Post-transcriptional regulation of gene expression and the intricate life of eukaryotic mRNAs

Abstract

In recent years, there has been a growing appreciation for how regulatory events that occur either co- or post-transcriptionally contribute to control of gene expression. Messenger RNAs (mRNAs) are extensively regulated throughout their metabolism in a precise spatiotemporal manner that requires sophisticated molecular mechanisms for cell-type specific gene expression, which dictates cell function. Moreover, dysfunction at any of these steps can result in a variety of human diseases including cancers, muscular atrophies, and neurological diseases. This review summarizes the steps of the central dogma of molecular biology with a focus on post-transcriptional regulation of gene expression.

Graphical Abstract

The central dogma of biology relies of numerous regulatory events to define the expression profile of any given cell. Created in https://BioRender.com.

Introduction

The expression of each gene is tightly controlled during the flow of genetic information from DNA, to RNA, to protein-- known as the central dogma of molecular biology (Crick, 1970). The central dogma describes the process by which DNA is transcribed into messenger RNA (mRNA) and subsequently translated into protein (Crick, 1970). Proteins produced work in concert to execute a vast array of distinct cellular functions necessary for cell maintenance and survival.

Importantly, DNA can also be transcribed into other classes of “non-coding” RNA (ncRNA) molecules including, but not limited to transfer RNA (tRNA), ribosomal RNA (rRNA), micro RNA (miRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), piwi-interacting RNAs (piRNA), long noncoding RNAs (lncRNAs), and circular RNA (circRNA) (Cable et al., 2021; Zhang et al., 2024). All these distinct types of RNAs have critical functions necessary for cellular maintenance and survival. Importantly “coding” RNA, or mRNA, only makes up about ~2% of the transcriptome, whereas the remaining ~98% consists of ncRNA, which is mostly represented by rRNA (Carninci & Hayashizaki, 2007; Costa et al., 2010; Costa et al., 2013). This review will primarily focus on processing of mRNA.

Precise spatiotemporal regulation of gene expression is critical for cellular differentiation and establishment of cell-type specific functions for proper tissue development (Schieweck et al., 2021). Although much of this regulation is achieved at the level of gene-specific transcription, there are numerous post-transcriptional events that dictate cell-type specific expression patters to govern cellular function. Post-transcriptional regulation of gene expression includes a multitude of distinct pre-mRNA processing events including 5’ capping, splicing, epitranscriptomic modification, polyadenylation, nuclear export, translation, and eventually mRNA decay in the cytoplasm (Figure 1) (Corbett, 2018; Gilbert & Nachtergaele, 2023; Moore, 2005; Schieweck et al., 2021). Each of these steps is intricately coordinated by numerous RNA binding proteins (RBPs) that associate with RNA molecules throughout the lifecycle of the RNA.

Figure 1. The Steps Involved in Gene Expression.

Regulation of gene expression occurs both in the nucleus and the cytoplasm. Transcription of the nascent pre-mRNA molecule occurs in the nucleus where the nascent transcript undergoes co-transcriptional 5’ capping, splicing, and cleavage followed by polyadenylation. The mature mRNA is then exported from the nucleus to the cytoplasm, through the nuclear pore complex. Within the cytoplasm, the mRNA may be transported, translated, and eventually undergoes decay. The nucleus is denoted by the purple background and the cytoplasm is denoted by the light-yellow background. The large purple cylinders indicate the nuclear envelope, and the cage structures represent the nuclear pore complex. Created in https://BioRender.com

1.1. Regulation of gene expression in the Nucleus

Transcription

Transcription is the first step in gene expression, in which a DNA sequence is transcribed into a molecule of RNA by a multiprotein RNA polymerase (RNAP) complex. In eukaryotic organisms, transcription of protein-coding genes is carried out by RNA polymerase II (RNAPII/Pol II) with the help of a multitude of other proteins that act in concert to transcribe specific genes in response to intra- and extracellular signals (Archuleta et al., 2024; Farnung, 2024; Kuldell & Kaplan, 2024). Transcription is critical for almost every cellular process, allowing constitutive production of “housekeeping” genes as well as the dynamic production of regulatory genes in response to specific cellular requirements. There are three intricately regulated phases of transcription: initiation, elongation, and termination (Cramer, 2019; Farnung & Vos, 2022; Kuehner et al., 2011; Lopez Martinez & Svejstrup, 2024; Mohamed et al., 2022; Rodríguez-Molina et al., 2023) (Figure 2).

Figure 2. Transcription.

Initiation: Hypophosphorylated RNA polymerase II (RNAPII) is recruited to a gene promoter into the preinitiation complex (not pictured) and is phosphorylated on serine 5. Elongation: RNAPII synthesizes an RNA molecule from the template DNA and serine 2 is phosphorylated. Termination: RNAPII halts RNA synthesis and both RNAPII and RNA are released from the template DNA and serine 2 phosphorylation is more prevalent (Kuehner et al., 2011). Created in https://BioRender.com

Transcription initiation by RNAPII requires several general transcription factors (GTFs) to properly position RNAPII at the transcription start site (TSS) and separate the DNA strands. These GTFs are designated TFIIA, -B, -D, -E, -F, and -H and together form the preinitiation complex (PIC) on promoter DNA (Farnung & Vos, 2022; Lodish, 2013). The promoter DNA strands are separated, allowing RNAPII to access the template strand of the DNA and begin pre-mRNA synthesis. Once the pre-mRNA strand is approximately ~20–50 nucleotides in length, RNAPII pauses and other elongation factors bind RNAPII (Lodish, 2013). After a stable elongation complex is formed, transcription of the gene can proceed. Once RNAPII has transcribed ~200nts from the TSS, elongation is highly processive through most genes (Farnung, 2024). RNAPII does not terminate transcription until after a sequence is transcribed that directs cleavage and polyadenylation (Lodish, 2013; Lopez Martinez & Svejstrup, 2024). After transcription of the poly(A) addition site, transcription termination can occur at multiple sites within the following 0.5–2kb (Lodish, 2013; Rodríguez-Molina et al., 2023).

The largest subunit of RNAPII, RPB1, contains a carboxyl terminal domain (CTD) comprised of heptapeptide (Y-S-P-T-S-P-S) repeats (Bentley, 2014; Payne et al., 1989; Sun & Fisher, 2024). Vertebrate RPB1 proteins contain 52 of these repeats which are essential for transcriptional regulation of gene expression (Bartolomei et al., 1988). The RPB1 CTD is involved in transcription, as well as 5’ capping and splicing of RNAs (discussed in detail in later sections). Phosphorylation of serine 5 (Y-S-P-T-S-P-S) is required for transcription initiation, RNAPII pausing, and co-transcriptional capping of the 5’ terminus of the pre-mRNA (Kuehner et al., 2011; Lodish, 2013; Payne et al., 1989). On the other hand, phosphorylation of serine 2 (Y-S-P-T-S-P-S) is necessary for attachment of the spliceosome for co-transcriptional splicing of the newly synthesized pre-mRNA and transcription termination (Gu et al., 2013; Kuehner et al., 2011; Lodish, 2013). Thus, the CTD of RPB1 is critical for the precise coordination of transcription and co-transcriptional pre-mRNA processing events (Bentley, 2014).

5’ Capping

Among the first steps of pre-mRNA regulation in the nucleus is the co-transcriptional addition of an N7-methylguanosine (m⁷G) cap structure onto the emerging pre-mRNA transcript as soon as the incorporation of the first 25–30nts by RNA polymerase II (Moteki & Price, 2002; Shatkin & Manley, 2000). In this step, termed 5’ capping, an m⁷G is linked to the first nucleotide of the nascent RNA via a reverse 5’ to 5’ triphosphate linkage (Wei et al., 1975) (Figure 2).

The 5’ cap is an evolutionarily conserved modification that plays both protective and functional molecular roles. Functionally, the 5’ cap is essential for coordination of downstream RNA processing events such as splicing, polyadenylation, nuclear export, and cap-dependent initiation of translation. The 5’ cap is also required to protect the transcript from 5’-3’ exonuclease degradation (Fresco & Buratowski, 1996; Inoue et al., 1989; Izaurralde & Adam, 1998; Jiao et al., 2013; Konarska et al., 1984; Moteki & Price, 2002; Ohno et al., 1987). Failure of the nascent pre-mRNA transcript to be capped causes transcript instability, as removal of the cap precedes exonuclease cleavage and decay through the major RNA degradation pathways (Losh & van Hoof, 2015; Meyer et al., 2004). Thus, the addition of the m⁷G cap is necessary for transcript stability and downstream RNA processing events.

Splicing

The pre-mRNA transcript is comprised of regions known as exons and introns. Exons are the nucleotide sequences that exit the nucleus, whereas introns are sequences that are removed before an mRNA exits the nucleus (Figure 3) (Hellen, 2018). A common misconception, even displayed this way in some textbooks, is that untranslated regions (UTRs) of mRNA are not exons, and that exons are limited to sequences that encode protein. This is not the case. Both UTRs and coding sequences (CDS) exit the nucleus and are therefore defined as exons (Figure 3). As depicted in Figure 3, both the 5’UTR and the 3’UTR can play important functions in regulating key steps in gene expression.

Figure 3. Untranslated Regions Play Critical Regulatory Roles.

Exons (light green and dark green) are sequences that exit the nucleus including untranslated regions (UTRs; light green) and the coding sequence (CDS). Introns (blue) are removed during splicing and do not exit the nucleus. Both the 5’- and 3-UTRs can play key roles in regulation of gene expression as indicated. Created in https://BioRender.com

For proper protein synthesis to occur, introns must be removed from the pre-mRNA transcripts and exons must be joined together in a process known as splicing. Splicing of pre-mRNA is a highly ordered process, which primarily occurs co-transcriptionally, and is mediated by the spliceosome–a macromolecular machine comprised of a multitude of various proteins and RNAs that orchestrate intron removal (Cvitkovic & Jurica, 2013; Schmidt et al., 2014; Wilkinson et al., 2020). Small nuclear ribonucleoproteins (snRNPs) of the spliceosome are responsible for both recognition of degenerate canonical splice site sequences at the 5’ and 3’ ends of the intron and catalysis (Wilkinson et al., 2020). Accurate removal of the introns is accomplished by a two-part transesterification reaction (Wilkinson et al., 2020) (Figure 4A). First, a lariat is formed via a nucleophilic attack of the phosphate group of the 5’ splice site by the 2’ hydroxyl group of the branch point adenosine located ~18–40 nucleotides from the 3’ splice site (Black, 2003; Collins & Guthrie, 2000; Wilkinson et al., 2020). Next, the free hydroxyl of the detached exon attacks the 3’ splice site, thus forming a lariat intron and ligating adjacent exons (Black, 2003; Collins & Guthrie, 2000; Wilkinson et al., 2020).

Figure 4. RNA Splicing.

A. Pre-mRNA splicing occurs through a two-step transesterification reaction to remove introns. B. There are multiple different types of alternative splicing including constitutive splicing, exon skipping/inclusion, alternative 5’ or 3’ splice sites, intron retention, and mutually exclusive exons. C. Backsplicing of a single exon as shown or multiple exons (not shown) can lead to the production of circRNA. D. In recursive splicing, introns can be removed in a multi-step process. Created in https://BioRender.com

In addition to traditional splicing mechanisms, many pre-mRNA transcripts also undergo a process known as alternative splicing. Alternative splicing is a mechanism by which exons of a given pre-mRNA molecule are assembled in different ways (e.g., exon skipping or mutually exclusive cassette exons) (Figure 4B) (Lee & Rio, 2015; Ule & Blencowe, 2019). In this process, the spliceosome utilizes alternative splice sites allowing one gene to encode distinct proteins or include alternative regulatory sequences which can have disparate, or in some cases, even opposing functions (Schwerk & Schulze-Osthoff, 2005). Remarkably, over 90% of all human genes undergo alternative splicing, contributing to extraordinary transcriptome and proteome diversity (Pan et al., 2008; Sultan et al., 2008; Wang et al., 2008). This process, like many other processes involved in the regulation of gene expression, requires interactions of the pre-mRNA molecule with a diverse set of RBPs that orchestrate the precise selection of exons to be included in the mature mRNA. This process occurs in a highly regulated, cell type-specific manner (Fu & Ares, 2014; Lee & Rio, 2015). Decades of research as well as the advent of sequencing technologies such as single-molecule and long-read sequencing have also uncovered several noncanonical splicing mechanisms (Hrdlickova et al., 2017; Stark et al., 2019). These noncanonical splicing mechanisms include backsplicing (Figure 4C), recursive splicing (Figure 4D), and the splicing of microexons (Gehring & Roignant, 2021).

Backsplicing is a noncanonical form of splicing whereby the 5’ terminus of an upstream exon is covalently linked to the 3’ terminus of a downstream exon (Figure 4C) generating a circularized RNA (circRNA) molecule (Starke et al., 2015). The precise function of circRNAs remains elusive, however, recent evidence suggests a potential role for at least some circRNAs as RNA sponges, sequestering microRNAs (miRNAs) and/or RNA binding proteins (Hansen et al., 2013; Memczak et al., 2013; Ragan et al., 2019).

Recursive splicing is a mechanism by which long introns (>50kb) are removed in a multistep splicing process. This unique splicing mechanism was first described for the splicing of the 74kb intron found within the ultrabithorax gene of Drosophila melanogaster (Burnette et al., 2005; Hatton et al., 1998). For proper splicing to occur, the 5’ and 3’ splice sites must be in proximity, allowing the 2-step transesterification reaction to occur (Shepard et al., 2009). Thus, longer introns where the 5’ and 3’ splice sites are separated by thousands of bases or more, pose a unique challenge for the spliceosome. To solve this problem, the spliceosome utilizes recursive splice sites (also known as ratcheting sites) consisting of combined 3’ and 5’ splice sites to progressively remove the large intron in pieces (Figure 4D) (Burnette et al., 2005; Duff et al., 2015; Hatton et al., 1998; Shepard et al., 2009; Sibley et al., 2015). These recursive splice sites are highly enriched within long introns, illustrating their critical role in the accurate splicing of large intronic sequences (Burnette et al., 2005).

Finally, the splicing of microexons refers to the splicing of very small exons (3–27nts) which insert only 1–9 amino acids into the resulting protein (Ustianenko et al., 2017). Recent evidence suggests that microexon inclusion requires specific U/C repeats and UGC motifs forming intronic splicing enhancers located upstream of the 3’ splice site, necessary for recognition by spliceosomal proteins (Raj et al., 2011). To date, several reviews have been published that provide an in-depth discussion of these distinct splicing mechanisms (Blijlevens et al., 2021; Gehring & Roignant, 2021; Poulos et al., 2011).

Endonucleolytic Cleavage and Polyadenylation

Like the processing steps that take place at the 5’ end of the mRNA molecule, the 3’ end of an mRNA transcript also undergoes a series of processing and maturation steps. The 3’ ends of almost all eukaryotic mRNAs are generated in a two-step process: endonucleolytic cleavage followed by polyadenylation. Within the nucleus, pre-mRNAs are co-transcriptionally cleaved at a polyadenylation signal (PAS) – a conserved sequence (typically AAUAAA or a similar variant) found within the 3’ untranslated region (3’UTR) of the pre-mRNA (Edmonds, 2002). The PAS as well as downstream cis elements are responsible for the recruitment of cleavage and polyadenylation factors required to guide the formation of the poly(A) tail (Nicholson & Pasquinelli, 2019; Proudfoot, 2011; Tian & Graber, 2012). Poly(A) tails are non-templated additions of adenosine residues to the 3’ end of the mRNA that serve several critical roles for downstream mRNA processing and transcript regulation. Cleavage of the pre-mRNA molecule takes place co-transcriptionally, approximately 10–30nt downstream of the PAS, allowing poly(A) polymerase (PAP) to catalyze the addition of adenosine residues onto the 3’ end(Meyer et al., 2002). After the addition of 10–14 adenosine residues, a nuclear poly(A)-binding protein (PABN1) binds the growing poly(A) tail allowing for processive synthesis of a full-length poly(A) tail (~200–250nts in metazoans) by PAP (Brawerman, 1981; Meyer et al., 2002; Nicholson & Pasquinelli, 2019; Sheiness & Darnell, 1973).

The mRNA poly(A) tail facilitates mature mRNA export from the nucleus (Dower et al., 2004) and acts as a critical regulator of gene expression in the cytoplasm, contributing both to the translational status and the stability of the mRNA (Boreikaitė & Passmore, 2023). For instance, the poly(A) tail synergizes with the 5’ cap and associated factors to facilitate translation (Gallie, 1991) although recent studies raise questions about the validity of a long-standing a model, which typically depicts a physical interaction between the 5’ end and the 3’ end of transcripts during translation (Adivarahan et al., 2018). Transcripts with shorter poly(A) tails have reduced rates of translation in some specific cell types; thus deadenylation can slow translation triggering subsequent decay of the transcript (Passmore & Coller, 2022). Reciprocally, poly(A) tails can be elongated in the cytoplasm to stabilize and stimulate translation of translationally repressed mRNAs (Beck & Hurt, 2017; Passmore & Coller, 2022). Cumulatively, the dynamic nature of poly(A) tails is critical for fine-tuned regulation of gene expression and shapes the overall architecture of the cellular proteome.

1.2. Nucleocytoplasmic transport

Eukaryotic cells segregate their RNA and protein synthesis into two distinct cellular compartments i.e., the nucleus and cytoplasm. The genetic material within the nucleus is separated from the cytoplasmic contents by a double membrane known as the nuclear envelope. This compartmentalization necessitates nucleocytoplasmic exchange mechanisms which are mediated by the nuclear pore complex (NPC) – a cylindrical ring-like structure embedded within the nuclear envelope (Figure 5). The NPC is one of the largest macromolecular complexes (~120 MDa) consisting of ~1,000 protein subunits called nucleoporins (NUPs) (Beck & Hurt, 2017; Tran & Wente, 2006). NUPs contain phenylalanine-glycine repeat domains which create a permeability barrier preventing the passive diffusion of larger cargos (>60KDa). While some small molecules can diffuse into the nucleus via NPCs, even smaller proteins, such as histones, require active import into the nucleus in a carrier-mediated manner (Alber et al., 2007; Terry & Wente, 2007).

Figure 5. The Nuclear Pore Complex.

Schematic cross-section of the nuclear pore complex embedded within the nuclear envelope showing major components. Created in https://BioRender.com

Nucleocytoplasmic transport is a complex mechanism involving a multitude of protein-protein interactions, regulatory mechanisms, and signaling pathways to efficiently import and export proteins and ensure efficient export of properly processed mRNAs. Nucleocytoplasmic transport of most cargoes involves karyopherin-mediated receptors, where the directionality of transport is determined by a RanGTP gradient (reviewed in: (Ding & Sepehrimanesh, 2021; Madrid & Weis, 2006; Quimby & Corbett, 2001)). Unlike protein and other RNA cargoes, mRNA export is atypical and requires a mechanism distinct from other types of cargoes. The export of mRNA is performed in three steps (1) packaging of mRNAs into mRNP complexes; (2) targeting and translocation of the mRNP; (3) intracytoplasmic release of the mRNPs for translation. Prior to export, newly synthesized mRNAs are packaged into mRNP complexes comprised of heterogeneous nuclear ribonucleoproteins (hnRNPs). In humans, the NFX1-NXT1 heterodimer acts as an export receptor for the mRNP and physically interacts with NUPs to facilitate mRNP passage through the NPC (Aibara et al., 2015; Forler et al., 2004; Stewart, 2007). Once the mRNP reaches the cytoplasmic face of the NPC, NFX1-NXT1 is released, and new proteins associate with the mRNP complex (Kelly & Corbett, 2009).

The Untranslated Regions

Although a majority of the mature transcript encodes the resulting protein, some exons instead play a regulatory role making up a region of the transcript known as the untranslated region (UTR). As shown in Figure 3, UTRs are located at both the 5’ and 3’ ends of the mRNA molecule and play critical roles in post transcriptional regulation of gene expression. These transcribed, but not translated regions of the mRNA contain numerous cis regulatory elements that recruit trans-acting factors that govern critical steps in post-transcriptional gene expression including mRNA processing, stability, localization, and translation.

The 5’UTR can contain several cis-regulatory elements including upstream open reading frames (uORFs), internal ribosomal entry sites (IRES), microRNA (miRNA) binding sites, as well as structural elements critical for regulation of splicing, mRNA stability, and famously, translation initiation. Dysregulation of the 5’UTR cis-regulatory elements or secondary structures can cause changes in gene expression that underlie a multitude of diseases, including cancer and neurodevelopmental disorders (Boivin et al., 2021; Mor-Shaked & Eiges, 2016; Schuster & Hsieh, 2019). Moreover, the 5’UTR plays a crucial role in translation initiation (discussed in detail in the Translation section). Thus, precise regulation of the 5’UTR is critical for proper control of gene expression.

Like the 5’UTR, the 3’UTR is a critical regulator of gene expression, with important roles in modulating mRNA stability, localization, and translation (Mayr, 2019). Moreover, decades of work have identified functions for the 3’UTR in control of nuclear export (Davis et al., 1997), polyadenylation (Richter, 1999), subcellular localization (Gao, 1998), and transcript stability (Chen & Shyu, 1995; Morales et al., 1997; Wang et al., 1995). Alterations in 3’UTR-mediated functions can affect the expression of one or more genes and thereby underlie the pathogenesis of many diseases including myotonic dystrophy (DM) (Boucher et al., 1995), inflammatory diseases (Kontoyiannis et al., 1999), and cancers (Chagnovich et al., 1996; Hong & Jeong, 2023; Rimokh et al., 1994).

1.3. Regulation of Gene Expression in the Cytoplasm

Translation

Translation is the process through which a protein is synthesized from the information contained within a mRNA molecule. Specifically, nucleotides encoded within the mRNA molecule are “decoded” by a large macromolecular complex termed the ribosome, which directs the addition of amino acids onto an elongating polypeptide chain to generate a functional protein (Figure 6). Initiation of translation can occur via two distinct mechanisms: (1) a cap-dependent mechanism requiring a free 5’ end and 5’ cap, or (2) a cap-independent mechanism which utilizes an internal ribosome entry site (IRES), which is an RNA element that allows for translation initiation during conditions of cellular stress (e.g., hypoxia, apoptosis, starvation, or viral infection) (Stern-Ginossar et al., 2019; Wek, 2018; Yang & Wang, 2019).

Figure 6. Translation.

A. The ribosome is comprised of a 40S small subunit and a 60S large subunit that come together to form the translation-competent 80S ribosome. The ribosome contains three binding sites for tRNA designated: the A (aminoacyl) site, which accepts incoming aminoacylated tRNA; the P (peptidyl) site, which holds the tRNA with the nascent peptide chain; and the E (exit) site, which holds the deacylated tRNA before it leaves the ribosome B. Overview of translation elongation. Step 1: An aminoacylated tRNA binds to the codon in the A-site. Step2: With correct codon:anti-codon pairing, the existing polypeptide chain present on the tRNA in the P-site is linked to the amino acid carried by the aminoacylated tRNA in the A-site via a deacylation reaction. Step 3: Finally, mRNA is shifted one codon over in the ribosome, exposing a new codon and the deacylated tRNA leaves the ribosome from the E-site. Created in https://BioRender.com

Cap-dependent translation initiation is a four-step process, which is reviewed in detail in (Leppek et al., 2018; Lodish, 2013; Rodnina et al., 2020). This process begins with recruitment of the 40S small ribosomal subunit and the associated eukaryotic initiation factors (eIFs) to the 5’ cap. First, the cap-binding complex is assembled as cap-binding protein eIF4E interacts with the scaffolding initiation factor eIF4G and the RNA helicase eIF4A. eIF4G interacts with cytoplasmic poly(A) binding protein (PABP) located on the poly(A) tail, which may facilitate circularization of the mRNA molecule to aid in translational control. Subsequently, eIF4G, via an interaction with eIF3, recruits the 43S pre-initiation complex consisting of eIF3, the 40S ribosomal subunit, the ternary complex (eIF2-GTP-Met-tRNA_i), eIF1, eIF1A, and eIF5 (Step 1). After the 43S pre-initiation complex binds near the 5’ cap to form the 48S complex, the complex travels along the 5’UTR (an ATP-dependent process known as ‘scanning’), until it encounters a start codon (typically, AUG) (Step 2). The eIF4A RNA helicase unwinds inhibitory secondary structures within the 5’UTR. Recognition of the start codon triggers release of eIFs (Step 3). Finally, the 60S large ribosomal subunit joins the 40S small ribosomal subunit to form the elongation-competent 80S ribosome (Step 4), which then proceeds with translation elongation (discussed later in this section).

Cellular stress conditions such as starvation or hypoxia, can inhibit the formation of the ternary complex thereby suppressing cap-dependent translation (Wek, 2018). Thus, alternative translation mechanisms are required to synthesize proteins necessary for cellular survival under stress conditions. Non-canonical translation initiation mechanisms can vary with respect to cellular stress conditions. For example, ribosomal shunting requires the 5’ cap as well as canonical initiation factors to load the 40S ribosomal subunit onto the mRNA molecule before translocating the 40S subunit downstream to initiate translation (Morley & Coldwell, 2008). On the other hand, IRESs can recruit a ribosome internally without the need for a 5’ cap or a free 5’ end. IRES translation initiation mechanisms have a range of initiation factor requirements depending on the type of IRES. For example, the simplest IRES studied (the Dicistroviridae intergenic region) requires no initiation factors (Jan et al., 2003), while others require all the initiation factors required for canonical translation initiation (such as the hepatitis A viral IRES) (Avanzino et al., 2017).

Translation elongation mechanisms are evolutionarily conserved and have been extensively studied in bacteria, with the key steps shared between bacteria and eukaryotes (Rodnina, 2018). The ribosome contains three binding sites for tRNA (Figure 6A) designated: the A (aminoacyl) site, which accepts incoming aminoacylated tRNA; the P (peptidyl) site, which holds the tRNA with the nascent peptide chain; and the E (exit) site, which holds the deacylated tRNA before it leaves the ribosome. As summarized in Figure 6B, translation elongation in eukaryotes starts when MET-tRNA_i^Met is bound to the P site of the ribosome in the context of the 80S initiation complex, with an empty A site (Step 1). Aminoacylated tRNA is then brought to the A site as a ternary complex with eukaryotic elongation factor-1A (eEF1A) and GTP. Correct codon-anticodon interactions cause conformational changes in the ribosome that allow a peptide bond to be formed through deacylation of the P-site tRNA and transfer of the peptide chain to the A-site tRNA (Step 2). This step is followed by translocation of the A-site tRNA with the growing peptidyl chain from the A-site into the P-site. As this translocation occurs, the deacylated tRNA in the P-site is translocated to the E-site for exit from the ribosome. This process is repeated for multiple steps with translocation of the mRNA and the tRNA facilitated by eEF2 (Step 3). Finally, translation termination occurs when the ribosome encounters a stop codon (typically UGA, UAG, or UAA), codons with no corresponding tRNA. Translation termination is mediated by release factors eRF1 and eRF3 which form a ternary eRF1/eRF3-GTP complex where eRF1 recognizes the stop codon, and, after hydrolysis of GTP by eRF3, release of the nascent peptide occurs (Hellen, 2018). The translation complex is disassembled, and the new polypeptide chain is released. Both in the course of protein synthesis and following release of the polypeptide chain, chaperone proteins aid in the folding of the protein into the correct structure. The steps described above are reviewed in: (Dever et al., 2018; Hellen, 2018; Lodish, 2013)

Transport/Trafficking/Localization

Once in the cytoplasm, the mRNA molecule can be trafficked to distinct subcellular locations for translation. Localization of mRNA to specific subcellular compartments provides a highly orchestrated mechanism for spatiotemporal control of gene expression, which is critical within highly polarized, asymmetric cells (Martin & Ephrussi, 2009). For example, in Drosophila melanogaster the localization of mRNAs that regulate developmental signaling, such as bicoid, oskar, and nanos, to the anterior and posterior poles of the oocyte is critical to establish a morphogen gradient for proper embryonic development (Johnstone & Lasko, 2001). Similarly, mRNA encoding the S. cerevisiae transcriptional repressor Ash1 is transported to the bud tip of a dividing yeast cell such that only the daughter cell receives the ASH1 mRNA, ensuring mother and daughter cells have distinct mating types (Paquin & Chartrand, 2008). Thus, localization of mRNAs to distinct subcellular locations allows spatial restriction of gene expression which is critical for many cellular and developmental processes.

The neuron is one type of highly polarized cell that relies extensively on mRNA trafficking mechanisms to regulate synaptic transmission and cell-cell communication. mRNA localization and local translation in neurons is crucial for a multitude of processes including polarization (Ciolli Mattioli et al., 2019), neuronal development (Wong et al., 2017; Yoon et al., 2016), and synaptic plasticity (Donlin-Asp et al., 2021; Miller et al., 2002). Neurons contain four main parts: (1) dendrites, which receive messages from other neurons; (2) a cell body which contains the nucleus and cytoplasm; (3) an axon, which transmits information away from the nucleus; and (4) axon terminals, which transmit electrical and chemical signals to other neurons or effector cells (Figure 7). Unlike other cells in the body, many neurons must extend great lengths to perform their given functions, thus trafficking of mRNAs produced in the nucleus all the way to distal neurites for local protein synthesis is an intricate mechanism necessary for neuronal function. For example, the sciatic nerve is longest neuron in our body that stretches from our spinal cord all the way to our toes and can exceed one meter in length. Thus, our cells require intricate mechanisms to properly orchestrate the trafficking and localization of mRNA molecules across these great lengths.

Figure 7. Specialized Cells Such as Neurons Require RNA Localization and Local Translation.

The specialized structure of a neuron consists of dendrites, which receive messages from other neurons; a cell body which contains the nucleus and cytoplasm; an axon, which transmits information away from the nucleus; and axon terminals, which transmit electrical and chemical signals to other neurons or effector cells. In neurons, RNAs can be packaged into mRNA ribonucleoprotein particles and transported away from the cell body. These trafficked mRNAs can be locally translated in a signal-dependent manner. Created in https://BioRender.com

After post-transcriptional processing and export from the nucleus, mRNAs are trafficked through the cytoplasm as mRNP complexes containing RBPs that regulate stability, localization and translation (Doyle & Kiebler, 2011). In many cases, this mRNP complex forms a part of a larger structure called an RNA transport granule which is transported through association with motor proteins, such as kinesin, dynein, and myosin along the cytoskeleton to the intended destination (Kiebler & Bassell, 2006). Often the mRNA is silenced during transport (Wang et al., 2010). Once the mRNA has arrived at its destination, signal-dependent local translation can ensue providing the cell with a spatially distinct proteome to govern a multitude of regulatory and developmental processes.

Decay

mRNA degradation is a highly regulated process that plays a crucial role in post-transcriptional regulation of gene expression. Evidence for mRNA turnover is highlighted by early studies demonstrating that mRNA steady-state levels do not directly correlate with the rates of mRNA synthesis (Vogel & Marcotte, 2012). Moreover, mRNA half-lives can differ significantly between mRNA transcripts (Chen et al., 2008). For instance, studies show that mRNAs encoding housekeeping proteins tend to have longer half-lives than the average mRNA molecule (Yang et al., 2003). Thus, RNA decay pathways can contribute to both the regulation of gene expression and serve as quality control pathways.

Turnover of mRNA in the cytoplasm can occur via several decay pathways (Figure 8) (Garneau et al., 2007; Gong & Maquat, 2011; Łabno et al., 2016). Deadenylation, which has long been considered the first and rate-limiting step during mRNA turnover (Garneau et al., 2007; Labno et al., 2016), is crucial for activation of decay in both the 3’-5’ and 5’-3’ directions (Labno et al., 2016). The deadenylation complex (Ccr4-NOT or Pan2-Pan3) is often recruited to the mRNA molecule to help accelerate deadenylation and thereby stimulate RNA decay (Wahle & Winkler, 2013). Deadenylation also may disrupt the closed circle of the mRNA molecule, which has conventionally been thought to be required for translation. Therefore removal of the poly(A) tail leads to translational repression (Cooke et al., 2010). After deadenylation, decapping factors are recruited to the 5’ end of the mRNA molecule to efficiently remove the m⁷G-methylguanosine cap (Nishimura et al., 2015; Tharun, 2009). Next, the 5’-3’ exoribonuclease, XRN1, can recognize the 5’ monophosphate and degrade the mRNA transcript (Jinek et al., 2011; Jones et al., 2012; Muhlrad et al., 1994). Alternatively, the deadenylated mRNA can also be degraded in the 3’-5’ direction by a multisubunit complex known as the RNA exosome, via the 3’-5’ exonucleolytic subunit, DIS3L (Schaeffer et al., 2009; Schneider et al., 2007). In specific cases, mRNA decay can also be initiated via endonucleolytic cleavage to trigger decay. Few endonucleases have been described in mammalian cells, but those that have been defined include Pmr1, ZC3H12A, the Nonsense-mediated Decay (NMD)-specific endonuclease Smg6, and the siRNA/miRNA RNA-induced silencing complex (RISC) (Tomecki & Dziembowski, 2010). Each of these decay mechanisms is capable of endonucleolytic cleavage of the mRNA molecule to induce mRNA decay.

Figure 8. RNA Decay Pathways and Surveillance.

RNA decay is a regulated process that can occur by multiple mechanisms. Typically, deadenylation occurs, which can be followed by decapping and 5’->3’ decay medicated by Xrn1. Alternatively, decay can occur in a 3’->5’ direction, which is mediated by the RNA exosome complex. These cytoplasmic decay pathways are complemented by surveillance pathways that can detect aberrant RNAs, including: Nonsense-mediated Decay (NMD), which detects transcripts that contain a premature termination codon; Non-stop Decay, which detects transcripts that lack a stop codon; and No-go Decay, which detects stalled ribosomes. Created in https://BioRender.com

More conventional RNA decay pathways are complemented by surveillance pathways (Figure 8) that target RNA for destruction when the RNA is detected as aberrant (Wolin & Maquat, 2019). The classical surveillance pathways include nonsense-mediated decay (NMD) where mRNAs that contain a premature termination codon are targeted for decay (Isken & Maquat, 2007; Kurosaki et al., 2019). Non-stop decay was identified as a way to destroy RNAs that lack a stop codon within the proper context (Klauer & van Hoof, 2012; van Hoof et al., 2002). No-go decay occurs when ribosomes stall on mRNAs, to allow recycling of the stalled ribosomes (Doma & Parker, 2006). There are likely other mechanisms that can target RNAs via interactions with specific RNA binding proteins. For example, Staufen-mediated decay (SMD) in mammalian cells is triggered when the RNA binding protein STAU1 binds to a STAU1-binding site within the 3’-UTR of target mRNAs (Gong & Maquat, 2011; Park & Maquat, 2013). Cytoplasmic surveillance pathways are also supported by a number of nuclear surveillance pathways that degrade defective RNAs before they exit the nucleus (Houseley & Tollervey, 2009; Rambout & Maquat, 2024).

1.4. Epigenetic modifications: N6-methyladenosine

In recent years, there has been a growing appreciation for the critical and diverse roles that epigenetic modifications play in post-transcriptional regulation of gene expression. Epigenetic modifications of the pre-mRNA molecule chemically alter cis regulatory sequences to control a multitude of downstream processing events including splicing, localization, translation, and RNA stability. Presently, over 160 different types of chemical modifications of RNA have been identified (Arzumanian et al., 2022), among which RNA methylation is the most prevalent (Zhao et al., 2020). To date, the most well-studied chemical modification on eukaryotic mRNA is N6-methyladenosine (m⁶A)–a methylation modification deposited co-transcriptionally onto the N6 position of select adenosine (A) residues (Figure 9A). This modification constitutes the most abundant internal modification on eukaryotic mRNAs (Arzumanian et al., 2022; Yang et al., 2018). Intriguingly, m⁶A is enriched within the brain and nervous system highlighting a key regulatory role for m⁶A in nervous system development (Ma et al., 2018).

Figure 9. N6-methyladenosine and the m⁶A mRNA Pathway.

A. Molecular structure of Adenosine residue with m⁶A methylation modification on the N6 position (pink circle). B. The methyltransferase complex (“writer”) is composed of five factors and deposits a methyl group onto target adenosine residues. YTH domain-containing m⁶A “reader” proteins recognize m⁶A methylated RNA and regulated downstream RNA fate. “Eraser” proteins mediate the removal of m⁶A methylation on target RNAs. Created in https://BioRender.com

The m⁶A methylation of mRNA was first observed in 1974 (Desrosiers et al., 1974), and decades of research following the discovery of this modification have uncovered a multitude critical functional roles for this epigenetic modification. Molecularly, m⁶A modifications are deposited within DRACH sequence motifs (D=Uracil, Guanine, or Adenine, R=Guanine or Adenine, Adenosine, Cytosine, H=Uracil, Adenine, or Cytosine) (Luo & Tong, 2014; Theler et al., 2014; Xu et al., 2014) and are enriched near stop codons and in 3’UTRs (Dominissini et al., 2012; Ke et al., 2015; Meyer et al., 2012). Critically, m⁶A has key post-transcriptional regulatory roles in mRNA splicing (Haussmann et al., 2016; Lence et al., 2016; Xiao et al., 2016), alternative polyadenylation site selection (Ke et al., 2015), nuclear export (Roundtree et al., 2017), translation (Meyer et al., 2015; Wang et al., 2015), and stability (Uzonyi et al., 2023; Wang et al., 2014; Zaccara & Jaffrey, 2020). The spatiotemporal regulation of m⁶A modifications is coordinated by three types of proteins called writers, readers, and erasers (Figure 9B) (Meyer & Jaffrey, 2017).

The m⁶A writer machinery, also known as the methyltransferase complex, is responsible for catalyzing the addition of a methyl group from the donor S-adenosylmethionine (SAM) onto the N6 position of target adenosine residues (Bokar et al., 1997). The methyltransferase complex is a multi-subunit complex consisting of methyltransferase-like protein 3 (METTL3; previously known as IME4 or MT-A70) (Bokar et al., 1997; McGraw et al., 2007), methyltransferase-like protein 14 (METTL14) (Liu et al., 2014), Wilms’ tumor 1-associated protein (WTAP) (Ping et al., 2014), vir-like m⁶A methyltransferase-associated protein (VIRMA, also known as KIAA1429) (Yue et al., 2018), and RNA binding motifs protein 15/15B (RBM15/15B) (Patil et al., 2016). METTL3 is the highly conserved core, catalytic subunit of the methyltransferase complex, which binds SAM (Lin et al., 2019). Genetic depletion of METTL3 in mammalian cells as well as plants, Drosophila melanogaster and yeast leads to complete or near complete loss of m⁶A in polyadenylated RNA (Agarwala et al., 2012; Geula et al., 2015; Kan et al., 2021; Zhong et al., 2008), defining METTL3 as the major m⁶A forming enzyme for mRNA transcripts. METTL14 on the other hand is highly homologous to METTL3 and is required to support METTL3 structurally and enhance METTL3 catalytic activity (Liu et al., 2014). WTAP is also a core component that associates with METTL3-METTL14 to enhance the catalytic activity of METTL3 (Ping et al., 2014; Zhong et al., 2008), while VIRMA and RBM15/15B participate in regulating the catalytic activity of the complex and help aggregate the core components (Yue et al., 2018).

Protein readers, defined as m⁶A recognition proteins, function through recognizing and binding m⁶A modifications to modulate downstream RNA processing and fate (Meyer & Jaffrey, 2017; Wu et al., 2017). Reader proteins recognize m⁶A modifications via a YTH (YT521B homology) domain that selectively binds m⁶A-modified RNA. Mammalian genomes contain five different YTH domain containing proteins that can bind m⁶A: YTHDF1, YTHDF2, YTHDF3, YTHDC1, and YTHDC2 (Patil et al., 2016). The YTHDF proteins are highly similar to one another and are primarily localized within the cytoplasm. There have been conflicting studies seeking to define the precise function of the YTHDF family proteins. However, studies clearly demonstrate that YTHDF1 promotes translation and interacts with translation initiation factors (such as eIF3) (Wang et al., 2015). On the other hand, YTHDF2 is the most abundant YTHDF family member in most cell types and has a well-established role as a regulator of mRNA stability (Wang et al., 2014). Previous work illustrates that mRNAs in YTHDF2-depleted cells have increased half-lives, indicating a key role for YTHDF2 in mediating mRNA decay(Batista et al., 2014; Dominissini et al., 2012; Geula et al., 2015). In contrast to YTHDF proteins, YTHDC1 is primarily nuclear and is the major m⁶A reader protein within the cell (Meyer & Jaffrey, 2017). YTHDC1 was originally identified as a regulator of splicing and subsequent studies have focused on elucidating the function of YTHDC1 in m⁶A-mediated splicing (Xiao et al., 2016). On the other hand, YTHDC2 is a nucleocytoplasmic protein with poorly defined functions (Meyer & Jaffrey, 2017; Patil et al., 2016). Several groups have resolved the structure of a YTH domain complexed with m⁶A (Luo & Tong, 2014; Theler et al., 2014; Xu et al., 2014). Intriguingly, these studies demonstrated that the affinity of YTH domain binding for m⁶A is relatively weak (~1–2 mM) (Luo & Tong, 2014; Theler et al., 2014; Xu et al., 2014), suggesting that YTH-domain containing proteins may require additional factors to form a stable complex with m⁶A.

Finally, m⁶A eraser proteins are responsible for the demethylation of m⁶A-modified RNAs. Two different enzymes have been identified with the ability to demethylate m⁶A: fat mass and obesity-associated protein (FTO) (Jia et al., 2011) and AlkB homolog H5 (ALKBH5) (Zheng et al., 2013). However, conflicting evidence raises questions about the contribution of these proteins to regulating m⁶A methylation.. For instance, FTO was the first enzyme linked to demethylation of m⁶A, however, more recent studies suggest that FTO instead preferentially targets m⁶A_m (a different highly prevalent methylation modification on mRNA) (Mauer et al., 2017). Moreover, analysis of m⁶A levels in Fto knockout mice showed little to no change in m⁶A levels across the transcriptome with the exception of a small subset of m⁶A peaks (Hess et al., 2013), and FTO did not show a preference for m⁶A in its physiological consensus site (Jia et al., 2011). Similarly, the degree to which ALKBH5 targets and demethylates m⁶A-modifed RNAs remains unclear, and characterization of the enzymatic catalytic activity of ALKBH5 reported slow kinetics towards m⁶A (Zheng et al., 2013).

1.5. RNA binding proteins

RNA Binding Proteins (RBPs) are critical for nearly every aspect of RNA processing and regulation. RBPs associate with RNA molecules to form dynamic ribonucleoprotein (RNP) complexes that regulate every step of RNA processing from synthesis to decay. The specific RBPs that associate with a given RNA molecule change depending on cellular context or the functional state of the RNA.

RBPs bind mRNAs via evolutionarily conserved, modular RNA-binding domains (RBDs) including RNA recognition motifs (RRMs), zinc finger domains (ZnF), hnRNP K homology (KH) domains, and RGG-motifs among other binding domains (Lunde et al., 2007). The RRM is the most common and well-characterized RNA binding domain (Lunde et al., 2007). Over 9,000 RRMs have been identified with functions in most steps of gene expression (Finn et al., 2006). Intriguingly, ~0.5–1% of human genes encode an RRM, and there are typically multiple RRMs within a given protein (Finn et al., 2006). Unlike RRMs, ZnFs are classical DNA-binding protein domains that can also bind RNA (Carballo et al., 1998; Hudson et al., 2004; Lee et al., 2006; Lu et al., 2003; Picard & Wegnez, 1979). ZnFs, which are comprised of Cysteine and Histidine residues are classified based on the pattern of the Cysteine and Histidine residues that coordinate zinc binding: Cysteine2Histidine2 (C2H2), Cysteine3Histidine (CCCH), or Cysteine2HistidineCysteine (CCHC). Like RRMs, these ZnF domains are typically present in multiple repeats within a single RBP. Similar to ZnFs KH-domains are typically found in multiple copies within a single RBD and can associate with DNA or RNA to regulate transcription and translation (Valverde et al., 2008). Finally, RGG-motifs are arginine-glycine-rich domains that bind RNA and are found in proteins critical for many cellular processes including translation, splicing, and apoptosis (Thandapani et al., 2013). These RBDs as well as others not mentioned here associate with RNA in a sequence- and structure-dependent manner (Lunde et al., 2007) and are critical for mediating the function of RBPs and their RNA targets to govern gene expression.

RBPs play critically important roles in governing RNA structure, regulatory functions, catalytic capacities (in the case of ncRNA), and all aspects of mRNA processing. A wealth of literature documents critical roles for RBPs in RNA processing often in a dynamic, cell type-specific manner. Given the diverse functional roles for RBPs throughout mRNA processing, it is not surprising that RBP loss or dysfunction has been linked to numerous diseases including neurological disorders, muscular atrophies, metabolic diseases, and cancer (Cooper et al., 2009; Darnell, 2010; Lukong et al., 2008). Thus, elucidating normal roles for RBPs during homeostasis or development will reveal important aspects of RBP function that are directly related to pathogenetic mechanisms underlying disease.

1.5.a. RBPs in disease

RBPs are evolutionarily conserved and widely distributed across different tissues, consistent with their frequent housekeeping roles. However, despite the ubiquitous nature of RBP expression, loss of function (LOF) mutations within genes encoding RBPs often results in tissue-specific diseases (Brinegar & Cooper, 2016). The tissue-specific nature of RBP-associated disease is attributed to multiple factors. First, RBPs can act on RNA targets or with protein partners that display tissue-specific expression patterns (Gebauer et al., 2021). Second, RBPs can bind target RNAs with a wide range of affinities and specificities leading to the formation of cell-type specific RNA regulatory complexes (Achsel & Bagni, 2016; Beckmann et al., 2016; Iadevaia & Gerber, 2015; Pique et al., 2008). Finally, RBPs form extensive networks with their RNA targets and other regulatory proteins that are characterized by redundancy, as well as feedback and feedforward control (Gebauer et al., 2021). Together, this complex network provides robust regulatory control such that RBP dysfunction may have distinct consequences in different cell types. Considering that RBPs coordinate elaborate networks of RNA-protein and protein-protein interactions that regulate key aspects of RNA processing, RBP dysfunction can negatively influence many different cellular pathways underlying disease phenotypes. A multitude of different diseases are associated with RBP dysfunction including muscular atrophies, cancer, and neurological disease (Lukong et al., 2008; Parra & Johnston, 2022; Prashad & Gopal, 2021).

The nervous system requires an intricate level of spatial and temporal regulation of gene expression for proper neuronal function. A neuron must rapidly modify synaptic function and connectivity to properly respond to external stimuli (van Oostrum & Schuman, 2024). However, the size, polarity, and structural complexity of neurons (Figure 7) presents unique challenges for fulfilling critical functions in development and plasticity. Thus, robust post-transcriptional regulatory mechanisms are required to enable rapid adaptation to the ever-changing demands of the nervous system. Activity-dependent control of gene expression within the neuron is established through intricate post-transcriptional regulation of mRNA expression via splicing, stability, trafficking, local translation, and degradation- all of which require the function of RBPs to coordinate these diverse processing events in space and time.

RBPs regulate every step of mRNA processing and profoundly impact neurodevelopment and neuronal function. The importance of RBP function in neurons is underscored by the prevalence of neurological diseases that have been linked to defects within RBPs causing aberrant processing of RNAs that are necessary for proper neuronal function (Brinegar & Cooper, 2016; Parra & Johnston, 2022; Prashad & Gopal, 2021). Moreover, transcriptomic analysis of human brain tissue indicates RNA processing defects are a common feature of neurological pathologies (Irimia et al., 2014; Raj et al., 2018; Tollervey et al., 2011; Voineagu et al., 2011). Thus, understanding the mechanisms by which RBPs impact the precisely regulated expression of neuronal mRNAs is key to elucidating neuronal disease pathogenesis.

There are numerous examples of neurological disease linked to mutations in genes encoding RBPs. One of the classical examples is Fragile X Syndrome, which is caused by the loss of the Fragile X Mental Retardation Protein (FMRP) that is encoded by the FMR1 gene (Penagarikano et al., 2007). Fragile X Syndrome is an X-linked neurological disorder characterized by mild to moderate intellectual disability (Penagarikano et al., 2007). The FMRP RBP contains multiple KH RNA binding domains as well as an RGG motif (Athar & Joseph, 2020). Several studies have identified FMRP target RNAs (Ascano et al., 2012; Ashley et al., 1993; Darnell et al., 2005), but these studies revealed little overlap among the targets identified, raising questions about the sequence specificity of FMRP as well as the possibility of binding to specific RNA structures such as the G-quadruplex (Darnell et al., 2004; Suhl et al., 2014). A number of studies demonstrate that FMRP associates with ribosomes and regulates translation . Studies suggest that FMRP plays a critical role in regulating local translation in dendrites, highlighting the critical role of local translation for proper neuronal function (Banerjee et al., 2018).

Beyond neurological disorders, mutations in genes that encode ubiquitously expressed RBPs can also cause muscle disease. For example, the gene PABPN1 encodes an RRM-type poly(A)-binding protein that regulates nuclear polyadenylation via association with adenosine molecules of a growing poly(A) tail and stimulating poly(A) polymerase (PAP) (Banerjee et al., 2013). Abnormal expansion of a (GCG)_n trinucleotide repeat in exon 1 of PABPN1 leads to the adult onset, autosomal dominant, degenerative disorder known as oculopharyngeal muscular dystrophy (OPMD) (Banerjee et al., 2013). In unaffected individuals, (GCG)₆ codes for the first six alanine residues within a homopolymeric stretch of ten alanines (Abu-Baker & Rouleau, 2007). However, in individuals with OPMD, this (GCG)₆ is expanded to (GCG)_8–13 leading to a stretch of 12–17 alanines in mutant PABPN1 (Abu-Baker & Rouleau, 2007). The mutant PABPN1 then aggregates within the nuclei of skeletal muscle fibers causing progressive muscle weakness (Abu-Baker & Rouleau, 2007; Banerjee et al., 2013).

As RBPs are key regulators of cell growth, differentiation, and proliferation, RBP dysfunction is implicated in a range of different cancers (Wang et al., 2022). Cancer is a heterogeneous disease caused by mutations, chromosomal rearrangements, and gene amplifications resulting in altered activity of genes encoding tumor suppressors or oncogenes. One example of an RBP implicated in tumorigenesis is the altered expression of mRNA cap binding protein eIF4E (Sonenberg & Hinnebusch, 2007; Wendel et al., 2007). eIF4E is a key translation factor that is highly expressed within different tumor types (Mars et al., 2024; Wendel et al., 2007). Behaving as a proto-oncogene, overexpression of eIF4E causes malignant transformation leading to cancer (Wendel et al., 2007).

Conclusions

The steps and molecular mechanisms of post-transcriptional regulation presented here provide a brief overview of eukaryotic mRNA metabolism. In reality, each of these steps are much more intricate than detailed in this review and rely on sophisticated mechanisms to govern gene expression in a tightly controlled spatiotemporal manner. A detailed mechanistic understanding of each of these steps is necessary to unravelling how dysfunction of mRNA metabolism leads to disease as well as to develop targeted therapeutics to combat these conditions.

Data Availability Statement

Data sharing is not applicable to this article as no new data were created or analyzed in this study.