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Key Points Although long non-coding RNAs (lncRNAs) and mRNAs share many common features, several types of lncRNAs are distinguished from mRNAs by unique features of biogenesis, form and function. lncRNAs exhibit more highly specific expression patterns than mRNAs. Many lncRNAs undergo special processing events, such as backspliced circularization, 5′- and 3′-bookending by processed small nucleolar RNAs (snoRNAs), and cleavage by RNase P. lncRNAs are more enriched in the nucleus than the cytoplasm relative to mRNAs, and although cytoplasmic lncRNAs associate with the ribosome, few are productively translated. Certain classes of lncRNAs are preferentially subject to degradation by nonsense-mediated decay and the nuclear exosome, and the elongation of divergent ncRNA transcripts is co-transcriptionally terminated by premature polyadenylation. lncRNAs are uniquely capable of cis action on the genome and chromatin. This feature of lncRNAs enables such biological phenomena as gene imprinting, dosage compensation of sex chromosomes, transcriptional enhancement, chromosome looping and antisense regulation. Long non-coding RNAs (lncRNAs) are a class of RNAs with great molecular and regulatory diversity. This Review discusses how, beyond their lack of protein-coding potential, some types of lncRNAs are known to exhibit features that are distinct from mRNAs, including their transcriptional regulation, localization, processing, biological capabilities and degradation. Such properties underlie many of the key cellular functions of lncRNAs. Long non-coding RNAs (lncRNAs) are a diverse class of RNAs that engage in numerous biological processes across every branch of life. Although initially discovered as mRNA-like transcripts that do not encode proteins, recent studies have revealed features of lncRNAs that further distinguish them from mRNAs. In this Review, we describe special events in the lifetimes of lncRNAs — before, during and after transcription — and discuss how these events ultimately shape the unique characteristics and functional roles of lncRNAs.

Key Points The EJC is deposited 24 nucleotides upstream of spliced junctions during splicing. It accompanies mRNAs from the nucleus to the cytoplasm, where it is removed by the first round of translation, and recycled back into the nucleus. The core of the EJC consists of four proteins. Structural studies revealed that the DEAD-box RNA helicase eIF4A3 functions as a clamp that binds RNA in a sequence-unspecific manner. MAGOH and Y14 form a heterodimer to lock eIF4A3 onto the mRNA, whereas MLN51 contacts eIF4A3 and the mRNA and provides further stability. The core complex acts as a binding platform for peripheral factors involved in splicing, transport, translation and nonsense-mediated decay (NMD). The composition of peripheral factors depends on the different stages of mRNA processing. The EJC has several functions in regulating different post-transcriptional processes, including splicing, cellular localization, translation and NMD. The EJC is not present at every exon junction, and it does not always bind at the canonical position. This differential loading could impact the composition and functions of different EJCs. The EJC acts as a central node of post-transcriptional gene regulation, and changes in EJC protein expression levels lead to several developmental defects and diseases. In addition to its known roles in nonsense-mediated mRNA decay, recent findings show that the exon junction complex (EJC) participates in diverse mRNA maturation processes, including splicing, transport and translation. This multi-functionality is reflected by an increasing number of EJC-related disorders being discovered. The exon junction complex (EJC) is deposited onto mRNAs following splicing and adopts a unique structure, which can both stably bind to mRNAs and function as an anchor for diverse processing factors. Recent findings revealed that in addition to its established roles in nonsense-mediated mRNA decay, the EJC is involved in mRNA splicing, transport and translation. While structural studies have shed light on EJC assembly, transcriptome-wide analyses revealed differential EJC loading at spliced junctions. Thus, the EJC functions as a node of post-transcriptional gene expression networks, the importance of which is being revealed by the discovery of increasing numbers of EJC-related disorders.

Discoveries in the past decade have highlighted the potential of mRNA as a therapeutic target for cancer. Specifically, RNA sequencing revealed that, in addition to gene mutations, alterations in mRNA can contribute to the initiation and progression of cancer. Indeed, precursor mRNA processing, which includes the removal of introns by splicing and the formation of 3′ ends by cleavage and polyadenylation, is frequently altered in tumours. These alterations result in numerous cancer-specific mRNAs that generate altered levels of normal proteins or proteins with new functions, leading to the activation of oncogenes or the inactivation of tumour-suppressor genes. Abnormally spliced and polyadenylated mRNAs are also associated with resistance to cancer treatment and, unexpectedly, certain cancers are highly sensitive to the pharmacological inhibition of splicing. This Review summarizes recent progress in our understanding of how splicing and polyadenylation are altered in cancer and highlights how this knowledge has been translated for drug discovery, resulting in the production of small molecules and oligonucleotides that modulate the spliceosome and are in clinical trials for the treatment of cancer.Precursor mRNA processing, which includes the removal of introns by splicing and the formation of 3′ ends by cleavage and polyadenylation, is frequently altered in tumours. This Review describes small molecules and oligonucleotides that modulate the spliceosome and are now in clinical trials for the treatment of cancer.

Many steps of RNA processing occur during transcription by RNA polymerases. Co-transcriptional activities are deemed commonplace in prokaryotes, in which the lack of membrane barriers allows mixing of all gene expression steps, from transcription to translation. In the past decade, an extraordinary level of coordination between transcription and RNA processing has emerged in eukaryotes. In this Review, we discuss recent developments in our understanding of co-transcriptional gene regulation in both eukaryotes and prokaryotes, comparing methodologies and mechanisms, and highlight striking parallels in how RNA polymerases interact with the machineries that act on nascent RNA. The development of RNA sequencing and imaging techniques that detect transient transcription and RNA processing intermediates has facilitated discoveries of transcription coordination with splicing, 3′-end cleavage and dynamic RNA folding and revealed physical contacts between processing machineries and RNA polymerases. Such studies indicate that intron retention in a given nascent transcript can prevent 3′-end cleavage and cause transcriptional readthrough, which is a hallmark of eukaryotic cellular stress responses. We also discuss how coordination between nascent RNA biogenesis and transcription drives fundamental aspects of gene expression in both prokaryotes and eukaryotes.Methodological advances have enabled discoveries of RNA polymerase interactions with RNA processing machineries, such as the splicing and 3′-end cleavage machineries. This Review discusses the roles of these interactions in gene regulation and eukaryotic cellular stress responses, and highlights parallels between co-transcriptional RNA processing in eukaryotes and prokaryotes.

Internal bases in mRNA can be subjected to modifications that influence the fate of mRNA in cells. One of the most prevalent modified bases is found at the 5′ end of mRNA, at the first encoded nucleotide adjacent to the 7-methylguanosine cap. Here we show that this nucleotide, N 6 ,2′- O -dimethyladenosine (m 6 A m ), is a reversible modification that influences cellular mRNA fate. Using a transcriptome-wide map of m 6 A m we find that m 6 A m -initiated transcripts are markedly more stable than mRNAs that begin with other nucleotides. We show that the enhanced stability of m 6 A m -initiated transcripts is due to resistance to the mRNA-decapping enzyme DCP2. Moreover, we find that m 6 A m is selectively demethylated by fat mass and obesity-associated protein (FTO). FTO preferentially demethylates m 6 A m rather than N 6 -methyladenosine (m 6 A), and reduces the stability of m 6 A m mRNAs. Together, these findings show that the methylation status of m 6 A m in the 5′ cap is a dynamic and reversible epitranscriptomic modification that determines mRNA stability. Fat mass and obesity-associated protein (FTO) preferentially demethylates m 6 A m , a modified adenosine that, when present at the 5′ end of certain mRNAs, positively influences mRNA stability by preventing DCP2-mediated decapping. m 6 A m is a novel epitranscriptomic mark Recent studies have highlighted the role of reversible modifications, such as the addition of a methyl group to adenosines (m 6 A), on RNA function. Samie Jaffrey and colleagues show that a dimethyl-modified base (m 6 A m ) at the 5′ end of certain mRNAs, next to the 7-methylguanosine cap structure, can positively influence mRNA stability by preventing their DCP2-mediated decapping. This modification is itself regulated by the fat mass and obesity-associated protein FTO, a demethylase that exhibits a preference for m 6 A m over m 6 A. This work provides insight into the biological importance of FTO, which has been implicated in body weight regulation.

RNA methylation to form N6-methyladenosine (m6A) in mRNA accounts for the most abundant mRNA internal modification and has emerged as a widespread regulatory mechanism that controls gene expression in diverse physiological processes. Transcriptome-wide m6A mapping has revealed the distribution and pattern of m6A in cellular RNAs, referred to as the epitranscriptome. These maps have revealed the specific mRNAs that are regulated by m6A, providing mechanistic links connecting m6A to cellular differentiation, cancer progression and other processes. The effects of m6A on mRNA are mediated by an expanding list of m6A readers and m6A writer-complex components, as well as potential erasers that currently have unclear relevance to m6A prevalence in the transcriptome. Here we review new and emerging methods to characterize and quantify the epitranscriptome, and we discuss new concepts — in some cases, controversies — regarding our understanding of the mechanisms and functions of m6A readers, writers and erasers.

Therapeutic mRNAs and vaccines are being developed for a broad range of human diseases, including COVID-19. However, their optimization is hindered by mRNA instability and inefficient protein expression. Here, we describe design principles that overcome these barriers. We develop an RNA sequencing-based platform called PERSIST-seq to systematically delineate in-cell mRNA stability, ribosome load, as well as in-solution stability of a library of diverse mRNAs. We find that, surprisingly, in-cell stability is a greater driver of protein output than high ribosome load. We further introduce a method called In-line-seq, applied to thousands of diverse RNAs, that reveals sequence and structure-based rules for mitigating hydrolytic degradation. Our findings show that highly structured “superfolder” mRNAs can be designed to improve both stability and expression with further enhancement through pseudouridine nucleoside modification. Together, our study demonstrates simultaneous improvement of mRNA stability and protein expression and provides a computational-experimental platform for the enhancement of mRNA medicines. The authors develop an RNA sequencing-based platform, PERSIST-seq, to simultaneously delineate in-cell mRNA stability, ribosome load, and in-solution stability of a diverse mRNA library to derive design principles for improved mRNA therapeutics.

Eukaryotic gene expression is constantly controlled by the translation-coupled nonsense-mediated mRNA decay (NMD) pathway. Aberrant translation termination leads to NMD activation, resulting in phosphorylation of the central NMD factor UPF1 and robust clearance of NMD targets via two seemingly independent and redundant mRNA degradation branches. Here, we uncover that the loss of the first SMG5-SMG7-dependent pathway also inactivates the second SMG6-dependent branch, indicating an unexpected functional connection between the final NMD steps. Transcriptome-wide analyses of SMG5-SMG7-depleted cells confirm exhaustive NMD inhibition resulting in massive transcriptomic alterations. Intriguingly, we find that the functionally underestimated SMG5 can substitute the role of SMG7 and individually activate NMD. Furthermore, the presence of either SMG5 or SMG7 is sufficient to support SMG6-mediated endonucleolysis of NMD targets. Our data support an improved model for NMD execution that features two-factor authentication involving UPF1 phosphorylation and SMG5-SMG7 recruitment to access SMG6 activity. Degradation of nonsense mediated mRNA decay (NMD) substrates is carried out by two seemingly independent pathways, SMG6-mediated endonucleolytic cleavage and/or SMG5-SMG7-induced accelerated deadenylation. Here the authors show that SMG5-SMG7 maintain NMD activity by permitting SMG6 activation.

Key Points Genetic and functional data indicate that RNA splicing factors can act as oncoproteins as well as tumour suppressors. A subset of RNA splicing factors are recurrent targets of specific point mutations in cancer. Many other splicing factors exhibit dysregulated expression in cancer. In many cases, recurrent spliceosomal mutations alter splice site or exon recognition preferences to cause abnormal RNA splicing. Spliceosomal mutations are sufficient to impair myeloid differentiation in mouse models. In the case of serine/arginine-rich splicing factor 2 ( SRSF2 ), impaired differentiation has been linked to a specific splicing change in a downstream gene (enhancer of zeste homologue 2 ( EZH2 )). Spliceosomal mutations may affect cellular processes, including epigenetic regulation, the DNA damage response and nonsense-mediated decay, in addition to regulation of RNA splicing. Small molecules that disrupt splicing catalysis and/or targeted correction of specific splicing changes may provide novel therapeutic opportunities for cancers bearing spliceosomal mutations. This Review discusses the current genetic and functional links between dysregulated and/or mutated RNA splicing factors and cancer, as well as the therapeutic opportunities presented by alterations in alternative splicing in cancer. The recent genomic characterization of cancers has revealed recurrent somatic point mutations and copy number changes affecting genes encoding RNA splicing factors. Initial studies of these 'spliceosomal mutations' suggest that the proteins bearing these mutations exhibit altered splice site and/or exon recognition preferences relative to their wild-type counterparts, resulting in cancer-specific mis-splicing. Such changes in the splicing machinery may create novel vulnerabilities in cancer cells that can be therapeutically exploited using compounds that can influence the splicing process. Further studies to dissect the biochemical, genomic and biological effects of spliceosomal mutations are crucial for the development of cancer therapies targeted at these mutations.

Although 5-methylcytosine (m 5 C) is a widespread modification in RNAs, its regulation and biological role in pathological conditions (such as cancer) remain unknown. Here, we provide the single-nucleotide resolution landscape of messenger RNA m 5 C modifications in human urothelial carcinoma of the bladder (UCB). We identify numerous oncogene RNAs with hypermethylated m 5 C sites causally linked to their upregulation in UCBs and further demonstrate YBX1 as an m 5 C ‘reader’ recognizing m 5 C-modified mRNAs through the indole ring of W65 in its cold-shock domain. YBX1 maintains the stability of its target mRNA by recruiting ELAVL1. Moreover, NSUN2 and YBX1 are demonstrated to drive UCB pathogenesis by targeting the m 5 C methylation site in the HDGF 3′ untranslated region. Clinically, a high coexpression of NUSN2, YBX1 and HDGF predicts the poorest survival. Our findings reveal an unprecedented mechanism of RNA m 5 C-regulated oncogene activation, providing a potential therapeutic strategy for UCB. Chen et al. provide an m 5 C landscape in bladder cancer and show m 5 C enrichment at oncogene mRNAs that promotes tumour progression. They identify YBX1 as the m 5 C ‘reader’ that recruits ELAVL1 to stabilize mRNAs.