleic acids, and exhibit diverse functions. Structure and functions of most lncRNAs remain unknown. Given the complexity and diversity of lncRNAs, several classification systems have been proposed based on different criteria (Figure 3A and B). According to genomic location and context, there are primarily four types of lncRNAs: Intergenic lncRNAs (lincRNAs), Intronic lncRNAs, Sense lncRNAs and Antisense lncRNAs.^{3, 9-12, 135, 138, 149, 152-158, 171-176}

LincRNAs are located in the intergenic regions between protein-coding genes. They are transcribed independently and often possess their promoter regions and transcribed by RNA polymerase II machineries.^177-179 Some well-known lincRNAs include HOTAIR, XIST, and MALAT1. HOTAIR is known for its role in gene silencing and chromatin remodelling. It represses gene expression by interacting with Polycomb Repressive Complex 2 (PRC2) and the LSD1/CoREST/REST complex.^{155-158, 180-182} Intronic lncRNAs are transcribed entirely from the introns of protein-coding genes.^183-188 They may regulate the expression of their host genes or other nearby genes, either through transcriptional interference or by acting as cis-regulatory elements. Examples of intronic lncRNA include ANRIL, GOMAFU, BC200, etc. LncRNA ANRIL is involved in gene regulation of the INK4b-ARF-INK4a gene cluster, which plays a critical role in cell cycle control and tumor suppression. It exerts its function through chromatin remodelling, particularly via polycomb repressive complexes, PRC1 and PRC2.

Sense lncRNAs overlap with exons of a protein-coding gene on the same strand. ^{153, 189-192} They can influence the transcription or post-transcriptional processing of the overlapping genes, such as alternative splicing, RNA stability, and translation. Examples of sense lncRNAs include KCNQ1OT1, TUG1, UCA1, HOTTIP, etc. UCA1 is implicated in the regulation of cell proliferation, migration, and apoptosis. It acts as a molecular sponge for certain microRNAs and can modulate the Wnt/β-catenin signalling pathway. Antisense lncRNAs are transcribed from the opposite strand of a protein-coding gene. ^{3, 138, 183, 193} They can form RNA-RNA hybrids with the sense mRNA, modulate transcription, or affect mRNA stability and translation. Examples include ZEB2-AS1, BACE1-AS, ADORA2A-AS1, etc. ZEB2-AS1 antisense RNA regulates the expression of ZEB2, a transcription factor important in embryonic development and cancer progression. ZEB2-AS1 increases ZEB2 expression by preventing the binding of the splicing factor SRSF1 to the ZEB2 pre-mRNA, which enhances the inclusion of an alternative splice site.

LncRNAs play a multitude of functions and there are mainly four categories of lncRNAs such as signal lncRNAs, decoy lncRNAs, guide lncRNAs and scaffold lncRNAs (Figure 3B). ^{4, 10-12} Signal lncRNAs act as molecular signals involved in the transcriptional regulation. They can be induced or repressed under specific cellular conditions or developmental stages, thus providing a temporal or spatial cue for gene regulation. Examples include H19, TUG1, MEG3, etc. H19 is involved in cell growth control and is highly expressed during embryonic development. It can function as a molecular sponge for certain microRNAs, particularly in the context of the miR-675, and plays a role in imprinting and growth regulation.^{3, 11, 12, 153, 155, 171, 194-197} Decoy lncRNAs bind and sequester regulatory proteins, such as transcription factors or chromatin modifiers, preventing them from acting on their target genes. One example is the GAS5 lncRNA, which binds to the glucocorticoid receptor and inhibits its activity.^{198, 199} Guide lncRNAs recruit chromatin-modifying complexes to specific genomic loci.^{155-158, 180-182} They can function in cis, targeting nearby genes on the same chromosome, or in trans, targeting genes on other chromosomes. The HOTAIR lncRNA is an example of a guide lncRNA that recruits the PRC2 complex to specific loci to repress gene expression.^{155-158, 180-182} Scaffold lncRNAs serve as a platform for the assembly of protein complexes or as a structural component, thereby facilitating the formation of functional molecular machinery. For example, the NEAT1 lncRNA forms the core of the paraspeckle nuclear bodies, which are involved in the regulation of gene expression.²⁰⁰ Apart from the common modes of lncRNA classification, these non-coding RNAs can also be categorized based on their conservation across species. Conserved lncRNAs show high sequence conservation across species, suggesting functional importance. ^{4, 10-12} Conserved lncRNAs are often involved in essential biological processes, such as X-chromosome inactivation by the XIST lncRNA. On the other hand, non-conserved lncRNAs display lower sequence conservation, which may reflect species-specific functions.

Recent study from our lab has identified a series of novel long non-coding RNAs, termed as LinfRNAs (Long inflammation-associated non-coding RNAs) which are significantly upregulated under inflammation in human macrophages.²⁰¹ Though the structure-functions of most hLinfRNAs (human LinfRNAs) remain elusive, they appear to be important and functional. Their knockdown alters the cytokine expression and metabolism in macrophages regulating inflammation and macrophage activation. Even if it is hard to believe, with respect to molecular evolution perspective, there is no obvious conservation of most of these lncRNAs across species. However, independent of the sequence conservation, there may be structural and functional analogs present in different species.

LncRNAs fold in three-dimensional structure

LncRNA being long stretch of nucleotides, may fold into different secondary and tertiary structures. Increasing studies suggest that these secondary and tertiary structures may have functional roles in binding and recognition of different proteins and nucleic acids and that may contribute to different signalling pathways or modulation in gene expression and so on.^{7, 9, 10, 12, 133, 153, 155, 156, 178, 202, 203} Secondary structures can include stem-loops, hairpins, bulges, internal loops, and other non-canonical motifs. The secondary structure of a lncRNA can play important roles in RNA stability, localization, and interaction with other molecules. At present, there is not much structural information on most lncRNAs, however, there are computation tools to predict RNA structure and functions. For example, using an online tool RNAfold Web server (http://rna.tbi.univie.ac.at//cgi-bin/RNAWebSuite/RNAfold.cgi), we predicted the secondary structure of lncRNA HOTAIR (Figure 4). Our analysis revealed there are different secondary structure conformations such as minimal energy structure and centroid secondary structure possible for HOTAIR. The structural complexity may be involved in the binding of spliceosome complex proteins and formation of repressor complexes in coordination with proteins like EZH2, CoREST, PRC2, SUZ12, etc. Beyond the secondary structure, lncRNAs may also fold in tertiary and quaternary structures which may influence their interactions with other biomolecules regulating cellular functions.^{202, 203} Some lncRNAs, such as XIST and HOTAIR, act as molecular scaffolds, bringing together multiple proteins or nucleic acids to perform specific functions.^{3, 4, 6, 155-157, 202, 203} LncRNAs also contain functional domains or motifs, which are specific sequences within the RNA that are critical for its biological function. For example, the lncRNA XIST, which is involved in X chromosome inactivation, contains a repeat motif called the “A-repeat” that is important for its interaction with the chromatin remodelling complex PRC2.^{152, 203, 204}

Figure 4. Predicted Secondary structure of HOTAIR and Putative RNA-protein interaction.

Using an online tool RNAfold Web server (http://rna.tbi.univie.ac.at//cgi-bin/RNAWebSuite/RNAfold.cgi), the secondary structure of lncRNA HOTAIR was predicted. Panel (A) displays the array of secondary structure motifs found within HOTAIR, emphasizing the structural complexity that contributes to its functional versatility, contrasting the minimal energy structure with the centroid secondary structure. These models demonstrate how structural variations may influence HOTAIR's interactions with other biomolecules. Panel (B) explores the functional implications of HOTAIR's secondary structures, focusing on their role in mediating interactions with protein complexes involved in gene repression and chromatin modification. Highlighted is the binding of HOTAIR to components of the spliceosome complex and repressor proteins such as EZH2, CoREST, PRC2, and SUZ12, illustrating how structural features of HOTAIR facilitate the formation of these repressive complexes.

In addition to the primary, secondary, and tertiary structures, lncRNAs can also undergo post-transcriptional modifications, such as RNA splicing, polyadenylation, and RNA-methylation. ^{4, 205} These modifications can affect RNA stability, localization, and interaction with other molecules. For example, the structure of lncRNAs can influence their recognition and modification by methyltransferases like METTL3/METTL14 and demethylases (erasers) like FTO and ALKBH5.^206-208 The accessibility of specific sites within the lncRNA sequence to these enzymes is often contingent on the RNA's secondary and tertiary structures. The structures play a crucial role in RNA methylation, particularly N6-methyladenosine (m6A), the most common modification in eukaryotic RNAs. The m6A modification typically occurs at specific consensus sequences (like RRACH, where R = A or G, and H = A, C, or U). The folding of lncRNAs can expose or hide these motifs, thus influencing the efficiency and location of methylation. For example, MALAT1, a highly conserved nuclear lncRNA, is involved in gene regulation, alternative splicing, and the metastasis of several cancers.^208-210 It undergoes m6A methylation, which influences its interaction with splicing factors. The structure of MALAT1 dictates the accessibility of m6A sites and thus modulates its role in splicing and cancer progression. Methylation can affect the stability, splicing, export, and degradation of lncRNAs. The structure of lncRNAs can determine how methylation alters these properties. For instance, m6A marks can promote the binding of specific proteins (readers) like YTHDF2, which can lead to altered localization or accelerated decay of the lncRNA.²¹¹ The structure of lncRNAs influences their interaction with RNA-binding proteins and other RNAs (including miRNAs and mRNA). Methylation can either enhance or inhibit these interactions depending on how it alters the lncRNA's conformation. The structure-mediated methylation of lncRNAs is implicated in a wide range of biological processes, such as differentiation, development, and response to stress. Overall, the structure of lncRNAs is diverse and complex, and their biological function is tightly regulated by their specific sequence, secondary and tertiary structure, and post-transcriptional modifications.

LncRNAs in regulation of ubiquitination and proteasomal degradation

While the functions of many lncRNAs remain unknown, there is increasing evidence to suggest that they play important roles in a variety of biological processes, including the regulation of gene expression and protein modification such as ubiquitination. For example, some lncRNAs have been shown to interact with components of the ubiquitin system, including E3 ubiquitin ligases and deubiquitinating enzymes, and to influence the ubiquitination status of target proteins. One well-studied example is the lncRNA HOTAIR, which has been shown to regulate the ubiquitination of Ataxin-1 by Dzip3 and Snurportin-1 by Mex3b in cells and accelerates their degradation.^{180, 181, 212, 213} This interaction leads to the recruitment of RNF2 to specific target genes, resulting in the ubiquitination and repression of those genes. Another example is the lncRNA RMRP (RNA component of mitochondrial RNA processing endoribonuclease), which has been shown to interact with the E3 ubiquitin ligase MDM2 and to regulate the ubiquitination and degradation of p53 via nuclear SNRPA1.^{214, 215} LncRNAs can also affect the stability of proteins through their interaction with other regulatory RNAs. Finally, lncRNAs can also regulate the expression of genes involved in ubiquitination and proteasomal degradation. For example, the lncRNA GAS5 has been shown to inhibit the expression of the E3 ubiquitin ligase MDM2, leading to the stabilization of the tumor suppressor protein p53 and increased proteasomal degradation.^{198, 199, 216} Overall, these studies suggest that lncRNAs play critical roles in the regulation of ubiquitination and proteasomal degradation, and their dysregulation can lead to various diseases such as cancer and neurodegeneration. Overall, these studies suggest that lncRNAs can play important roles in the regulation of ubiquitination, and that they may represent a novel class of regulators in the ubiquitin system (Table 2).

Table 2:

List Long Non-coding RNAs associated with ubiquitination and proteasomal degradation.

LncRNA (Chromosome location)	Full name	Function in Ubiquitination and Proteasomal Degradation	REF
H19 (11p15.5)	H19 Imprinted Maternally Expressed Transcript	Regulates HIF-1α	237, 238, 240, 241
UCA1 (19p13.12)	Urothelial Carcinoma Associated 1	Regulates the stability of the tumor suppressor PTEN	242, 243, 246, 251
MALAT1 (11q13.1)	Metastasis Associated Lung Adenocarcinoma Transcript 1	Modulates the expression and activity of E3 ubiquitin ligases	252, 253, 261, 262
HOTAIR (12q13.13)	HOX Transcript Antisense Intergenic RNA	Interacts with different E3 ligases to promote degradation of specific target proteins involved in cancer progression	182, 224, 225, 230-233
NEAT 1 (11q12.1)	Nuclear Enriched Abundant Transcript 1	Might have functions in assembly of ubiquitin ligase and their substrates	264-266
HULC (6p24.3)	Highly Upregulated in Liver Cancer	Predicted to modify ubiquitination status of proteins	295-297
PVT1 (8q24.21)	Plasmacytoma variant translocation 1	Prevents E3 ubiquitin ligase SKP2 from ubiquitination and proteasomal degradation	282
BACE1-AS (11q23.3)	BACE 1 Antisense RNA	Enhances proteasomal degradation by binding to miR-485-5p and increasing the expression of proteasomal subunit PSMB2	3, 4, 7, 33, 171
LNC473 (6q27)	LINC00473	Inhibits ubiquitination of survivin via deubiquitinase USP9X and thus enhances cell proliferation and invasion in hepatocellular carcinoma	279
PTENP1 (9p13.3)	Phosphatase and Tensin Homolog Pseudogene 1	Acts as a sponge for some of the PTEN-targeting miRNAs and protects it from degradation	319
LINC00963/MetaLnc9 (9q34.11)	Long Intergenic Non-Protein Coding RNA 963	It interacts with PGK1 kinase and prevents its ubiquitination, thereby fostering AKT/mTOR signalling	320
MEG3 (14q32.2)	Maternally Expressed 3	Promotes the phosphorylation of EZH2 at Thr-345 and Thr-487, which lowers protein levels of EZH2 by promoting its ubiquitination and degradation	321
BDNF-AS (11p14.1)	Brain-Derived Neurotrophic Factor Antisense RNA	Promotes breast cancer progression by functioning as a scaffold RNA supporting RNH1 ubiquitination by the E3 ligase TRIM21 and thus RNH1 degradation which leads to cancer progression	322

HOTAIR in ubiquitination and proteasomal degradation

Long non-coding RNA HOTAIR (HOX Transcript Antisense Intergenic RNA) is an essential regulatory RNA molecule that plays a crucial role in various cellular processes, including chromatin remodelling, gene expression, and epigenetic regulation.^{155-158, 180-182, 216} Discovered in 2007, HOTAIR has drawn considerable attention due to its role in chromatin dynamics and gene expression. ²¹⁷ It spans approximately 2,200 nucleotides in length, contains 6 exons and is located within the HOXC gene cluster on chromosome 12 in humans. It is conserved across all mammalian species, the sequence conservation is relatively low in mammals, but high among primates. ¹⁸² Exon 6 in particular is a highly conserved domain in HOTAIR and it has been traced back to kangaroos, confirming the presence of HOTAIR in marsupials. ²¹⁸ Despite poor sequence conservation level of HOTAIR the lncRNA has several secondary structural elements that may be significant in conservation. ²¹⁹ The similarity in HOTAIR sequence among mouse and human is 58% and about 50% in rats. Human HOTAIR has 6 exons, but rat and mouse HOTAIR have 5 exons as the exon 2 is absent in both. ¹⁸⁰ The detail functions of different structural domains need further investigation.

Recently, studies have reported the involvement of HOTAIR in ubiquitination and proteasomal degradation, particularly in the context of cancer. HOTAIR has been found to modulate the ubiquitination process by interacting with various E3 ubiquitin ligases, thereby promoting the proteasomal degradation of their target proteins. One such example is its interaction with the E3 ubiquitin ligase Mex3b in colorectal cancer cells. HOTAIR upregulates Mex3b expression and enhances its activity, promoting the ubiquitination and degradation of the tumor suppressor PTEN (Phosphatase and Tensin Homolog) and thereby contributing to cancer progression.^{156, 157, 180-182, 212}

HOTAIR in Cancer.

Despite extensive research on the role of HOTAIR in epigenomic, transcriptional, and translational regulation, recent studies have expanded our understanding of HOTAIR's post-translational capabilities within the cytoplasm. Notably, a current study uncovered HOTAIR's function in promoting ubiquitin-dependent proteolysis, serving as a scaffold that facilitates E3 ubiquitin ligase-mediated ubiquitination and subsequent proteasomal destruction of specific proteins. This process involves HOTAIR's interaction with E3 ligases such as Dzip3 and Mex3b, directing the degradation of Ataxin-1 and Snurportin-1, crucial for cellular processes.²¹² In the realm of cellular senescence, HOTAIR plays a pivotal role by interacting with the human antigen R (HuR) protein.^{220, 221} Normally, HuR destabilizes HOTAIR by attracting the let7-Ago2 complex, but in senescent cells, an increase in HuR levels reverses this effect, stabilizing HOTAIR and accelerating the breakdown of Ataxin-1 and Snurportin-1. This action helps avert premature senescence. Interestingly, this pathway also has oncogenic potential, as Ataxin-1 can stimulate the E-cadherin promoter, an essential tumor suppressor gene. In the context of hepatitis B virus-induced liver cancer, HOTAIR operates as a crucial intermediary for ubiquitination, linking PLK1 with targets like SUZ12 and ZNF198 for their degradation, facilitated by Mex3b's E3 ligase activity.^{222, 223} Furthermore, recent findings illustrate HOTAIR's interaction with RUNX3, leading to its degradation via MEX3B-dependent ubiquitination in gastric cancer.^{224, 225} This interaction between HOTAIR and RUNX3 opens potential therapeutic avenues for combating metastatic gastric cancer. Other investigations have shown HOTAIR's ability to inhibit ubiquitination, such as the recent discovery of its role in advancing castration-resistant prostate cancer by binding to the androgen receptor, enhancing its stability, and shielding it from MDM2-mediated degradation. This diverse spectrum of functions underscores HOTAIR's intricate involvement in cellular regulation and its potential as a target for therapeutic intervention.

HOTAIR has been recognized for playing a facilitative role in androgen receptor (AR)-mediated transcriptional responses, thereby aiding in the progression of castration-resistant prostate cancer (CRPC).^{226, 227} Additionally, HOTAIR's influence on CRPC progression extends to its reciprocal modulation of EZH2 and DNMT1, leading to an inhibition of CRPC progression by polyphylline-1.²²⁸ Furthermore, HOTAIR has been observed to protect AR from degradation mediated by the E3 ubiquitin ligase MDM2, achieved through direct interaction with AR (Figure 5). As a result, when HOTAIR is overexpressed, it leads to an increase in the expression of AR target genes independently of androgens, thereby accelerating the advancement of CRPC. Indeed, HOTAIR can attach to the N-Terminal Domain (NTD) of the AR protein, thereby preventing the E3 ubiquitin ligase MDM2 from associating with AR, as both interact with the same domain. Similarly, the long non-coding RNA (lncRNA) known as suppressor of cytokine signaling 2-antisense transcript 1 (SOCS2-AS1) aids in the AR-mediated transcriptional process. By binding to the AR protein, SOCS2-AS1 enhances the transcriptional suppression of the tumor suppressor gene TNFSF10.^{229, 230}

Figure 5: Role of HOTAIR in AR protein degradation.

LncRNA HOTAIR modulates the stability of the androgen receptor (AR) protein, thereby influencing AR-mediated gene expression. The AR protein, when bound by dihydrotestosterone (5-DHT), typically activates a cascade of AR-responsive genes (ARG). However, this activation can be attenuated by the E3 ubiquitin ligase MDM2, which interacts with the AR protein to promote its degradation through the proteasomal pathway. HOTAIR intervenes in this process by binding to the AR protein, obstructing the association between MDM2 and AR. This protective action of HOTAIR impedes the ubiquitination and subsequent proteasomal degradation of AR, sustaining AR protein levels and enhancing the expression of AR-responsive genes. The AR is a pivotal regulator of various physiological processes, and its modulation by HOTAIR represents a crucial post-translational regulatory mechanism that can impact AR signaling pathways.

Additionally, HOTAIR regulates the ubiquitination of lysine-specific demethylase 1 (LSD1), a histone demethylase that plays a key role in gene expression and cancer development.^{182, 231} The study found that HOTAIR recruits the E3 ubiquitin ligase DZIP3 to LSD1 and promotes its ubiquitination and degradation. The study also showed that HOTAIR is overexpressed in breast cancer and that it promotes breast cancer cell invasion and metastasis by downregulating LSD1. Overall, these studies suggest that HOTAIR plays a key role in the regulation of ubiquitination and that dysregulation of HOTAIR expression may contribute to the development and progression of cancer.

HOTAIR in neurological disorder.

Research indicates that HOTAIR may play a significant role in modulating neuronal protein stability, interactions, and functionalities through its involvement in ubiquitin-mediated proteolysis. Specifically, HOTAIR has been identified to bind with the RNA-binding domains of certain E3 ubiquitin ligases, such as DAZ-interacting zinc finger 3 (Dzip3) and Mex-3 RNA-binding family member B (Mex3b).^{212, 224, 232} This interaction promotes the ubiquitination and subsequent degradation of proteins associated with cellular senescence, namely ataxin-1 and snurportin-1. Importantly, ataxin-1 is a vital player in neurodevelopmental pathways and is linked to a range of neurological conditions, including spinocerebellar ataxia type 1. Furthermore, the engagement between HOTAIR and Mex3b is believed to influence the activity of key molecules such as the suppressor of mothers against decapentaplegic family member 4 (SMAD-4) and nucleoside diphosphate linked moiety X-type motif 3 (NUDT-3), underlining HOTAIR's significant impact on neuronal function and health.

HOTAIR in inflammation and immune response.

The NF-κB transcription factor, crucial for cell proliferation and survival, acts as a pivotal regulator of numerous genes vital for an appropriate inflammatory response. ^{156, 157, 180} Its balanced activation and timely termination are essential to avoid pathological states. Dysregulation of NF-κB, observed in various diseases including nearly all forms of cancer, contributes to tumor progression through mechanisms like enhanced cell proliferation, angiogenesis, and epithelial-to-mesenchymal transition (EMT), while also inhibiting cell death.

Our recent investigations have revealed that HOTAIR plays a significant role in modulating NF-κB activity by reducing levels of its inhibitor, IκBα. ^{156, 157, 180} This reduction in IκBα appears to facilitate NF-κB activation, with HOTAIR itself being transcriptionally activated by NF-κB following lipopolysaccharide (LPS) exposure in macrophages.^{156, 157, 180} The suppression of HOTAIR through siRNA-mediated knockdown diminishes LPS-induced activation of pro-inflammatory cytokines and genes, such as IL-6 and iNOS, primarily through the downregulation of NF-κB. Although the precise mechanisms remain to be fully elucidated, it's hypothesized that HOTAIR may influence the modulation of ubiquitin ligases or kinases that target IκBα, thereby governing the degradation of IκBα and subsequent NF-κB activation (Figure 6).¹⁵⁷

Figure 6: HOTAIR regulates NF-κB activation via degradation of IκB during inflammation.

NF-κB activation plays a central role in cytokine expression under inflammation. In the absence of inflammation NF-κB interacts with its negative regulator IκBα and remain inactive. However, under inflammation, IκBα gets phosphorylated followed by polyubiquitination and its proteasomal degradation. This results in release of NF-κB leading to its activation. Activated NF-κB translocate to the nucleus and binds to its target gene (such as cytokines) promoters resulting in their activation. LncRNA HOTAIR, which is overexpressed under inflammation, regulates (facilitate) IκBα degradation and hence control NF-κB activation and cytokine expression. The exact mechanisms underlying HOTAIR-mediated regulation of IκBα degradation and NF-κB activation remain to be fully elucidated, but it is proposed that HOTAIR may influence the activity of ubiquitin ligases or kinases targeting IκBα.

Furthermore, our research underscores the critical involvement of HOTAIR in metabolic reprogramming during macrophage activation, inflammation, and immune responses.¹⁵⁸ HOTAIR has been shown to regulate the expression of the glucose transporter Glut1 and metabolic processes in macrophages under inflammatory conditions, with LPS-induced NF-κB binding to the Glut1 promoter being notably decreased upon HOTAIR knockdown.¹⁵⁸ This reduction correlates with a significant decrease in glucose uptake, which typically experiences a tenfold increase under LPS stimulation.

Additionally, HOTAIR's regulation extends to the suppression of PTEN expression during LPS-induced inflammation in macrophages, highlighting its significant influence on glucose metabolism through the modulation of Glut1 expression and glucose uptake.¹⁵⁸

Beyond HOTAIR, our research has identified a series of long noncoding RNAs, termed LinfRNAs (long noncoding inflammation associated RNAs), that play roles in inflammation and immune response across higher eukaryotes, including humans.²⁰¹ Independent research further supports HOTAIR's involvement in NF-κB-mediated cytokine expression and activation during DNA damage responses, such as those induced by platinum-based chemotherapy. This creates a feedback loop that potentially diminishes chemotherapeutic effectiveness and promotes cellular aging.²³³ Intriguingly, HOTAIR's upregulation, driven by NF-κB activation in the context of obesity and a sedentary lifestyle in gluteofemoral fat, suggests a novel link between these conditions and an increased risk of colorectal cancer.²³⁴

In summary, HOTAIR plays a crucial role in regulating ubiquitination and proteasomal degradation by interacting with E3 ubiquitin ligases and modulating their activity or stability. This involvement in protein degradation pathways has implications in various pathological conditions, particularly in cancer progression and metastasis.

H19 in ubiquitination and proteasomal degradation

The H19 lncRNA, encoded by the H19 gene, is a maternally expressed imprinted gene. H19 is highly conserved in mammals, plays an instrumental role in mammalian development and growth control and has been implicated in various pathophysiological conditions.^{195, 196, 235} H19 is located at chromosome 11p15.5, typically ranging from 2.3 to 2.8 kilobases in length. This region is known for its significant role in growth control and development, harboring both paternally and maternally expressed genes, including the insulin-like growth factor 2 (IGF2).²³⁵ The H19 gene is exclusively maternally expressed due to imprinting mechanisms that silence the paternal allele. This genomic imprinting is intricately regulated by differential methylation patterns within the IGF2/H19 shared regulatory region, ensuring a tight control over H19 expression.

The unique feature of H19 is its capacity to function both as an RNA molecule and as a molecular scaffold to accommodate specific binding proteins, thereby affecting various cellular processes, including genomic imprinting, embryonic development, and tumorigenesis.^{235, 236} H19 acts as a regulator of gene expression, both at the transcriptional and post-transcriptional levels. It can modulate the expression of various genes, particularly those involved in growth and development. H19 is crucial in genomic imprinting, a process where only one allele of a gene is expressed depending on its parental origin. H19 is typically expressed from the maternally inherited chromosome and plays a significant role in embryonic development and growth control.^{235, 236} H19 has a dual role in cancer. It can function as a tumor suppressor in certain contexts, while in others, it may act as an oncogene promoting cancer progression and metastasis.²³⁷ H19 can function as a molecular sponge, sequestering microRNAs (miRNAs) and affecting their regulatory functions. It also interacts with various proteins to influence cellular processes like cell cycle, apoptosis, and metastasis.

Recent studies have also suggested a role for H19 in regulating ubiquitination and proteasomal degradation. For example, H19 is involved in the regulation of HIF1α (Hypoxia-Inducible Factor 1-alpha) under hypoxic conditions is an intricate and significant aspect of cellular response to low oxygen levels. ^{194-197, 238} HIF1α is a key transcription factor that mediates cellular adaptation to hypoxia, a condition where there is a deficiency of oxygen in the tissue. Under hypoxic conditions, H19 is upregulated, and this upregulation plays a crucial role in modulating the response of cells to low oxygen levels. H19 achieves this by influencing the stability and activity of HIF1α.^{194-197, 238} Normally, in the presence of oxygen, HIF1α is rapidly degraded. However, under hypoxic conditions, HIF1α becomes stabilized and activates the transcription of various genes that help the cell adapt to low oxygen levels. H19 contributes to this process by interacting with various molecular partners that affect HIF1α stability.²³⁸ One of the mechanisms involves the regulation of HIF1α mRNA. H19 can bind to HIF1α mRNA, thereby protecting it from degradation. This interaction enhances the stability of HIF1α mRNA, leading to increased levels of HIF1α protein under hypoxic conditions. Furthermore, H19 can also interact with proteins that are involved in the ubiquitination and degradation of HIF1α. By inhibiting these interactions, H19 helps in maintaining the stability of HIF1α protein²³⁸ (Figure 7). For instance, H19 might interfere with the function of VHL (Von Hippel-Lindau tumor suppressor), a protein that targets HIF1α for degradation under normoxic conditions. Additionally, H19 may also be involved in the epigenetic regulation of HIF1α. By altering the chromatin structure around the HIF1α gene, H19 could affect the transcriptional efficiency of HIF1α, thus influencing its overall expression and activity.^236-238 The role of H19 in regulating HIF1α under hypoxia has significant implications, particularly in the context of cancer. Many tumors are characterized by hypoxic regions, and the H19-HIF1α pathway can contribute to the adaptation of cancer cells to these low oxygen conditions, promoting tumor progression and resistance to therapy.

Figure 7: Role of H19 in protein degradation during hypoxic conditions.

Under conditions of low oxygen availability, H19 expression is elevated, which significantly impacts the cellular adaptation mechanisms to hypoxia. Typically, HIF1α undergoes rapid proteasomal degradation in normoxic conditions. However, hypoxia triggers the stabilization of HIF1α, allowing it to activate the transcription of a variety of genes that equip the cell to cope with decreased oxygen levels. Under hypoxia, elevation of H19 enhances HIF1α stability. There are two potential modes through which H19 may facilitate HIF1α stabilization. Firstly, H19 binds directly to HIF1α mRNA, shielding it from degradation pathways, which results in increased HIF1α protein synthesis during hypoxic stress. Secondly, H19 interacts with proteins that promote the ubiquitination and subsequent degradation of HIF1α protein. By interrupting these interactions, H19 effectively contributes to the persistence of HIF1α under hypoxic conditions.

Additionally, H19 is associated with the regulation of Huwe1, an E3 ligase containing HECT domain.²³⁹ Study reports that genetic deletion of Huwe1 in the mouse inhibits transformation of ovary surface epithelium cells without significantly affecting cell survival and apoptosis. In Huwe1-deficient cells, expression of histone H1.3 increased, inhibiting the expression of noncoding RNA H19. H19 silencing phenocopied the effects of Huwe1 deficiency, whereas H1.3 silencing partially rescued the expression of H19 and the Huwe1-null phenotype.²³⁹

H19 lncRNA can be processed to produce miR-675, a microRNA that plays a crucial role in regulating the UPS.^{240, 241} The miR-675 derived from H19 directly targets FADD, associated with liver necroptosis. It also results in progression of epithelial-mesenchymal transition of human cutaneous squamous cell cancer. Overall, these studies suggest that H19 plays an important role in regulating ubiquitination and proteasomal degradation, and dysregulation of H19 expression may contribute to the development of various diseases, including cancer.

UCA1 in ubiquitination and proteasomal degradation

The long non-coding RNA UCA1 (Urothelial Carcinoma-Associated 1) is a lncRNA approximately 1.4 kilobases in length.^{242, 243} UCA1 was initially identified in bladder cancer. ²⁴⁴ Comparative genomics studies indicate that UCA1 is present in mammals, with homologous sequences identified in primates, rodents, and other vertebrates. ²⁴⁵ The evolutionary conservation of UCA1, particularly in mammals, suggests it has been subject to selective pressures that preserve its function in regulating cellular processes. ²⁴⁵

In human, UCA1 gene is located on chromosome 19p13.12 and this locus is notable for its association with several genes involved in cancer and other diseases. Itis a well-studied lncRNA implicated in cell proliferation, migration, and invasion, particularly in cancer progression.^{242, 243} UCA1 has been found to interact with microRNAs (miRNAs) during the pathogenesis of cancer. This interaction can influence various aspects of cancer progression, including cell proliferation, invasion, drug resistance, and metabolism. ²⁴⁶ UCA1 is often overexpressed in many types of cancer and is involved in carcinogenesis. Its overexpression has been linked to poor prognosis in several cancers, including bladder cancer, breast cancer, and colorectal cancer.²⁴²

UCA1 (lncRNA UCA1) has been implicated in the modulation of the phosphatidylinositol-3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling axis and its associated molecular mediators.^{242, 247, 248} Notably, overexpression of lncRNA UCA1 is linked to elevated levels of several pivotal proteins within this signaling pathway. These proteins include AKT serine/threonine kinase 3 (AKT3), the phosphorylated form of the mammalian target of rapamycin (p-mTOR), and the ribosomal protein S6 kinase (S6K), alongside a reduction in the levels of the eukaryotic translation initiation factor 4E (EIF4E) in gastric cancer (GC) cells.^{242, 247, 248} This complex regulatory network fosters GC cell growth and proliferation by fine-tuning the expression and activity of key signaling molecules involved in cellular metabolism and growth processes.

Furthermore, research by Wang and colleagues delineates a mechanism whereby specificity protein 1 (SP1) augments lncRNA UCA1 expression in GC cells. ^{247, 248} This upregulation occurs via SP1's binding to the core promoter region of UCA1. The resultant overexpressed lncRNA UCA1 engages in a critical interaction with enhancer of zeste homolog 2 (EZH2), a histone methyltransferase, thereby modulating the AKT/GSK-3B/cyclin D1 signaling pathway. This interaction not only boosts EZH2 levels but also upregulates cyclin D1 expression, facilitating cell cycle progression. Such findings underscore the role of lncRNA UCA1 in epigenetic modulation, particularly histone modification, further corroborating its significance in gene regulation and cancer progression.

Recent studies have also shown the involvement of UCA1 in the regulation of ubiquitination and proteasomal degradation. LncRNA UCA1 has been identified as a key regulator of gastric cancer metastasis through its influence on G protein-coupled receptor kinase 2 (GRK2) and Casitas B-lineage Lymphoma (Cbl-c)-mediated ubiquitination.^{247, 248} By interacting directly with GRK2, lncRNA UCA1 exposes ubiquitination sites on GRK2, earmarking it for Cbl-c-mediated degradation. This action results in the activation of the extracellular-signal-regulated kinase (ERK)/matrix metalloproteinase-9 (MMP-9) signaling pathway, leading to elevated MMP-9 levels that degrade cell membrane components, thereby enhancing cancer cell migration and invasion capabilities. In the realm of chemoresistance, lncRNA UCA1 plays a pivotal role through miRNA signaling pathways. Silencing of lncRNA UCA1 has been shown to upregulate miR-27b mRNA levels, resulting in lowered half maximal inhibitory concentration (IC50) values for chemotherapeutic agents such as doxorubicin, cisplatin, and 5-fluorouracil, alongside an increase in doxorubicin-induced apoptosis in doxorubicin-resistant SGC-7901 GC cells.²⁴⁹ This suggests that downregulation of lncRNA UCA1 expression may enhance the chemosensitivity of gastric cancer cells to certain chemotherapy drugs. Supporting this, a later study has reported that silencing lncRNA UCA1 augments the chemosensitivity of gastric cancer cells to cisplatin by modulating the expression of miR-513a-3p and Cytochrome P450 1B1 (CYP1B1), providing a potential therapeutic avenue to combat chemoresistance in gastric cancer.²⁵⁰

An independent study found that UCA1 can alleviate arsenic induced G2/M phase arrest in human liver cells.²⁵¹ This is achieved through UCA1's interaction with the enhancer of zeste homolog 2 (EZH2), a protein involved in cell cycle regulation. UCA1 accelerates the turnover rate of EZH2 protein under normal and arsenic-exposure conditions, thereby influencing the cell cycle progression. The study also found that UCA1 enhances the interaction between cyclin-dependent kinase 1 (CDK1) and EZH2, leading to increased phosphorylation of EZH2 and its subsequent degradation.

These findings suggest that UCA1 plays a multifaceted role in cellular processes, particularly in the context of protein degradation and cancer progression. However, the exact mechanisms and full range of functions of UCA1 are still being explored.

MALAT1 in ubiquitination and proteasomal degradation

MALAT1 (Metastasis-associated lung adenocarcinoma transcript 1), also recognized as Nuclear Enriched Abundant Transcript 2 (NEAT2), was initially discovered through a microarray analysis of non-small cell lung cancer tumors, highlighting its upregulation in more metastatic cases.²⁵² This gene resides on human chromosome 11q13.1 and its mouse counterpart on chromosome 19qA, situated within a gene-dense area of high syntenic evolutionary conservation. Despite its length approximately 8.7 kilobases in humans and 6.7 in mice and being transcribed by RNA polymerase II, MALAT1 is confirmed as non-coding due to its low protein-coding potential.^{252, 253} A notable aspect of MALAT1 is its exceptional sequence conservation across vertebrates, with over 50% overall conservation and more than 80% at the 3′ end, a rarity among long non-coding RNAs (lncRNAs), as less than 10% exhibit such significant evolutionary conservation. MALAT1's high degree of sequence conservation is crucial for its function, as significant deviations might disrupt its ability to interact with target molecules or fold into necessary secondary structures. ^{254, 255} In particular, certain regions within MALAT1, such as the 3' end triple helix structure, are highly conserved, indicating their critical role in stabilizing the RNA molecule and protecting it from exonucleolytic degradation. ²⁵⁵ Beyond the sequence, the secondary and tertiary structures of MALAT1 are also conserved. The formation of secondary structures such as hairpins, loops, and the triple helix is vital for MALAT1’s interaction with proteins and other RNAs. These structures facilitate its localization to nuclear speckles and its role in modulating splicing and gene expression. ²⁵⁶ The conservation of these structural motifs across species suggests that these elements are essential for the RNA’s function, allowing it to maintain its regulatory roles in diverse biological contexts. The evolutionary conservation of MALAT1 is not merely a matter of sequence and structure but extends to its functional roles in cellular processes. MALAT1’s involvement in the regulation of alternative splicing, gene expression, and chromatin dynamics appears to be conserved across species, indicating that these functions are fundamental to eukaryotic cellular biology. ^{257, 258} Studies in model organisms such as mice have shown that MALAT1 deletion affects gene expression patterns, particularly those related to cell cycle regulation and metabolism, highlighting its conserved role in these essential processes. ²⁵⁹ Alignment of the human MALAT1 sequence with orthologous sequences in other vertebrates reveals a high degree of conservation, particularly in regions corresponding to functional domains. ²⁶⁰ These findings support the notion that MALAT1’s regulatory functions have been preserved through evolutionary time due to the selective advantage they confer.

MALAT1's expression is ubiquitously high across all tissues, comparable to housekeeping genes like β-Actin, with the highest expression observed in ovaries.^{252, 261} This widespread expression, attributed to its strong promoter activity and RNA stability, contributes to its cellular abundance. MALAT1’s complexity extends to its alternatively spliced isoforms and interaction with a natural antisense transcript, TALAM1, which seems to enhance MALAT1’s stability and cellular levels. Notably, simultaneous knockdown of MALAT1 and TALAM1 in breast cancer cells has shown a significant reduction in cell migration, invasion, and metastasis, underscoring its potential role in cancer progression.^{252, 261}

MALAT1 is involved in multiple signaling pathways, including those related to cancer progression, cell growth, and survival.^{252, 261} It can modulate signaling by interacting with key proteins or RNA molecules. In various cancers, MALAT1 is associated with tumor growth, metastasis, and poor prognosis. It promotes cancerous behaviors in cells, such as proliferation, migration, and invasion. MALAT1 plays a role in the formation of blood vessels (angiogenesis), essential for both normal physiological processes and in tumor growth. MALAT1 contributes to the organization of nuclear speckles, subnuclear structures involved in RNA processing and transport.^{262, 263}

MALAT1 has been implicated in the modulation of ubiquitination and proteasome degradation pathways. A recent study sheds light on the pivotal role of MALAT1 in promoting hepatic lipid accumulation and insulin resistance, a critical concern in obesity and type 2 diabetes management.²⁵³ Excessive levels of palmitate were shown to elevate MALAT1 expression in hepatocytes and in the livers of genetically obese (ob/ob) mice, leading to the activation of SREBP-1c, a key transcription factor that drives lipid biosynthesis, resulting in increased intracellular lipid storage. Notably, suppressing MALAT1 not only reduced SREBP-1c levels and lipid accumulation both in lab cultures and live models but also enhanced insulin sensitivity in ob/ob mice. A fascinating aspect of this study is the mechanism of action of MALAT1, particularly its regulation of SREBP-1c through ubiquitination.²⁵³ The findings indicate that MALAT1 directly interacts with nuclear SREBP-1c, stabilizing it and preventing its ubiquitination, thereby increasing lipid synthesis enzymes' expression. This post-transcriptional regulation underscores the intricate ways in which lncRNAs influence metabolic pathways. The study underscores the potential of targeting MALAT1 as a therapeutic strategy to mitigate hepatic steatosis and improve insulin sensitivity, pointing towards a new direction in the treatment of obesity-related metabolic disorders. The interaction between MALAT1 and SREBP-1c, particularly through the modulation of ubiquitination, highlights a novel regulatory mechanism of lipid metabolism that could be pivotal for future interventions.

These examples demonstrate the involvement of MALAT1 in ubiquitination and proteasome degradation pathways. Further studies are needed to elucidate the molecular mechanisms underlying these interactions and explore the potential of MALAT1 as a therapeutic target in various human diseases.

LncRNA NEAT1 in ubiquitination and proteasomal degradation

LncRNA NEAT1 (Nuclear Enriched Abundant Transcript 1, also known as Nuclear Paraspeckle Assembly Transcript 1) is encoded by the NEAT1 gene, which is situated on human chromosome 11q13.1.²⁶⁴ This lncRNA has emerged as a key player in the orchestration of nuclear body formation, particularly paraspeckles, which are essential in gene expression regulation, nuclear retention of defective RNAs, and various stress responses. Yet, as the breadth of NEAT1's influence becomes more apparent, intriguing connections between this lncRNA and the processes of ubiquitination and proteasomal degradation have begun to surface. Initially, it's prudent to understand NEAT1's canonical function. Serving predominantly as scaffolds, lncRNAs, like NEAT1, facilitate the assembly of diverse protein complexes.²⁶⁴ Such scaffolding roles have placed lncRNAs at the nexus of multiple processes. Intriguingly, a hypothesized function for NEAT1 is in the assembly of ubiquitin ligases and their substrates. While the specifics remain to be elucidated, a direct mediation of ubiquitination by NEAT1 would represent a convergence of its scaffolding function with protein turnover processes.²⁶⁴ Moreover, the molecular characteristics of NEAT1 render it a potential candidate for direct interactions with proteins, possibly modifying their ubiquitination status. Some lncRNAs are known to protect or even promote the degradation of specific proteins, and NEAT1's broad interactome might position it similarly. If NEAT1 were to bind certain proteins, this could change the accessibility of lysine residues, modifying ubiquitination sites, and thus protein stability.

Upon proteasome inhibition, NEAT1 transcription is significantly upregulated, leading to a marked elongation of paraspeckles.^264-266 This was demonstrated through luciferase-reporter assays, which indicated heightened NEAT1 promoter activity under these conditions. Interestingly, despite their elongation, these paraspeckles were not found to be enriched in ubiquitin-conjugated aggregates, nor did they show an increased density of paraspeckle proteins (PSPs). However, they did sequester a larger proportion of the total PSP pool due to their increased length. This enlargement of paraspeckles is attributed primarily to the elevated levels of NEAT1, aligning with previous findings of NEAT1 up-regulation and paraspeckle enlargement during myotube differentiation and sustained NEAT1 transgene expression. These observations also reinforce the idea that NEAT1 synthesis is a crucial factor in paraspeckle formation.^264-266 In HeLa cells, paraspeckle width appears to be constant, possibly reflecting NEAT1's molecular size, and there is a correlation between paraspeckle length and the amount of NEAT1 transcribed.²⁶⁶ The mechanism governing the establishment of the optimal length of newly formed paraspeckles remains unclear, but proteasome inhibition might delay their detachment from chromatin, resulting in significant elongation. Additionally, NONO and SFPQ, known NEAT1-associated proteins, are shown to be sequestered within these elongated paraspeckles following proteasome inhibition, leading to a reduction in their nucleoplasm availability (Figure 8).²⁶⁶ The intensity of this sequestration suggests that this stress-induced mechanism could significantly impact gene expression, considering the potential trapping of various other PSPs within paraspeckles under these conditions. An alternate avenue to consider is NEAT1's potential indirect impact on the ubiquitin-proteasome system. It is conceivable that NEAT1 may influence the transcription or stability of key proteins involved in this system. Interactions with transcription factors, chromatin remodelers, or RNA-binding proteins could subtly tilt the balance of ubiquitination regulators. Thus, NEAT1 might exert effects that cascade down to influence ubiquitination and degradation without direct involvement in the process. Further, NEAT1's dysregulation in diseases, especially in cancers, offers compelling circumstantial evidence of its role in protein degradation.

Figure 8: Role NEAT1 and paraspeckle formation in proteasomal degradation.

This illustrates the relationship between NEAT1 expression and paraspeckle morphology, revealing that the expansion of paraspeckles is closely tied to increased levels of NEAT1, as observed during myotube differentiation. In HeLa cells, while the width of paraspeckles is constant, suggesting a link to NEAT1's structure, their length varies with the amount of NEAT1 RNA. Proteasome inhibition is shown to extend paraspeckles, sequestering NEAT1-binding proteins like NONO and SFPQ and reducing their presence in the nucleoplasm. Although the precise mechanisms that determine the optimal length of newly formed paraspeckles are not yet fully understood, it is postulated that proteasome inhibition could impede their release from chromatin, leading to pronounced elongation.

Along with the Paraspeckle Assembly, NEAT1 is also involved in ubiquitination of phosphatase and tensin homolog (PTEN)-induced kinase 1 (PINK1).²⁶⁷ It is reported that elevated NEAT1 level leads to an increase in PINK1 level by influencing its ubiquitination and preventing its degradation. Notably, the overexpression of PINK1 reversed the beneficial effects of NEAT1 silencing on cell survival. Overall, the finding established link between inflammation and NEAT1 involved ubiquitination.

The lncRNA NEAT1 is found to be regulating inflammation in the peripheral system and the regulation of cytokine expression, activation of microglia, expression of lipid.²⁶⁸ Nong et al. demonstrated that silencing NEAT1 enhanced cell viability and mitigated inflammatory responses, primarily through modulation of the TLR4/NF-κB signaling pathway.²⁶⁹ They also found that TNF receptor-associated factor 6 (TRAF6), an E3 ubiquitin ligase crucial for signaling in the IL-1 receptor/TLR family and TNFR superfamily, is implicated in NF-κB activation. Yamamoto et al., utilizing TRAF6-deficient mice, showed the indispensable role of TRAF6 in numerous pathophysiological processes via NF-κB pathway activation.²⁷⁰ Previous studies suggested that reduced NEAT1 expression interacts with TRAF6, diminishing its levels and markedly suppressing anti-inflammatory cytokine expression. ²⁷¹

It is also found that NEAT1 blocks PGK1 degradation via the ubiquitin–proteasome pathway where silencing NEAT1 leads to downregulation of PGK1.²⁷² Notably, the PGK1 (phosphoglycerate kinase 1) is an enzyme playing important role in glucose metabolism and coordinates between glycolysis and TCA cycle.

In summation, the emerging story of NEAT1's interplay with the ubiquitin-proteasome system underscores the multifaceted roles lncRNAs play within cellular circuitry. Although our understanding remains in its infancy, it is apparent that NEAT1, and lncRNAs in general, may hold the keys to unlocking novel regulatory mechanisms that govern protein turnover, with significant implications for health and disease.

LncRNA LINC01138 in ubiquitination and proteasomal degradation

LINC01138, positioned at chromosome 1q21.2, plays a crucial role in hepatocellular carcinoma (HCC), showing marked amplification and heightened expression in HCC tissues.^{273, 274} Its expression levels are strongly correlated with the clinical and pathological characteristics of HCC, making it a valuable prognostic biomarker due to its association with reduced survival rates among patients. Furthermore, LINC01138's oncogenic activities extend to prostatic cancer, suggesting its utility as a broad-spectrum oncogene.²⁷³ The research investigates LINC01138's biological roles, pinpointing its interactions with IGF2BP1, IGF2BP3, and PRMT5. These proteins, by binding to specific regions of LINC01138, form an oncogenic axis that enhances the stability of LINC01138 RNA, promoting cellular processes such as proliferation, colony formation, and migration in HCC. This axis delineates LINC01138's complex involvement in cancer progression and underscores its therapeutic potential.

Further, the study delves into the multi-layered regulation of LINC01138 expression, encompassing genomic, transcriptional, and post-transcriptional aspects, which underscores its intricate role in HCC. Among the regulatory mechanisms, the activation by androgen receptor (AR) and stabilization by IGF2BP1 and IGF2BP3 are particularly noteworthy, presenting LINC01138 as a potential target for therapeutic intervention.²⁷³ PRMT5, identified as a downstream target of LINC01138, is crucial for the methylation of arginine residues on various protein substrates, influencing NF-κB-dependent gene expression. This interaction signifies a novel regulatory pathway, with PRMT5 acting as a mediator of LINC01138's oncogenic effects, further contributing to cancer cell proliferation, cell cycle progression, and metastasis.²⁷³ The study also proposes the potential therapeutic benefits of targeting PRMT5 with specific inhibitors, such as PJ-68 and HLCL-61, which disrupt its association with LINC01138, offering a promising strategy for treating HCC patients with elevated LINC01138 levels. This approach highlights the therapeutic significance of the LINC01138-PRMT5 axis in HCC progression.

In osteoarthritis (OA), LINC01138 is similarly implicated in disease progression, particularly through its regulation of the hsa-miR-1207-5p/KIAA0101 axis. ²⁷⁵ OA's hallmark, ECM degradation, involves the diminishing synthesis of key components like aggrecan and Col2α1, alongside increased MMP-13 production. LINC01138 downregulation counters ECM degradation and inflammatory cytokine production, showcasing a pathway for OA management.

LINC01138 functions as miRNA sponges, regulating various cellular processes. For instance, LINC01138's interaction with hsa-miR-1207-5p and its downstream target KIAA0101, a protein involved in cell proliferation and DNA repair, elucidates a regulatory mechanism in OA.²⁷⁵ Silencing KIAA0101 negates the effects of hsa-miR-1207-5p inhibition on chondrocytes, emphasizing its role in OA pathology. Notably, several signaling pathways such as MAPK, PI3K/Akt, and JAK/STAT are involved in OA, with KIAA0101 modulating the JAK/STAT and Wnt pathways. This modulation by KIAA0101, especially in inhibiting these pathways, underscores a novel therapeutic angle in OA treatment, focusing on the molecular interactions between LINC01138, hsa-miR-1207-5p, and KIAA0101.

LINC01138 has been identified as a crucial regulator in the cellular environment, particularly influencing the protein levels of PRMT5 without altering its mRNA levels. ^{273, 274} This lncRNA exerts its effects through a specific mechanism where it prevents the interaction between PRMT5 and the E3 ubiquitin ligase CHIP, thereby inhibiting PRMT5 degradation. Gene set enrichment analysis has revealed that LINC01138 and PRMT5 share similar downstream signalling pathways, suggesting that PRMT5 acts as a critical intermediary in LINC01138's contribution to oncogenesis.

In summary, LINC01138 emerges as a significant molecular player in HCC and OA, involved in complex regulatory networks that influence cancer progression and joint degeneration. Its interactions with specific proteins and miRNAs highlight potential therapeutic targets, offering insight into novel treatment strategies for these conditions. The multi-dimensional regulation of LINC01138, coupled with its broad oncogenic potential, underscores the need for further research to fully understand its role in disease progression and therapy.

Other lncRNAs linked to protein degradation

Over recent years, the spotlight has increasingly turned to long non-coding RNAs (lncRNAs) as critical regulators of a plethora of cellular processes. While many have started uncovering their significant roles in cellular functions, there remains a myriad of lncRNAs whose functions are just beginning to be elucidated. Among these, several have been shown to have intricate links to the ubiquitination-proteasomal degradation pathway, a core mechanism that governs protein turnover and maintains cellular homeostasis. Understanding the interaction between these minor lncRNAs and the ubiquitin-proteasome system provides deeper insights into the layered complexities of cellular regulation and the development of various pathologies.

Gemcitabine resistance poses a significant challenge in PDAC treatment, with lncRNAs emerging as crucial players in cancer progression. SLC7A11-AS1 is identified as being overexpressed in PDAC tissues and resistant cell lines, contributing to chemoresistance by reducing intracellular ROS levels through NRF2 stabilization. ²⁷⁶ Mechanistically, SLC7A11-AS1 interacts with β-TRCP1, preventing NRF2 degradation and maintaining low ROS levels necessary for cancer stemness. Targeting SLC7A11-AS1 could potentially overcome gemcitabine resistance in PDAC, suggesting it as a promising therapeutic target.

In a diabetic cerebral ischemia/reperfusion (I/R) model, LncRNA Fendrr expression was notably increased alongside heightened levels of NLRC4 inflammatory complex and pyroptosis-mediated inflammatory factors. ²⁷⁷ Knockdown of Fendrr in high glucose-treated hypoxia/reoxygenation (H/R)-induced microglia led to decreased NLRC4 and associated inflammatory cytokines linked to pyroptosis. Fendrr interacted with HERC2 protein, facilitating the inhibition of NLRC4 ubiquitination, while HERC2 promoted NLRC4 ubiquitination. Moreover, HERC2 overexpression reversed the effects of Fendrr overexpression in the diabetic cerebral I/R model of microglia, indicating Fendrr's role in protecting against NLRC4 protein degradation, thus promoting microglial pyroptosis.

In colorectal cancer, there is an upregulation of lncRNA SNHG15 which works to sustain the stability of Slug by inhibiting its ubiquitination and subsequent degradation through its interaction with the zinc finger domain of Slug. ²⁷⁸ In hepatocellular carcinoma, LINC00473 interacts with the oncoprotein survivin, modulating its stability.²⁷⁹ Additionally, LINC00473 recruits deubiquitinase USP9X to curtail survivin ubiquitination, thereby augmenting survivin expression. Furthermore, lncRNA NBAT1 binds to PSMD10, facilitating its degradation, consequently diminishing PSMD10 and HSF1 occupancy at the ATG7 promoter and suppressing ATG7 transcription. ²⁸⁰

The long non-coding RNA ANCR is implicated in modulating EZH2 stability. ²⁸¹ EZH2 functions as a crucial epigenetic regulator and inducer of epithelial-to-mesenchymal transition (EMT), contributing to metastasis across multiple cancer types. Specifically, ANCR facilitates the association between CDK1 and EZH2, resulting in heightened EZH2 phosphorylation, subsequent ubiquitination, and eventual degradation.

LncRNA PVT1 has been observed to contribute to the degradation of the tumor suppressor p15 by safeguarding the E3 ubiquitin ligase SKP2 from ubiquitination and subsequent proteasomal degradation.^{3, 282} The implications are manifold, especially when considering that this process can support oncogenesis by reducing the levels of tumor suppressors in the cellular environment.

LncRNA ZNNT1 is pivotal in promoting the tumorigenic properties of colon cancer cells harboring wild-type p53. Additionally, ZNNT1 contributes to the degradation of p53 by disrupting its interaction with the SART3-USP15 complex.²⁸³ These insights could be invaluable for the development of cancer therapies.

In conclusion, the emerging research surrounding lncRNAs and their relationship with the ubiquitination-proteasomal degradation pathway represents a frontier in cellular biology that promises to reshape our understanding of intracellular regulatory mechanisms. As these lncRNAs are further studied, it is likely that more intricate pathways and networks will be discovered, broadening the horizon of potential therapeutic targets, especially in the context of pathological conditions where ubiquitination and degradation play pivotal roles. A deeper dive into these lesser-known lncRNAs is not only imperative for the academic enrichment of the field but also for the development of future diagnostic and therapeutic tools that can be harnessed in the fight against a variety of diseases.

MicroRNAs in Protein Degradation

MicroRNAs (miRNAs) are small non-coding RNA molecules that play a critical role in the regulation of gene expression.^284-287 They are involved in various cellular processes, including protein degradation. MiRNAs primarily function by binding to the 3' untranslated regions (3' UTRs) of target messenger RNAs (mRNAs), leading to either degradation of the mRNA or inhibition of its translation. This post-transcriptional regulation can indirectly affect protein degradation pathways. For instance, miRNAs can target mRNAs encoding for proteins that are involved in the ubiquitin-proteasome system (UPS) or autophagy-lysosome pathway (ALP), two major protein degradation systems in eukaryotic cells. MiRNAs have been shown to regulate various components of the UPS, including E3 ubiquitin ligases and deubiquitinating enzymes (DUBs). Emerging research underscores the role of microRNAs (miRNAs) in the regulation of muscle atrophy, acting through various mechanisms including the modulation of MuRF1 and MAFbx expression. For instance, miR-23a has been identified as a negative regulator of these E3 ligases, offering protection against glucocorticoid-induced muscle atrophy in transgenic mice models.^{288, 289} Similarly, muscle-specific miR-1 is upregulated in a dexamethasone (Dex)-induced mouse model of atrophy, enhancing the expression of MuRF1 and MAFbx through the HSP70/protein kinase B (Akt)/Forkhead box (Fox) O3 signaling pathway, contributing to Dex-induced muscle atrophy.^{288, 290} Furthermore, the miR-199/214 cluster has been implicated in the regulation of the ubiquitin-proteasome pathway, highlighting the complex interplay of miRNAs in muscle atrophy development.^{288, 291} (Table 3)

Table 3:

MicroRNAs in Ubiquitination and Proteasomal Degradation

MicroRNA	Target	Mechanism	REF
miR-1	MuRF1, MAFbx	Induces MuRF1 and MAFbx expression via the HSP70/protein kinase B(Akt)/forkhead box (Fox) O3 signaling pathway. Cause of Dex-induced muscle atrophy	284, 289, 290
miR-23a	MuRF1, MAFbx	Inhibits the translational activation of MuRF1 and MAFbx via binding with their 3′-UTR	284, 285, 288, 289, 291, 292
miR-199/214 cluster	MuRF1, MAFbx	Regulation of atrophic gene expression in a PI3K/Akt/FoxO-dependent manner	288, 289, 291-293
miR-182	Atrophic genes	Represses atrophic gene expression via inhibition of FoxO protein translation	284, 285, 288, 289, 291, 292
miR-486	PTEN	Suppressing PTEN expression enhances PI3K/Akt activation and promotes FoxO phosphorylation. Phosphorylated FoxO is located in the cytoplasm and is degraded by the proteasome.	284, 285, 288, 289, 291, 292
miR-71	ERAD	Promotes ubiquitin-dependent protein turnover to maintain proteostasis and longevity in nematodes	284, 285, 288, 289, 291, 292
miR-30	HD	Inhibits ubiquitin complex binding with proteasome	288, 289

MiRNAs also target a variety of proteins to regulate myogenesis.²⁹² Myostatin, a negative mediator of myogenesis, is regulated by miR-27a and miR-27b, which promote satellite cell proliferation and myogenesis by suppressing myostatin expression. Additionally, miRNAs such as miR-125b, miR-133, and miR-199a-3p are involved in the regulation of the insulin-like growth factor/insulin-like growth factor receptor signaling pathway, influencing cell differentiation and muscle regeneration.^{288, 292, 293} MiR-203 and miR-155 are known to suppress myoblast differentiation by targeting c-Jun and myocyte enhancer factor 2C (MEF2C) and MEF2A, respectively. Conversely, miR-29 acts as a pro-myogenic factor by downregulating Akt3 or RING1 and YY1-binding protein.^{288, 292, 293}

The phosphoinositide 3-kinase/Akt/FoxO (PI3K/Akt/FoxO) signaling pathway is crucial in muscle atrophy, where Akt inactivation leads to the promotion of muscle atrophy through various mechanisms, including the dephosphorylation and nuclear translocation of FoxO transcription factors, which are key regulators of muscle protein degradation.^{288, 292, 293} MiRNAs such as miR-486 and miR-182 have been shown to modulate this pathway by targeting PTEN and FoxO3a, respectively, thereby influencing muscle atrophy and regeneration.^{288, 292, 293} Specifically, miR-486 overexpression, observed in myostatin knockout mice and skeletal muscle, plays an essential role in maintaining skeletal muscle size through the Akt/mTOR signaling pathway. Additionally, muscle-specific miR-1 promotes muscle atrophy by dephosphorylating and activating FoxO3a in an HSP70/Akt-dependent manner.^{288, 292, 293}

Dysregulation of miRNAs can lead to aberrant protein degradation, contributing to the pathogenesis of various diseases. In cancer, for instance, miRNA dysregulation can result in the destabilization of tumor suppressor proteins or the overexpression of oncogenic proteins. In neurodegenerative diseases like Alzheimer's, altered miRNA expression can affect the degradation of amyloid-beta peptides, influencing disease progression. The intricate role of miRNAs in protein degradation underscores their importance in maintaining cellular homeostasis and their potential as therapeutic targets. Understanding the specific miRNAs involved in protein degradation pathways could lead to novel strategies for treating diseases associated with protein aggregation and degradation dysfunction.

Targeting LncRNA for treatment of abnormal protein degradation linked to human diseases

The understanding of cellular intricacies has witnessed a paradigm shift with the rise in appreciation of long non-coding RNAs (lncRNAs). While a substantial amount of research has focused on understanding the role of lncRNAs in various pathological states, one avenue that remains a subject of intense investigation is their involvement in abnormal protein degradation linked human diseases.^{3, 11, 13, 15, 171, 294} Their specific expression patterns and stability in body fluids make lncRNAs attractive as potential biomarkers, and their intricate regulatory functions deem them noteworthy therapeutic targets.

Aberrant protein degradation is at the epicenter of various human diseases. For instance, neurodegenerative diseases such as Alzheimer's, Parkinson's, and Huntington's are characterized by abnormal protein aggregations due to faulty degradation processes. In such contexts, lncRNAs have emerged as influential players. BACE1-AS is one such lncRNA whose expression is upregulated in Alzheimer's patients.^{3, 11, 171, 172, 294} It stabilizes BACE1 mRNA, leading to increased levels of BACE1 protein, an enzyme responsible for producing amyloid-beta plaques—a hallmark of Alzheimer's. Such findings highlight the potential of lncRNAs like BACE1-AS not only as biomarkers for disease progression but also as therapeutic targets to modulate disease pathophysiology. In the realm of cancer, dysregulation of protein homeostasis due to abnormal ubiquitination and degradation is frequently observed.^{3, 11, 171, 172, 294} In hepatocellular carcinoma (HCC), the upregulation of lncRNA HULC detected via microarray analysis was found to be linked to aberrant lipid metabolism, underscoring its potential as both a biomarker and therapeutic target.^295-297 Following discovery, validation using techniques such as qRT-PCR on larger patient cohorts becomes essential to confirm the specificity and sensitivity of the lncRNA as a biomarker. Apart from the aforementioned diseases, cardiac pathologies also exhibit connections with lncRNAs and abnormal protein degradation.^{11, 12, 294} In myocardial infarction, the lncRNA MIAT is upregulated, and its knockdown has been shown to reduce myocardial apoptosis, suggesting its potential as a therapeutic target. Similarly, in conditions like muscular dystrophy, where protein degradation is detrimental, the lncRNA linc-MD1 acts as a ceRNA, modulating miRNAs and thus influencing the dystrophic phenotype.^{11, 12, 294}

Among diseases associated with defects in protein degradation, Von Hippel Lindau (VHL) disease is a rare genetic disorder characterized by the formation of tumors and cysts in different parts of the body.^298-302 It's caused by mutations in the VHL gene, which is a tumor suppressor gene. The VHL protein, encoded by the VHL gene, plays a crucial role in cellular responses to oxygen levels and in protein degradation pathways, particularly through the ubiquitin-proteasome system.^298-302 In normal cells, the VHL protein is part of a complex that targets certain proteins for degradation. One of its key functions is to regulate the levels of hypoxia-inducible factor (HIF), a protein that helps cells respond to low oxygen conditions. Under normal oxygen conditions, VHL binds to HIF and targets it for degradation via the ubiquitin-proteasome pathway. However, in low oxygen conditions, or when the VHL protein is dysfunctional due to mutation, HIF is not properly degraded. The dysfunction in the VHL protein leads to an accumulation of HIF, even under normal oxygen conditions.^298-302 This accumulation results in the overactivation of various genes that HIF regulates, including those involved in cell proliferation, angiogenesis (the formation of new blood vessels), and metabolism. This overactivation is a significant factor in the development of tumors and cysts characteristic of VHL disease. The direct link between VHL disease and protein degradation is this dysregulation of the HIF pathway due to impaired function of the VHL protein.^298-302 This impairment means the cell behaves as if it is constantly in a low oxygen environment, leading to the uncontrolled growth of cells and formation of tumors. Understanding this link has been crucial for developing treatments for VHL disease. For example, drugs that can inhibit HIF or its downstream effects are being explored as potential therapies. Additionally, insights into the VHL pathway have broader implications for understanding and treating other forms of cancer, where similar mechanisms of dysregulated protein degradation and hypoxia responses may play a role.

While the prospect of utilizing lncRNAs as biomarkers is promising, challenges persist. One of the significant challenges is the often-cited lack of conservation of lncRNAs across species, which makes it difficult to study their functions using conventional model organisms. Additionally, the cellular and tissue-specific expressions of lncRNAs necessitate the collection of precise samples for accurate biomarker analysis. Furthermore, in the therapeutic realm, targeting lncRNAs efficiently without off-target effects remains a considerable hurdle. Yet, the potential rewards of harnessing lncRNAs in the biomarker and therapeutic landscape are immense. The advent of novel technologies, such as CRISPR-Cas9 systems for targeted lncRNA modulation and nanoparticle-based delivery systems for lncRNA mimics or inhibitors, provides a beacon of hope.^{1, 4, 9, 11, 171, 172, 294} With these advancements, it becomes conceivable to think of a future where lncRNAs, intertwined with the ubiquitination-proteasomal degradation pathway, stand at the forefront of diagnostic and therapeutic strategies against a multitude of human diseases.

To summarize, lncRNAs, with their intricate involvement in cellular processes and pathologies related to abnormal protein degradation, have cemented their position as potential powerhouses in the realms of biomarkers and therapeutic targets. While challenges remain, continued research in understanding their functions, coupled with advancements in biotechnological tools, promises a new era where lncRNAs play central roles in managing and treating human diseases.

Conclusion

Long non-coding RNAs (lncRNAs) have emerged as important regulators of gene expression and cellular processes.^{4, 9, 11, 171} This review discusses the role of several lncRNAs, including HOTAIR, H19, UCA1, and MALAT1, and others in the regulation of ubiquitination and proteasomal degradation. The structures and functions of most lncRNAs remain uncharacterized and understanding them remains an ongoing challenge, necessitating a considerable investment of time and effort. Nevertheless, emerging studies suggest that lncRNAs are pivotal to cellular machinery and contribute to cellular and physiological functions including the regulation of protein degradation and turnover. Beyond protein degradation and protein turnover, lncRNA are implicated in regulation of various other post-translational modifications (PTMs) of proteins and nucleic acids modifications. These include phosphorylation ³⁰³, acetylation ³⁰⁴, glycosylation ³⁰⁵, methylation ²⁰⁵, SUMOylation ³⁰⁶, etc. For instance, lncRNA UASR1 promotes breast cancer cell growth and migration via the AKT/mTOR pathway by upregulating phosphorylation of AKT, TSC2, 4EBP1, and p70S6K. ³⁰⁷ H19, overexpressed in colorectal cancer, acts as a competitive endogenous RNA (ceRNA), sponging miRNAs to regulate mRNAs like AKT3 and MET, and inhibits mTORC1-mediated 4E-BP1 phosphorylation. ^{308, 309} In oral squamous cell carcinoma, lncRNA CASC9 activates the AKT/mTOR pathway, enhancing cell aggressiveness. ³¹⁰ Conversely, lncRNA FER1L4 suppresses lung cancer cell proliferation and metastasis by downregulating PI3K and AKT. ³¹¹ Apart from that, acetylation is a type of PTM significant in the regulation of RNA binding proteins (RBPs). LncRNAs have emerged as mediators of RBP acetylation. For example, MALAT1 decreases the acetylation of p53 by competing with sirtuin 1 (SIRT1) and deleted in breast cancer 1 (DBC1) for interaction, thereby freeing SIRT1 and modulating its activity. ³¹² In case of protein methylation, the stability of the protein is often dependent on the function of lncRNAs. For example, the stability of EZH2 protein relies on the lincRNA lncRNA-p21 which not only disrupts the PRC2 complex, leading to the release of EZH2, but also promotes the interaction between EZH2 and STAT3, resulting in STAT3 methylation. ³¹³ During protein glycosylation, lncRNAs mediate several steps in the glycan synthesis. For instance, lncRNA LINC00467 acts as an oncogene in esophageal squamous cell carcinoma by sequestering miR-485-5p, which normally down-regulates DPAGT1, a key mediator in the initial step of N-glycans biosynthesis involving the addition of GlcNAc to dolichol phosphate. ³⁰⁵ As a type of PTM, SUMOylation can alter the stability and localization of proteins by the attachment of polypeptide sequences and other chemical moieties. ³⁰⁶ A recent study demonstrated the oncogenic activity of lncRNA RMST by inhibiting glioma cell mitophagy and discovered that RMST enhances FUS SUMOylation, particularly boosting SUMO1 modification at K333, which promotes the interaction between FUS and hnRNPD, stabilizing their expression and cell mitophagy, highlighting RMST as a promising prognostic factor for glioma patients. ³¹⁴

Targeting these lncRNAs may hold promise as a therapeutic strategy for human diseases. The implications of dysregulated lncRNA-mediated protein modifications including the degradation pathways in various diseases further underscore the significance of this research area. Challenges remain, including the lack of conservation of lncRNAs across species, which complicates functional studies, and the difficulty of targeting lncRNAs without off-target effects. However, advancements in technologies like CRISPR-Cas9 for targeted lncRNA modulation or developing small molecular inhibitors of lncRNAs may offer promising solutions. As the field continues to evolve, further investigations into the precise molecular mechanisms and functional consequences of lncRNA-mediated regulation of protein degradation will undoubtedly uncover new insights and therapeutic opportunities for a wide range of diseases.