Biology of RNA Surveillance in Development and Disease

Abstract

The ‘RNA world’, in which RNA molecules stored information and acquired enzymatic properties, has been proposed to have preceded organism life. RNA is now recognized for its central role in biology, with accumulating evidence implicating coding and noncoding (nc)RNAs in myriad mechanisms regulating cellular physiology and disequilibrium in transcriptomes resulting in pathological conditions. Nascently synthesized RNAs are subjected to stringent regulation by sophisticated RNA surveillance pathways. In this review, we integrate these pathways from a developmental viewpoint, proposing RNA surveillance as the convergence of mechanisms that ensure the exact titration of RNA molecules in a spatiotemporally controlled manner, leading to development without the onset of pathological conditions, including cancer.

Shaping the Developmental Transcriptome

The RNAs that ensue from the transcription of coding and ncRNAs (see Glossary) are essential for life and contribute toward diverse known and unknown biologies. Cellular development, with the accompanying rapid synthesis of a plethora of RNAs involved in cell division and differentiation, presents a challenge for quality control, necessitating comprehensive RNA surveillance. Proper orchestration of development depends upon rapid changes in transcriptomic programs between developmentally evolving cells, with the transcriptome being constantly supervised by various pathways. Failure can lead to developmental diseases and malignancies (a list of defects in RNA-processing events that lead to pathological conditions is provided in Table 1). While mRNA processing and mRNA surveillance have been intensively studied for many decades [1], we understand less regarding the surveillance of the noncoding transcriptome. However, a significant portion of the mammalian genome can be transcribed as ncRNAs [2] (Box 1), and it is likely that these noncoding transcripts and/or ncRNA transcription contributes to cellular differentiation during development.

Table 1.

Overview of RNA Surveillance-Linked Pathologies

Pathology/syndrome	Mechanism	Note on study	Refs
Acute myeloid leukemia	METTL14 and METTL3-dependent RNA methylation	Human patients and mouse models	Vu et al., 2017 Nature medicine; Weng et al., 2018, Cell stem cell
Aicardi–Goutières syndrome	Mutations in ADAR1, upregulating interferon-regulated genes and inducing inflammation	Human patients	Rice et al., 2012, Nature Genetics
	TREX1 mutations activate cGAS/STING through LINE-1 expression, leading to inflammation	Human primary cells	Thomas et al., 2017, Cell Stem Cell
	MDA5 mutations lead to its constitutive activation by cellular dsRNA (mainly endogenous retroelement Alu:Alu hybrids)	Biochemistry	Ahmad et al., 2018, Cell
	RNase H2 gene mutations activate cGAS/STING pathway and the interferon pathway in an untimely manner	RNaseh2b mutant knock-in mouse model	Mackenzie et al., 2016, EMBO
Amyotrophic lateral sclerosis and frontotemporal lobar degeneration	Defect in siRNA silencing and TE expression	Expression of human TDP-43 protein in Drosophila	Krug et al., 2017, Plos genetics
Asymmetric DNA mutagenesis	DNA asymmetric mutations commonly observed in cancers; one class is transcription-coupled	Human patients	Haradhvala et al., 2016, Cell [136]
	MTR4 and Senataxin helicases in cooperation with RNA exosome prevent R-loop formation and DNA asymmetric mutations	KO cell lines, mouse primary B cells (conditional RNA exosome KO)	Lim et al., 2017, Cell
Cancer	Primary piRNA pathway reactivation coincident with oncogenic transformation of somatic cells	Drosophila model	Fagegaltier et al., 2016, Genes and development [137]
	Alterations of RNA modifications (m6A and others)	Review	Dai et al., 2018, Cell death and disease
	Increase mRNA stability by ARE and GU-rich elements	Reviews	Khabar, 2017 [138], WIREs RNA; Vlasova-St-Louis and Bohjanen, 2017, Cytokines and grow factor review
Cerebellar and corpus callosum hypoplasia	EXOSC8 (RNA exosome) mutations, altering mRNA metabolism	Human patients and zebrafish model	Boczonadi et al., 2014, Nat com
Fragile X syndrome	Loss of FMRP protein implicated in human fragile X syndrome. This study showed how FMRP cooperates with ADAR2 to regulate editing of neuronal circuit formation genes	Zebrafish model	Shamay-Ramot et al., 2015, Plos genetics
Human Mendelian diseases	Mutations in RNA-binding protein genes	Review	Castello et al., 2013, Trends in genetics
Inflammatory myofibroblastic tumors	UPF1 mutations and upregulation of NIK, inducing NF-kB activation and inflammation	Human patients	Lu et al., 2016, JCI
Lung adenocarcinoma	ADAR-mediated editing increases FAK kinase mRNA stability and expression, correlating with cancer invasiveness	Human patients	Amin et al., 2017, Science signaling
Mild myopathy	APOBEC2 RNA-editing enzyme preferentially expressed in muscles; deficiency leads to decrease in body mass and mild myopathy	APOBEC2-KO mouse model	Sato et al, 2010, JBC
Multiple myeloma	Dis3 mutations associated with chromosomal translocations at immunoglobulin heavy chain locus	Human patients	Lionetti et al., 2015, Oncotarget
Myotonic dystrophy	RNAs containing microsatellite expansions sequester MBLN2 proteins, perturbing splicing and polyadenylation in brain	Human primary tissues, ‘RNA toxicity hypothesis’	Goodwin et al., 2015, Cell Rep
	MBLN3 expression during embryogenesis and muscle regeneration with binding to 3′UTR of cell growth and proliferation genes. Defects in muscle regeneration and function in MBLN3-KO mice	MBLN3-KO mouse model	Poulos et al., 2013, Hum Mol genetics
Myotonic dystrophy type 1	Alternative splicing and abnormal polyadenylation in muscles due to trinucleotide expansions in RNAs, altering activities of RNA-processing factors, including MBNL proteins	Human samples and KO mouse models	Thomas et al., 2017, Genes and development
Pancreatic adenosquamous carcinoma	Somatic mutations of UPF1 (NMD factor) upregulate NMD substrate mRNAs	Human patients	Liu et al., 2014, Nat medicine
Perlman syndrome and Wilms tumor susceptibility	DIS3L2 germline mutations, mitotic abnormalities, dysregulated expression of mitotic control proteins	Human patients	Astuti et al., 2012, Nat genetics
Pontocerebellar hypoplasia 1	EXOSC3 (RNA exosome) mutations found in patients. Knockdown experiments in zebrafish perturb embryonic development and brain formation	Human patients and zebrafish model	Wan et al., 2011, Nature Genetics
Pontocerebellar hypoplasia 1 like	RBM7 (NEXT complex) mutations, altering gene expression. Knockdown of rbm7 in zebrafish induced defects in motor neurons and cerebellum	Human patients and zebrafish model	Giunta et al., 2016, Human mol genetics
R-loop stabilization and genomic instability	EXOSC3 deficiency increases R-loop formation at DNA translocation hotspots	Mouse primary B cells (conditional Exosc3 KO)	Pefanis et al., 2014, Nature
R-loop-associated pathologies	Mutations in genes involved in R-loop removal or formation	Review	Richard and Manley, 2017, J Mol Biol.
Retinitis pigmentosa, hearing loss, premature aging, short stature, mild intellectual disability and distinctive gestalt	EXOSC2 (RNA exosome) mutations, probably altering RNA metabolism	Human patients	Di Donato et al., 2016, J med genetics
RNA-editing and ADAR1-linked pathologies	Implication of editing in different pathologies, cancers, and neuropathies	Review	Song et al., 2016, Genes
Systemic lupus erythematosus	RNase H2 gene mutations associated with accumulation of ribonucleotides in genomic DNA and DNA damage, culminating with upregulation of IFN-stimulated genes	Human patients	Günther et al., 2015, J Clin Invest.
	TREX1 mutations in patients with SLE, with two mutations affecting TREX1 protein subcellular targeting, and probably deregulating its activity	Human patients	Lee-Kirsh et al., 2007, Nat Genet.

Box 1. The Different Classes of Coding and Noncoding RNAs.

Coding RNAs are processed from pre-mRNAs to mature mRNAs and translated into proteins to fulfill cellular functions, thereby linking information in DNA to active proteins. By contrast, most RNAs do not produce proteins and are referred to as ncRNAs. While transcription from ‘junk DNA’ was initially considered a byproduct and generally treated as ‘transcriptional noise’, it appears that ncRNAs have crucial roles in cellular physiology.

The major categories of ncRNAs are ribosomal RNA (rRNA), transfer RNA (tRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), very small RNA (miRNA, siRNA, and piRNA), and long ncRNA (lncRNA). Recent advances in biology have demonstrated unexpected functions for lncRNAs, which regulate diverse physiological events. LncRNAs include various RNAs, such as enhancer RNA (eRNA), promoter-associated antisense RNA, intergenic RNA, and intragenic RNA. Circularization of RNAs is another important aspect of RNA processing, opening the field of circular RNAs, described as potential miRNAs sponges, with critical implications in physiological and pathological processes. Many transposable elements (TEs) are integrated in higher eukaryote genomes and can eventually be expressed. These elements have to be tightly regulated by RNA surveillance because they can be deleterious by introducing genomic instability. In addition to endogenous RNAs, mammalian cells are also challenged by exogenous RNAs that are either transferred by extracellular vesicle cargos resulting from intercellular communication, or from microorganisms, with the latter being mainly viral RNAs.

Cells have to regulate both the transcription and processing of all these different RNAs, with strong quality control ensured by RNA surveillance. Although ‘pervasive’ transcription of genomes is widely reported, tight regulation of gene expression in space and time, culminating in the control of developmental schemes, makes more evolutionary sense.

The recent development of coding RNA and ncRNA transcriptomics and epitranscriptomics [3] has provided evidence for a higher level of RNA diversity. How RNA diversity meaningfully contributes to organism development is the focus of this review. Here, we propose to extend the definition of RNA surveillance beyond its role in quality control to include its implication in post-transcriptional regulation, describing RNA surveillance as the convergence of mechanisms that ensure the exact titration of any RNA molecule in a spatiotemporally controlled manner, leading to development without the onset of pathological conditions.

RNA Diversity and the RNA Surveillance Machinery

RNA surveillance functions to sieve out short-lived, long-lived, and nonfunctional RNAs as per cellular requirements and could be a central mode of gene regulation (Figure 1), culminating in the control of developmental schemes. RNA surveillance pathways, ensuring strong control of their target RNAs (Box 2), have been extensively reviewed elsewhere [1,4] and are briefly described here.

Figure 1. Cellular Context of RNA Processing and Surveillance.

Integrated view of RNA maturation and the different levels of RNA quality control, processing, and surveillance. (A) Nuclear surveillance: maturing mRNAs can undergo various modifications (editing, deamination, or chemical modifications) that affect their stability, decay, splicing, and export. Noncoding (nc)RNAs are also susceptible to chemical modifications and degradation. Circular RNAs can also be produced and potentially exported from the nucleus. (B) mRNA fate is dependent on its modification and interaction with other proteins and/or RNAs. 5′ end decapping leads to RNA degradation by the Xrn1 exonuclease; deadenylation and uridylation trigger 3′ end decay by the RNA exosome or Dis3L2 exonucleases, while other endonucleases can directly cut RNA. This level of cytoplasmic surveillance regulates the concentration of coding RNAs and ncRNAs, which eventually can be delocalized in subcellular compartments. RNAi (e.g., miRNA) fine-tunes gene expression. Nonfunctional transcripts are processed by the nonsense-mediated decay (NMD), nonstop-mediated decay (NSD), or no-go decay (NGD) pathways.

Box 2. The RNA Surveillance Machinery.

RNA surveillance pathways are unusually redundant in terms of their control of their target RNA outcome. Several layers of RNA surveillance work in concert to fine-tune the RNA concentration of any given RNA molecule. This process starts at the chromatin level, where DNA-associated RNAs are unwound and degraded to avoid genomic instability, while nuclear and cytoplasmic surveillance completes this monitoring to control the titration of ncRNA and mRNA levels.

RNA decay is ensured by exoribonucleases that degrade RNAs from their 5′ (XRN family) or 3′ (RNA exosome, Dis3L2) extremities following decapping, polyA shortening, polyuridylation, and so on, induced by specific stimuli or natural turnover. Endoribonucleases [ribonuclease (RNase) A, RNase H, Dis3, Dicer, etc.] can directly cleave the body of RNA molecules and participate in RNA processing. Many cofactors participate in RNA surveillance, including RNA helicases, which unwind RNA molecules, multiprotein complexes, RNA-binding proteins (RBPs), and adaptor proteins, all modulating RNA interactions and degradation. For example, the Ccr4-NOT complex has important roles in both the nucleus and cytoplasm, where it globally contributes to gene expression control, while the transcription and export (TREX) complex regulates mRNA maturation and export, and is essential for cell differentiation and organism development. The THO complex controls both mRNA processing and snoRNA expression in yeast. The Trf4/Air2/Mtr4p polyadenylation (TRAMP) complex interacts with the RNA exosome to monitor the RNA concentration of various substrates, while the SUPV3L1, SKIV2L (SKI) complex is important for cytoplasmic RNA degradation by the RNA exosome complex or the DIS3L2 enzyme. Other important mechanisms participate in mRNA quality control, including splicing-mediated decay, nonsense-mediated decay (NMD), nonstop-mediated decay (NSD), and no-go decay (NGD). RNAi contributes to RNA surveillance using various mechanisms to control mRNA expression and degrade exogenous and TE RNAs. The flow of RNA processing and RNA localization, including its sequestration, are also important parameters contributing to gene expression.

RNA Degradation

mRNAs are protected at their 5′ end by a cap that is removed by a decapping complex, following specific signals or natural turnover. Decapped mRNAs are degraded by the 5′ XRN exonuclease family of proteins, which are highly conserved and have critical functions in RNA surveillance, gene silencing, and nonsense-mediated mRNA decay (NMD) [5]. In yeast, Xnr1 participates in the degradation of antisense ncRNAs, regulating chromatin modifications and gene expression [6]. 5′ RNA processing by decapping and exoribonuclease activity can be initiated co-translationally [7] and is linked to NMD, which participates in the degradation of pervasive noncoding transcripts [8]. At the 3′ end, polyadenylated mRNAs are gradually deadenylated by poly(A) ribonucleases (e.g., PARN) and degraded by the RNA exosome complex or the DIS3L2 enzyme. Dis3L2 has 3′ end exoribonuclease activity, is cytoplasmic, and ensures the degradation of various uridylated RNAs [9]. The RNA exosome has major functions in RNA processing, with emerging functions in the maintenance of genome stability. The RNA exosome has different subunit and cofactor compositions that determine its differential ribonuclease activity [10] and it can degrade RNA unwound from DNA by the RNA helicases Setx and Mtr4 [11,12]. RNAs can be processed by endonucleases, such as RNase H and RNase A, which cleave directly in the body of these molecules. RNase H activity, which cleaves RNA when it is associated with DNA in a DNA/RNA hybrid configuration, is found in all kingdoms of life and is mandatory for DNA replication, DNA repair, and RNA splicing [13]. The Dis3 protein of the RNA exosome complex also has endonuclease activity [14]. Dicer also has endonuclease activity that is implicated in the generation of miRNAs and in RNAi [15], while other endonucleases participate in specific RNA surveillance pathways.

Various Mechanisms Used for RNA Surveillance

RNAs adopt many different secondary structures and interact with other molecules, potentially masking their degradation signals. Therefore, co-factors, in particular helicases, are necessary to unwind RNA for processing [16], while other multiprotein complexes interact with different ribonucleases to finely regulate their activity (e.g., Ccr4-NOT, TREX, THO, TRAMP, SKI, etc.). Other complexes, RNA-binding proteins (RBPs), and adaptor proteins contribute to specific interactions between RNA substrates and RNA decay factors [17]. Transcription generates mRNAs that can include premature termination codons. The NMD pathway detects and degrades these aberrant transcripts, and controls gene expression using UPF proteins along with RNA exo- and endonucleases [18,19]. Other mechanisms monitor transcripts that lack stop codons and remove them by nonstop-mediated decay (NSD) [20], while no-go decay (NGD) degrades transcripts on which ribosomes have stalled. Spliceosome-mediated decay is observed in the context of the expression of non-intronic genes in yeast [21]. Spliceosome-mediated RNA decay is implicated in regulating alternative splicing and intron retention, and occurs in most genes and all tissues due to a central role in cell differentiation [22–24]. RNA silencing is another mechanism that controls gene expression, or degrades exogenous RNAs. ncRNAs are cleaved by the RNase III Dicer [15] to produce miRNAs or small interfering (si)RNAs that are loaded onto the RNA-induced silencing complex (RISC) and the Argonaute (Ago) proteins, which induce inhibition of translation, mRNA cleavage, or degradation of their targets. Germ cells use a piwi-interacting (pi)RNA guide loaded onto PIWI proteins to cleave RNA and promote DNA methylation [25]. miRNAs are themselves subject to degradation by the Tudor-SN endonuclease in human cells [26]. Finally, RNA trafficking and localization participate in the fine-tuning of gene expression. Site-specific mRNA regulation was recently shown in neurons, where the N6-methyladenosine (m⁶A) eraser FTO is locally translated to regulate m⁶A RNAs [27].

Influence of RNA Sequence and Modifications on RNA Surveillance

RNA composition modulates their folding, secondary structures, and interactions (Box 3). AU-rich elements (AREs) interact with RBPs that regulate the processing and decay of mRNAs [28]. GU-rich elements are found in genes of signaling components and are upregulated in cancer cells [29]. 3′ untranslated region (UTR) miRNA binding sites are crucial for gene expression regulation and RNAi. mRNA interactions with trans-regulators have key functions in homeostasis, while disruptions lead to neurodevelopmental disorders [30]. RNA composition can be altered by diverse processes, including RNA editing, performed by ADAR proteins that change A to I, with critical implications in gene regulation and diseases [31]. ADAR1- and ADAR2-dependent editing appear to be tissue specific [32], and may be important to confer cell identity and lineage specificity. Some APOBEC proteins can deaminate RNA, resulting in C to U modifications [33], and could be important in the regulation of endogenous or exogenous RNAs by recruiting the RNA exosome to degrade these transcripts.

Box 3. Additional Layers Contributing to RNA Surveillance.

Additional parameters that contribute towards the fine titration of RNA molecules in the cellular milieu must be considered to complete our developmental overview of RNA surveillance. For mRNAs, different intrinsic factors, mainly localized in their 3′ UTR sequences, participate in their stability and surveillance (AU-rich elements, GU-rich elements, miRNA-binding sites, etc.). 3′ capping, 5′ end modifications, and RNA composition itself also strongly orientate RNA molecules to their respective fates. RNAs interact with different molecules, and improvements in biochemistry methods further extend the interactome list every day. A broad range of RNA-binding proteins with important biological functions have been identified, but many processes remain to be discovered. RNA–RNA interactions can be intra- or inter-molecular, with a huge number of possibilities and an interesting dynamic that can have important roles during differentiation and development. Finally, DNA-associated RNAs appear to be transitory, but could have important roles in chromatin remodeling and gene expression regulation.

Coding RNAs and ncRNAs are also subject to editing and chemical modifications. RNA editing by adenosine deaminases acting on RNA (ADAR) proteins directly changes the ribonucleotide sequence from adenosine to inosine (A to I), with emerging functions in physiology and pathology. Some APOBEC proteins can deaminate RNAs, resulting in cytosine to uracil (C to U) alteration and could be implicated in endogenous and exogenous RNA surveillance, participating in the fine-tuning of biological functions. Another expanding field has emerged recently with the study of epitranscriptomes by high-throughput sequencing, and identified more than 100 RNA modifications. The enzymes that write, read, or edit these modifications are now under study, demonstrating the crucial role of these chemical changes in a range of biological functions. Importantly, these RNA modifications are now clearly implicated in pathology, including a large number of cancers. This complex interplay between RNAs, their modifications, and interactions with other molecules, expands the complexity of RNA regulation and surveillance.

RNA modifications regulate their chemical and structural properties [34], with the most abundant m⁶A implicated in a range of biological processes, including development [35]. Epitranscriptomics [3] has led to the identification of many other modifications, greatly diversifying the RNA species. Specific enzymes catalyze (writers), decipher (readers), or edit (erasers) these RNA alterations, contributing to RNA stability. Uridylation by terminal uridylyl transferases (TUTases) is another modification that affects RNA stability and tags them for degradation [36]. RBPs with critical roles in biological systems have been characterized, suggesting that >10% of proteins have RNA-binding capacity [37]. RBPs regulate pluripotency [38], and mutations are observed in human Mendelian diseases, mainly neurological disorders and cancers [39]. Additionally, complex networks of intra- and intermolecular RNA–RNA interactions impact RNA stability and are dynamically remodeled during human embryonic stem cell (ESC) differentiation [40].

RNA Surveillance during Cellular Differentiation and Embryogenesis

Although RNA surveillance machineries were initially investigated in model organisms, their relevance to development and pathologies is particularly interesting in humans. Indeed, these mechanisms are used to fine-tune gene expression programs during embryogenesis and pluripotency (Figure 2).

Figure 2. Organism Development and Critical Points of RNA Surveillance.

Overview of RNA-associated events governing organism development. RNA processing and surveillance orchestrate physiological development, from fertilization to tissue and organ maturation. (A) Main steps of embryogenesis and RNA surveillance events. These examples illustrate how RNA surveillance contributes to physiological functions in developing organisms. (B) Different mechanisms of RNA surveillance regulate the development of germ cells, the nervous and immune systems, and skeletal muscles. Defects in these functions lead to developmental abnormalities and associated pathologies. Abbreviations: PGC, primordial germ cell; TE, transposable element; TF, transcription factor. For additional definitions, please see the main text.

Embryogenesis is a paradigm for RNA surveillance since it begins using maternal RNAs but quickly switches to the zygotic transcriptome, a phenomenon called the ‘maternal-to-zygotic transition’ (MZT). The maternal transcriptome is shaped by TUT4 and TUT7 enzymes and uridylation of mRNAs, which facilitates their degradation during mouse oocyte maturation, where these mRNAs have shorter poly(A) tails than those in somatic cells [41]. One-third of maternal RNAs are methylated and, after fertilization, m⁶A methylation facilitates the decay of these RNAs, which are bound by YTHDF2; this turnover is required for embryonic development in zebrafish [42] and mice [43]. miRNAs are strongly expressed after fertilization, with greater mono- and oligo-adenylation, potentially protecting them from degradation. miRNA function appears to be dynamic, activated at the two-cell stage to regulate developmentally important genes [44]. The CCR4/POP2/NOT deadenylase complex participates in maternal RNA degradation in coordination with the RBP Smaug in Drosophila embryos [45]. Notably, the recruitment of this complex to its RNA target for degradation depends on the piRNA pathway [46]. These events illustrate how RNA-processing pathways can converge to perform a specific task.

At the chromatin level, zygotic DNA is demethylated and, therefore, transposable elements (TEs) may be expressed and must be degraded to avoid genomic instability. Ago2 quickly binds small RNAs to interfere with TE RNAs, followed by the formation of repressive chromatin marks to ensure TE silencing in ESCs [47]. However, this dogma is challenged by evidence demonstrating cellular functions for TEs, such as long interspersed nuclear elements-1 (LINE-1), acting as scaffolding RNA to control gene expression in ESCs and pre-embryos [48]. Many promoters in human and mouse ESCs are transcribed bidirectionally, with coordinated changes during differentiation [49]. DNA sequences, expressing enhancer (e)RNAs, control gene expression and cellular differentiation in the context of chromosome looping and long-range DNA interactions in ESCs, where a superenhancer controls DNA looping at the Nanog locus to maintain pluripotency [50]. Finally, transcription of intragenic enhancers can directly decrease host gene expression by interfering with RNA polymerase II activity and leading to impaired ESC differentiation [51].

In addition to embryogenesis, RNA surveillance can also regulate pluripotency. Zinc finger TF217 (ZFP217) is a key player in maintaining pluripotency via epigenetic and epitranscriptomic regulation, where it interacts with, and sequesters, methyl-transferase-like 3 (Mettl3), decreasing transcript methylation. ZFP217 knockdown in ESCs increased m⁶A RNA levels, especially of Nanog, Sox2, Klf4, and c-Myc mRNAs, promoting their degradation and inducing cell differentiation [52]. Circular RNAs are themselves methylated; an atlas of these RNAs revealed a specific pattern of methylation in human ESCs, in that they were Mettl3 dependent and interacted with YTHDF1/YTHDF2 readers [53]. A comparison of pluripotent cell methylated transcriptomes revealed gene and cell specificity of m⁶A, whereby m⁶A promotes reprogramming to pluripotent cells and, importantly, the sequence specificity of these modifications is regulated by miRNAs in a pairing-dependent manner [54]. Interestingly, miRNA levels can be regulated by the expression of long (l)ncRNAs, as suggested for the ncRNA Cyrano and its complementary sequence, which both modulate the expression of master self-renewal factors and ESC maintenance [55]. Another regulatory network implicating Cyrano, miRNAs, and a circular RNA has been also described in the mammalian brain [56]. These data suggest a complex interplay between the different classes of ncRNA and mRNA in surveillance pathways.

CNOT3, a component of the Ccr4-Not complex, is required for embryonic development; its deficiency increases the poly(A) length, half-life, and steady-state level of differentiation gene mRNAs [57]. In human ESCs, NMD maintains pluripotency by targeting specific genes, including many signaling components, with the TGF-β and BMP axis being important for cell differentiation [58]. RNA exosome controls the redox status of pluripotent stem cells by degrading ARE-containing RNAs, including Gpx2, which is responsible for protection from reactive oxygen species [59], while m⁶A is crucial for regulating ESCs and pluripotent cells [60], acting in concert with other RNA surveillance mechanisms.

RNA Surveillance during Tissue Development

During development, the three germ layers (ectoderm, mesoderm, and endoderm) give rise to organs after somitogenesis, histogenesis, and organogenesis. Here, we focus on tissues in which RNA surveillance mechanisms have been described (the hematopoietic, nervous, and muscular systems) and address the situation of germ cells, which remodel their chromatin and transcriptional programs (Figure 2).

Immune System

Hematopoiesis generates common myeloid and lymphoid progenitors, which engender blood and immune system cells. A reservoir ensures both stem cell maintenance and differentiation of effector cells. The hematopoietic lineage derives from the endoderm layer, where this transition is controlled by m⁶A. Mettl3-deficient zebrafish embryos have fewer m⁶A transcripts, which delays YTHDF2-mediated RNA decay of nocth1a and rhoca [61], genes specifically implicated in this differentiation process. Hematopoietic stem cells (HSCs) express high levels of METTL14, which maintain pluripotency; its deletion induces cell differentiation in HSCs, as well as in acute myeloid leukemia cells, the pathological counterpart of myeloid cells [62]. A similar phenotype is observed with human HSCs, in which depletion of METTL3 impairs stem cell maintenance; by contrast, METTL3 is necessary for the proliferation of acute myeloid leukemia cells, where m⁶A promotes oncogene translation [63]. tRNAs pseudouridylation chemical modification controls ESCs and their hematopoietic commitment; its deficiency is observed in myelodysplastic syndromes [64]. Myeloid progenitors generate innate immune system cells, thrombocytes, and erythrocytes. The RNA exosome expression level (Exosc8 subunit) is downregulated by the master regulator of erythropoiesis GATA-1, inducing erythrocyte maturation [65]. RNA exosome is also necessary for the expression and signaling of the Kit receptor, preventing erythropoietin-induced differentiation in vivo [66]. Upon infection or development, Tet2 oxidizes 5-methylcytosines of RNAs, modulating the recruitment of the ADAR1 editing enzyme and the expression level of mRNAs, in particular Socs3 and the Jak/Stat axis, inducing an increase in myelopoiesis and mast cell production [67]. This specific level of gene expression regulation further extends the list of mechanisms involved in the control of cellular function.

Lymphoid progenitors produce natural killer cells and T and B lymphocytes, conferring adaptive immunity. Mettl3 has crucial roles in T cell homeostasis and differentiation, where SOCS1, SOCS3, and CISH mRNAs, inhibitors of the STAT signaling axis, are methylated to ensure their decay [68]. Antigen-experienced B cells produce high-affinity antibodies by transcription-coupled DNA alteration processes called class switch recombination (CSR) and somatic hypermutation (SHM) [69,70]. CSR and SHM require a series of steps of ncRNA transcription and coupled RNA processing by the RNA exosome complex in association with Mtr4 and senataxin helicases. In the absence of the RNA exosome complex, CSR and SHM mechanisms are perturbed, leading to weak and/or altered antibody gene diversification [11,71,72]. As a collateral effect of B cell activation, leading to increased transcription and RNA splicing, NMD has a role in degrading potentially deleterious mRNAs in B cells [73]. Finally, the immunoglobulin locus recombination is driven by a 3′ regulatory region (′RR) [74] superenhancer, and transcription of enhancer RNA and 3′RR activation is regulated by the RNA exosome complex [75].

Nervous System

The nervous system derives from the ectoderm, beginning with the formation of the neural tube, giving rise to the brain structures. Methyltransferase components and readers are enriched in the Drosophila nervous system, where they mediate m⁶A-dependent alternative splicing, which is important for neuronal function and fly behavior in vivo [76]. During cortical development, absence of Mettl14, Mettl3, and m⁶A prolongs the cell cycle of radial cells in mice. m⁶A is enriched at genes encoding transcription factors (TFs) and genes implicated in neurogenesis, promoting their degradation. m⁶A signaling also regulates human cortical neurogenesis in vitro and tags transcripts related to brain pathology risk [77].

The importance of RNA editing in human brain is emerging: different patterns of ADAR-dependent editing have been observed, associated with neuronal maturation and mRNA abundance, potentially influencing miRNA binding during cortical development. These transcripts are related to vesicle or organelle membranes and glutamate signaling, with some perturbations observed in spinal cord injury and glioblastoma [78]. While RNA surveillance usually represses the expression of endogenous retrotransposons, LINE-1 is expressed in the hippocampus during early life, linked to maternal care, and changing the neuronal genome [79]. Another study showed how an RNA splicing event could participate in long-term memory in Drosophila [80]. Finally, a recent study revealed the dynamics of RNA methylation in mouse brain following stress exposure, where an imbalance may be implicated in depressive disorders in humans [81].

Myogenesis

Muscle cells derive from the endoderm layer, where myogenesis requires the key TF MyoD for differentiation and fusion of myoblasts in mature skeletal muscle fibers. In progenitor cells, Mettl3 is required to methylate MyoD mRNAs at the 5′UTR region for processing and skeletal muscle differentiation [82]. During human myogenesis, the NMD factor UPF1 directly promotes the degradation of MyoD protein, linking the mRNA and protein decay mechanisms [83]. After birth, a pool of muscle stem cells, known as satellite cells, allows muscle regeneration in case of injury. The mRNA decay protein AUF1 specifically degrades ARE-containing mRNAs and contributes to the maintenance of these stem cells, while mutations may lead to human myopathies [84].

Germ Cells

Germ cells generate gametes by meiosis, switching from a diploid to haploid state; they are produced by the induction of primordial germ cells (PGCs) from embryonic cells in mammals. PGCs have specific features, including transcription repression and chromatin-state alteration. miRNAs and 3′ nucleotide addition control the RNA molecules necessary for PGC maturation, and the transition to gonadal development in mouse embryos [85]. Germ cells express both lineage-specific TFs and a distinct transcriptional profile, while DNA undergoes global demethylation [86]. This chromatin derepression is potentially perilous because some endogenous TEs can be expressed and must be controlled.

In oocytes, maternal RNAs mainly govern the first steps of zygote maturation and many are then degraded. Maternal RNA degradation is mediated by uridylation and decay [41], and by methylation in combination with their readers. YTHDF2 reader-deficient mice fail to accomplish transcript dosage during oocyte maturation, resulting in female infertility [43]. Mechanistically, YTH domain-containing proteins are important for regulating meiotic genes. In yeast, one YTH domain-containing protein (Mm1), both directly targets meiotic transcripts to the RNA exosome for degradation and tethers them to nuclear foci, enforcing translation inhibition [87]. In mice, YTFHDC2 controls the transition from mitosis to meiosis by binding mitotic transcripts as well as specific piRNA precursors and interacting with granule components [88]. siRNA production also contributes to meiotic control in mouse oocytes, where Ago2 regulates the oocyte transcriptome during meiosis, subsequent chromosome alignment, and, simultaneously, the expression of some transposable elements (TEs) [89].

In male germ cells TDRD6 is highly expressed, interacting with the spliceosome and one methyltransferase; its depletion leads to splicing defects in spermatocytes [90]. The demethylation enzyme ALKBH5 is necessary for male fertility to erase m⁶A and undertake subsequent accurate splicing, producing some long mRNAs during late meiosis [91]. A global shortening of mRNAs has been previously observed and is necessary during spermatogenesis. This effect is mediated by the NMD component UPF2, which targets and degrades longer 3′UTRs. UPF2 knockout (KO) spermatocytes again accumulate alternatively spliced transcripts, leading to global defects and infertility [92]. This noncanonical role of the NMD pathway could be important in other cells. The RNA exosome complex itself participates in spermatogenesis regulation, during which Exosc10 is dynamically expressed [93].

The CCR4-NOT complex is recruited by the RBP DND1, which binds a motif in the 3′ UTR of the mRNA and destabilizes its targets, regulating apoptosis, inflammation, and pluripotent gene expression in PGCs and spermatogonial stem cells (SSCs) [94]. The RNA methylome atlas revealed the dynamics of m⁶A modifications, with increased methylation at the pachytene/diplotene spermatocyte and round spermatid stages. Methylation affects the translation efficiency of important genes implicated in SSC proliferation and differentiation, and later for haploid-specific genes [95]. Taken together, these results show the complexity of RNA processing contributing to meiosis.

More globally, RNA surveillance is crucial during normal embryogenesis and development.

RNA Surveillance Deficiencies Leading to Pathologies

RNA surveillance defects lead to associated pathologies during development. Here, we describe some disorders and how RNA processing contributes to maintaining genome integrity (Figure 3 and Table 1).

Figure 3. RNA Surveillance of DNA-Associated RNAs.

(A) DNA transcription can create DNA:RNA hybrids, also called R-loops. These structures expose one DNA strand to environmental mutagenic agents (e.g., radiation, chemicals, and enzymes) and must be resolved by RNA surveillance mechanisms (e.g., RNase H or RNA exosome). R-loop persistence results in asymmetric DNA mutations and eventually DNA double-strand breaks and translocations. (B) Cofactors participate in R-loop resolution; the RNA helicase Mtr4 is directly associated with the RNA exosome to unwind DNA-associated RNAs, coupled with RNA decay. (C) Other RNA helicases, such as Senataxin, participate in R-loop unwinding to dissociate DNA-associated RNAs that are subsequently degraded by the RNA exosome.

Immunological Disorders and Autoimmunity

Aicardi–Goutières syndrome (AGS) is a disorder characterized by abnormal inflammation affecting the immune and nervous systems. AGS can be caused by mutations in TREX1, RNase H2 complex, or ADAR1, which upregulate interferon-regulated genes [96]. Mechanistic studies demonstrate that ADAR1 edits endogenous double-strand (ds) RNAs, preventing the activation of the cytosolic dsRNA sensor MDA5 and the interferon pathway [97]. Loss of tolerance is caused by Alu retroelements that form Alu:Alu dsRNA structures and activate MDA5 in the absence of ADAR1-dependent editing [98]. A mouse model of RNase H2 mutation suggested an involvement of the cGAS/STING pathway, with defects in ribonucleotide excision repair, increased DNA damage, and activation of interferon-stimulated genes [99]. RNase H2 mutations are also linked to systemic lupus erythematosus (SLE) [100], and TREX1 deficiencies or mutations have also been implicated in both AGS [101] and SLE [102]. This could be due to retrotransposon expression followed by reverse transcription and accumulation of cytoplasmic nucleic acids in the absence of the nuclease activity of TREX1, or directly linked to the ribonuclease function of this enzyme [103]. Mutations have been identified in the human polynucleotide phosphorylase gene PNPT1 that also converge on interferon pathway activation. This axis is implicated in the processing of mitochondrial dsRNAs, which are usually unwound by SUV3 helicases followed by polynucleotide phosphorylase activity [104]. Several diseases are linked to mutations in RNA exosome genes or its cofactors [105]. Plasma cell disorders, including multiple myeloma, present Dis3 loss-of-function mutations, preferentially associated with chromosomal translocations to the immunoglobulin locus [106], reminiscent of ncRNAs accumulation at translocation hotspots in mice [71].

Neurodegenerative Diseases

Defects in RNA editing are implicated in fragile X syndrome, a frequently inherited form of intellectual disability, caused by the absence of the fragile X mental retardation protein (FMRP). Under normal conditions, ADAR2 interacts with FMRP to regulate editing of genes implicated in neuronal circuit formation, although this editing is increased in Fmrp-deficient zebrafish [107]. RNA editing is also enriched in introns of human brain transcripts, suggesting a conserved role for this function [108]. Some repeat-expansion-associated diseases, such as myotonic dystrophy (neuromuscular), can induce ‘RNA toxicity’ and nervous system pathologies. In brain samples from patients with myotonic dystrophy, RNAs containing microsatellite expansions sequestered the MBLN2 protein and perturbed RNA splicing and polyadenylation, with the same effect observed in Mbln1 KO mice [109].

The RNA exosome is mutated in several human neuropathies: for example, EXOSC3 mutations are implicated in spinal motor neuron disease corresponding to pontocerebellar hypoplasia 1, with a similar phenotype seen in Exosc3-knockdown zebrafish [110]. EXOSC8 mutations induce a progressive disease involving cerebellar and corpus callosum hypoplasia, abnormal myelination, or spinal motor neuron disease. Knockdown experiments showed an increase in ARE mRNAs of myelin proteins related to the pathology [111]. An Exosc2 mutation was identified in a patient with retinitis pigmentosa and mild intellectual disability [112]. RBM7 (part of the NEXT complex) mutation alters RNA metabolism, and knockdown experiments revealed defects in motor neurons and the cerebellum, reminiscent of Exosc3 and Exosc8 [113]. TDP-43 is frequently mutated in human amyotrophic lateral sclerosis and frontotemporal lobar degeneration, inducing defects in siRNA silencing and concomitant TE expression, DNA breaks, and cell death in the Drosophila brain [114].

Myopathies

Myopathies are a group of diseases affecting muscular development and function. Myotonic dystrophy, a class of inherited muscular dystrophies, is caused by triplet expansions (in DMPK or CNBP genes) that create toxic RNAs sequestering MBNL alternative splicing factor proteins. Mbnl3 is expressed during embryogenesis and skeletal muscle regeneration (where relevant exons undergo a prenatal RNA isoform transition) and binds the 3′UTR of cell growth and proliferation genes. Accordingly, Mbnl3-KO mice have age-dependent defects in injury-induced muscle regeneration and muscle function [115]. Alternative splicing and abnormal poly(A) are also detected in the muscles of patients with congenital myotonic dystrophy. Mbnl protein deletions in mouse muscle recapitulated the human symptoms of congenital myopathy with specific defects in RNA splicing [116]. APOBEC2 is preferentially expressed in muscles, with an RNA-editing function. This deaminase is important for regulating muscle development, and APOBEC2-deficient mice have decreased body mass and mild myopathy [117].

Genome Integrity, RNA Surveillance, and Cancer

The emerging field of RNA surveillance provides new perspectives to approach cancer genesis. Transcription of some genomic regions due to sequence context or other reasons can induce transcriptional stalling and associated RNA:DNA hybrid accumulation, also known as R-loops. These structures regulate chromosomal organization, epigenetics, gene expression, DNA replication and repair, and class switch recombination [118]. They are implicated in human diseases, including neuropathies and cancers [119], where R-loops can expose a single-stranded DNA to mutagenic factors. The RNA exosome and its cofactors are necessary for regulating DNA:RNA hybrid levels to suppress asymmetric DNA mutagenesis [11] and associated genomic instability [71,120] (Figure 3). Other RNA-processing pathways participate in R-loop resolution in humans, such as the RNA helicases Aquarius and Senataxin, and topoisomerase I [121]. DNA:RNA hybrid stabilization and associated R-loop formation can lead to dsDNA breaks because of collisions between the stalled transcription and DNA replication machineries [122,123]. These collisions are more detrimental when the transcription–replication machineries collide in a head-on configuration [124], while convergent transcription increases R-loop formation, RNA polymerase stalling, and genomic instability in leukemic cells [71,125–127].

In cancer cells, differential RNA expression can affect any class of RNA, including lncRNAs, miRNAs, and mRNAs themselves, which are normally controlled by RNA surveillance. Somatic mutations in NMD factors have been reported, where UPF1 mutants upregulate NMD substrate transcripts in pancreatic adenosquamous carcinoma [128], and in myofibroblastic tumors. In these cancers, upregulated mRNAs, including NF-kB inducing kinase (NIK), induce NF-kB activation and contribute to inflammation [129]. Germline mutations in the Dis3L2 gene have been found in patients with Perlman syndrome and susceptibility to Wilms’ tumors. Loss of exonuclease activity induces mitotic abnormalities and dysregulated expression of mitotic control proteins [130]. Disequilibrium in m⁶A modifications has important roles, with oncogenic mutations in methyltransferase, demethylase, or reader genes [131]. The demethylase ALKBH5 is necessary for the proliferation of glioblastoma cells by maintaining the levels of certain mRNAs, in particular FOXM1. Interestingly, the specificity of this gene is conferred by FOXM1 antisense RNA, which works as a guide to target its own mRNAs and induce tumorigenicity [132]. Inversely, the FTO eraser is overexpressed in a subtype of acute myeloid leukemia, where it reduces the expression levels of important genes, contributing to both cell transformation and leukemogenesis [133]. In lung adenocarcinoma, ADAR-mediated editing increases the stability of the FAK kinase mRNA, enhancing its expression, and is correlated with cancer invasiveness [134]. RNA editing also contributes to the epitranscriptome diversity of cancer stem cells [135].

Concluding Remarks

Strong evidence shows that RNA surveillance is a key component of physiological development, regulating early stages of embryogenesis, pluripotency, lineage specification, organogenesis, and the balance of stem cell maintenance and differentiation.

At the cellular level, an intriguing concept is the flow and relationship between these different RNA-processing events (see Outstanding Questions). RNA surveillance starts at the chromatin level with the detection and removal of DNA-associated RNAs and, in addition to its participation in genomic stability, such mechanisms could contribute to gene expression regulation, in concert with epigenomic changes. Nuclear and cytoplasmic RNA surveillances then constantly adjust the RNA concentration using various combinations of mechanisms in different cells. RNA modification incorporation and removal are potentially faster events than transcriptional regulation, providing direct dynamic control of RNA levels. Differential marks and complex RNA surveillance pathways commit coding and ncRNAs to their relevant fates, affecting their kinetics, localization, interactions, and functions. The challenge now is to study these RNA surveillance mechanisms in every type of cell and their impact on homeostasis and differentiation. A more systematic analysis of RNA surveillance pathways in pathologies and cancers should also be considered to explain transcriptomic disorders.

Outstanding Questions.

How do RNA surveillance functions specifically regulate distinct steps of cellular differentiation and development by co- and/or post-transcriptionally acting on mRNAs or ncRNAs? Is this specificity provided by a combination of various RNA surveillance mechanisms? How are RNA surveillance mechanisms regulated during development?

How do RNA modifications specifically tag a subset of RNAs, influencing their processing and decay, but not others? How does a given RNA molecule have different marks depending on cell type?

How do the combinations of RNA modifications (RNA epigenetic code) influence RNA processing? What is the kinetics of RNA modifications?

What are the global functions of RNA-editing events? Does RNA editing also impact cell division and differentiation during organism development?

Is piRNA maturation germ cell specific or does it have a role in other cell types? Are piRNA-like pathways activated in cells to prevent the onset of cancer?

Intercellular RNA exchanges are observed both in physiological and pathological conditions. Does RNA surveillance determine intercellular RNA exchange during embryogenesis and organism development?

While recent data suggest a functional role of some TEs, how does RNA surveillance distinguish functional TEs from undesirable and harmful TEs?

The growing field of RNA surveillance provides great opportunities to better understand all these mechanisms, where RNA-associated events control genome stability, modulate epigenomes, transcriptomes, epitranscriptomes, RNA–RNA interactions, and RNA–protein networks, providing a unique universal system to regulate many biological levels of cellular physiology. The resulting strength and efficiency of these elegant mechanisms has been adopted by evolution to fine-tune global RNA expression during development and beyond.

Highlights.

Emerging functions for RNA surveillance pathways demonstrate their important role in physiological processes, from cellular proliferation to differentiation during development, while defects lead to pathologies.

Complex RNA surveillance pathways are working in concert to regulate RNA levels, with some redundancy and specificity depending on cell types.

RNAs are dynamically edited and remodeled by chemical modifications, strongly influencing their half-life, interactions, localizations, and functions, in particular during dynamic processes such as development.

Dysregulation of RNA surveillance pathways is observed in many cancers, leading to overexpression of different classes of coding RNA and ncRNA.

Glossary

Noncoding (nc)RNA

any class of RNA that does not produce a functional mRNA.

R-loops

DNA:RNA hybrids. These structures are usually created during transcription and RNA polymerase stalling at specific DNA sequences.

RNA exosome

an evolutionary conserved multiprotein complex with ribonuclease activity. In humans, it comprises 11 subunits, including two catalytic subunits (Exosc10 and Dis3) and has both 3′ to 5′ exoribonuclease and endoribonuclease activities. The RNA exosome interacts with various complexes and helicases, which fine-tune its activity in the nucleus and cytoplasm.

RNA processing

a set of processes controlling RNA maturation. These include RNA capping, polyadenylation, splicing, editing, RNA modifications, trafficking, localization, or any additional changes. These different steps are crucial for mRNA and gene expression control, as well as for ncRNAs.

RNA surveillance

set of processes controlling the RNA level. RNA surveillance is complementary to RNA processing and ensures the exact titration of any RNA molecule in both time and space.