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Jens Lykke-Andersen, Frank Baas, Joseph Gleeson and colleagues report that mutations in the 3′ exonuclease TOE1 cause pontocerebellar hypoplasia type 7. They further show that these mutations result in the accumulation of incompletely processed small nuclear RNAs, leading to severe, early-onset neurodegeneration. Deadenylases are best known for degrading the poly(A) tail during mRNA decay. The deadenylase family has expanded throughout evolution and, in mammals, consists of 12 Mg 2+ -dependent 3′-end RNases with substrate specificity that is mostly unknown 1 . Pontocerebellar hypoplasia type 7 (PCH7) is a unique recessive syndrome characterized by neurodegeneration and ambiguous genitalia 2 . We studied 12 human families with PCH7, uncovering biallelic, loss-of-function mutations in TOE1 , which encodes an unconventional deadenylase 3 , 4 . toe1 -morphant zebrafish displayed midbrain and hindbrain degeneration, modeling PCH-like structural defects in vivo . Surprisingly, we found that TOE1 associated with small nuclear RNAs (snRNAs) incompletely processed spliceosomal. These pre-snRNAs contained 3′ genome-encoded tails often followed by post-transcriptionally added adenosines. Human cells with reduced levels of TOE1 accumulated 3′-end-extended pre-snRNAs, and the immunoisolated TOE1 complex was sufficient for 3′-end maturation of snRNAs. Our findings identify the cause of a neurodegenerative syndrome linked to snRNA maturation and uncover a key factor involved in the processing of snRNA 3′ ends.

Protein-coding and noncoding genes in eukaryotes are typically expressed as linear messenger RNAs, with exons arranged colinearly to their genomic order. Recent advances in sequencing and in mapping RNA reads to reference genomes have revealed that thousands of genes express also covalently closed circular RNAs. Many of these circRNAs are stable and contain exons, but are not translated into proteins. Here, we review the emerging understanding that both, circRNAs produced by co- and posttranscriptional head-to-tail “backsplicing” of a downstream splice donor to a more upstream splice acceptor, as well as circRNAs generated from intronic lariats during colinear splicing, may exhibit physiologically relevant regulatory functions in eukaryotes. We describe how circRNAs impact gene expression of their host gene locus by affecting transcriptional initiation and elongation or splicing, and how they partake in controlling the function of other molecules, for example by interacting with microRNAs and proteins. We conclude with an outlook how circRNA dysregulation affects disease, and how the stability of circRNAs might be exploited in biomedical applications.

Key Points Analysis of the recent atomic-resolution cryo-electron microscopy structures of the spliceosome reveals that during splicing all spliceosomal complexes share a rigid core of 20 RNAs and proteins. The active site of the spliceosome is positioned in the catalytic cavity of pre-mRNA-splicing factor 8 (Prp8), is stabilized by surrounding proteins and remains static throughout the two steps of transesterification. The catalytic metals in the active site are coordinated by U6 small nuclear RNA and the catalytic triplex. The interconversion of the various spliceosomal complexes is driven by eight conserved, RNA-dependent ATPase/helicases. The spliceosome is a protein-directed metalloribozyme. Atomic-resolution structures have recently been obtained for the intact spliceosome at different stages of the splicing cycle. These structural data have proved that the spliceosome is a protein-directed metalloribozyme and have increased our understanding of pre-mRNA splicing mechanisms, explaining a large body of existing genetic and biochemical data. Precursor messenger RNA (pre-mRNA) splicing is an essential step in the flow of information from DNA to protein in all eukaryotes. Research over the past four decades has molecularly delineated the splicing pathway, including characterization of the detailed splicing reaction, definition of the spliceosome and identification of its components, and biochemical analysis of the various splicing complexes and their regulation. Structural information is central to mechanistic understanding of pre-mRNA splicing by the spliceosome. X-ray crystallography of the spliceosomal components and subcomplexes is complemented by electron microscopy of the intact spliceosome. In this Review, I discuss recent atomic-resolution structures of the intact spliceosome at different stages of the splicing cycle. These structures have provided considerable mechanistic insight into pre-mRNA splicing and have corroborated and explained a large body of genetic and biochemical data. Together, the structural data have proved that the spliceosome is a protein-directed metalloribozyme.

Key Points Spliceosomal snRNAs are transcribed from specialized promoters, which recruit RNA polymerase II cofactors that aid in proper 3′ end maturation of these non-polyadenylated transcripts. Like most non-coding RNAs, small nuclear RNAs (snRNAs) use cognate antisense elements to interact with their nucleic acid targets via base pairing. Assembly of functional small nuclear ribonucleoproteins (snRNPs) involves a series of non-functional intermediates that are often sequestered in subcellular compartments that are distinct from their sites of action. snRNP function requires multiple protein partners (such as DExD/H helicases or WD box proteins) the roles of which may include modulating RNA structure or tethering an enzyme. snRNPs recognize specific sequences in pre-mRNAs and assemble into the spliceosome in a stepwise manner. The splicing reaction itself is catalysed by U6/U2 snRNA complex that resembles a self-splicing ribozyme. Alternative splicing is typically regulated by multiple cis -elements and trans -factors, which form complex interaction networks that may provide a great deal of regulatory plasticity. Pre-mRNA splicing can be regulated throughout the entire spliceosomal assembly pathway, although the early steps are the main stages of regulation. The tight regulation of each step of spliceosome assembly from small nuclear RNAs and associated proteins requires coordination between distinct cellular compartments. This in turn dictates where and when alternative splicing occurs and is vital for normal gene expression control. One of the most amazing findings in molecular biology was the discovery that eukaryotic genes are discontinuous, with coding DNA being interrupted by stretches of non-coding sequence. The subsequent realization that the intervening regions are removed from pre-mRNA transcripts via the activity of a common set of small nuclear RNAs (snRNAs), which assemble together with associated proteins into a complex known as the spliceosome, was equally surprising. How do cells coordinate the assembly of this molecular machine? And how does the spliceosome accurately recognize exons and introns to carry out the splicing reaction? Insights into these questions have been gained by studying the life cycle of spliceosomal snRNAs from their transcription, nuclear export and re-import to their dynamic assembly into the spliceosome. This assembly process can also affect the regulation of alternative splicing and has implications for human disease.

Numerous substrates have been identified for Type I and II arginine methyltransferases (PRMTs). However, the full substrate spectrum of the only type III PRMT, PRMT7, and its connection to type I and II PRMT substrates remains unknown. Here, we use mass spectrometry to reveal features of PRMT7-regulated methylation. We find that PRMT7 predominantly methylates a glycine and arginine motif; multiple PRMT7-regulated arginine methylation sites are close to phosphorylations sites; methylation sites and proximal sequences are vulnerable to cancer mutations; and methylation is enriched in proteins associated with spliceosome and RNA-related pathways. We show that PRMT4/5/7-mediated arginine methylation regulates hnRNPA1 binding to RNA and several alternative splicing events. In breast, colorectal and prostate cancer cells, PRMT4/5/7 are upregulated and associated with high levels of hnRNPA1 arginine methylation and aberrant alternative splicing. Pharmacological inhibition of PRMT4/5/7 suppresses cancer cell growth and their co-inhibition shows synergistic effects, suggesting them as targets for cancer therapy. Arginine methyltransferases (PRMTs) are involved in the regulation of various physiological and pathological conditions. Using proteomics, the authors here profile the methylation substrates of PRMTs 4, 5 and 7 and characterize the roles of these enzymes in cancer-associated splicing regulation.

Comprehensive genomic characterization of prostate cancer has identified recurrent alterations in genes involved in androgen signaling, DNA repair, and PI3K signaling, among others. However, larger and uniform genomic analysis may identify additional recurrently mutated genes at lower frequencies. Here we aggregate and uniformly analyze exome sequencing data from 1,013 prostate cancers. We identify and validate a new class of E26 transformation-specific ( ETS )-fusion-negative tumors defined by mutations in epigenetic regulators, as well as alterations in pathways not previously implicated in prostate cancer, such as the spliceosome pathway. We find that the incidence of significantly mutated genes (SMGs) follows a long-tail distribution, with many genes mutated in less than 3% of cases. We identify a total of 97 SMGs, including 70 not previously implicated in prostate cancer, such as the ubiquitin ligase CUL3 and the transcription factor SPEN . Finally, comparing primary and metastatic prostate cancer identifies a set of genomic markers that may inform risk stratification. Meta-analysis of exome sequencing data identifies new recurrently mutated driver genes for prostate cancer. Comparison of primary and metastatic tumors further identifies genomic markers for advanced prostate cancer that may inform risk stratification.

Myelodysplastic syndromes and related disorders (myelodysplasia) are a heterogeneous group of myeloid neoplasms showing deregulated blood cell production with evidence of myeloid dysplasia and a predisposition to acute myeloid leukaemia, whose pathogenesis is only incompletely understood. Here we report whole-exome sequencing of 29 myelodysplasia specimens, which unexpectedly revealed novel pathway mutations involving multiple components of the RNA splicing machinery, including U2AF35 , ZRSR2 , SRSF2 and SF3B1 . In a large series analysis, these splicing pathway mutations were frequent (∼45 to ∼85%) in, and highly specific to, myeloid neoplasms showing features of myelodysplasia. Conspicuously, most of the mutations, which occurred in a mutually exclusive manner, affected genes involved in the 3′-splice site recognition during pre-mRNA processing, inducing abnormal RNA splicing and compromised haematopoiesis. Our results provide the first evidence indicating that genetic alterations of the major splicing components could be involved in human pathogenesis, also implicating a novel therapeutic possibility for myelodysplasia. RNA-splicing defects in blood disorders Exome sequencing and analysis of myelodysplasia specimens identified frequent non-overlapping alterations in multiple components of the RNA splicing machinery, including mutations in U2AF35 , ZRSR2 , SRSF2 and SF3B1 . Most affected genes are involved in recognition of the 3′ splice site during pre-messenger RNA processing, and are thought to cause abnormal RNA splicing and compromised haematopoiesis. The results demonstrate the role of aberrant splicing in human pathogenesis.

SF3B1 is the most commonly mutated RNA splicing factor in cancer 1 – 4 , but the mechanisms by which SF3B1 mutations promote malignancy are poorly understood. Here we integrated pan-cancer splicing analyses with a positive-enrichment CRISPR screen to prioritize splicing alterations that promote tumorigenesis. We report that diverse SF3B1 mutations converge on repression of BRD9, which is a core component of the recently described non-canonical BAF chromatin-remodelling complex that also contains GLTSCR1 and GLTSCR1L 5 – 7 . Mutant SF3B1 recognizes an aberrant, deep intronic branchpoint within BRD9 and thereby induces the inclusion of a poison exon that is derived from an endogenous retroviral element and subsequent degradation of BRD9 mRNA. Depletion of BRD9 causes the loss of non-canonical BAF at CTCF-associated loci and promotes melanomagenesis. BRD9 is a potent tumour suppressor in uveal melanoma, such that correcting mis-splicing of BRD9 in SF3B1 -mutant cells using antisense oligonucleotides or CRISPR-directed mutagenesis suppresses tumour growth. Our results implicate the disruption of non-canonical BAF in the diverse cancer types that carry SF3B1 mutations and suggest a mechanism-based therapeutic approach for treating these malignancies. A range of SF3B1 mutations promote tumorigenesis through the repression of BRD9, a core component of the non-canonical BAF complex, and correcting BRD9 mis-splicing in these SF3B1 -mutant cells suppresses tumour growth.

Key Points The alternative splicing regulatory network is modulated by functional coupling between transcription and RNA processing. The transcription machinery can influence alternative splicing decisions by affecting the time in which cis -regulatory elements are transcribed (kinetic model) or by assisting in the recruitment of trans -acting regulatory proteins (recruitment model). Kinetic coupling, which requires changes in the elongation rate of RNA polymerase II (Pol II), can be induced by the presence of transcriptional roadblocks in specific intragenic regions or by modification of the Pol II complex such as phosphorylation of the carboxy-terminal domain (CTD) of its core catalytic subunit. Chromatin structure is a major regulator of splicing, affecting several steps of its coupling with transcription. These include the modulation of transcriptional properties through chromatin conformation and chromatin marks, the recruitment of splicing factors through adaptor proteins that recognize specific histone modifications and specific pausing at exons through preferential nucleosome positioning. Alternative splicing provides multicellular organisms with an extended proteome, the possibility of cell type- and species-specific protein isoforms without increasing the gene number, and the possibility of regulating the production of different proteins through specific signalling pathways. Its importance is supported by the increasing number of diseases associated with alternative splicing misregulation. Emerging evidence indicates that there are common structural and functional features of the polypeptide sequences encoded by alternative cassette exons in comparison to those encoded by constitutive exons. Such features include an increased flexibility and higher number of post-translational modifications. Several gene therapy strategies are being designed to cure hereditary disease by targeting misregulated alternative splicing events. In one of the most advanced studies the use of modified oligonucleotides has proved to be effective in restoring normal levels of a protein defective in spinal muscular atrophy. The prevalence and physiological importance of alternative splicing in multicellular eukaryotes has led to increased interest in its control. Much has been learnt about how transcription and chromatin structure influence splicing events, as well as the effects of signalling pathways, and this understanding may hold promise for the development of gene therapies. Alternative splicing was discovered simultaneously with splicing over three decades ago. Since then, an enormous body of evidence has demonstrated the prevalence of alternative splicing in multicellular eukaryotes, its key roles in determining tissue- and species-specific differentiation patterns, the multiple post- and co-transcriptional regulatory mechanisms that control it, and its causal role in hereditary disease and cancer. The emerging evidence places alternative splicing in a central position in the flow of eukaryotic genetic information, between transcription and translation, in that it can respond not only to various signalling pathways that target the splicing machinery but also to transcription factors and chromatin structure.

Genomic sequencing reveals similar but limited numbers of protein-coding genes in different genomes, which begs the question of how organismal diversities are generated. Alternative pre-mRNA splicing, a widespread phenomenon in higher eukaryotic genomes, is thought to provide a mechanism to increase the complexity of the proteome and introduce additional layers for regulating gene expression in different cell types and during development. Among a large number of factors implicated in the splicing regulation are the SR protein family of splicing factors and SR protein-specific kinases. Here, we summarize the rules for SR proteins to function as splicing regulators, which depend on where they bind in exons versus intronic regions, on alternative exons versus flanking competing exons, and on cooperative as well as competitive binding between different SR protein family members on many of those locations. We review the importance of cycles of SR protein phosphorylation/dephosphorylation in the splicing reaction with emphasis on the recent molecular insight into the role of SR protein phosphorylation in early steps of spliceosome assembly. Finally, we highlight recent discoveries of SR protein-specific kinases in transducing growth signals to regulate alternative splicing in the nucleus and the connection of both SR proteins and SR protein kinases to human diseases, particularly cancer.