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In cancer, recurrent somatic single-nucleotide variants—which are rare in most paediatric cancers—are confined largely to protein-coding genes 1 – 3 . Here we report highly recurrent hotspot mutations (r.3A>G) of U1 spliceosomal small nuclear RNAs (snRNAs) in about 50% of Sonic hedgehog (SHH) medulloblastomas. These mutations were not present across other subgroups of medulloblastoma, and we identified these hotspot mutations in U1 snRNA in only <0.1% of 2,442 cancers, across 36 other tumour types. The mutations occur in 97% of adults (subtype SHHδ) and 25% of adolescents (subtype SHHα) with SHH medulloblastoma, but are largely absent from SHH medulloblastoma in infants. The U1 snRNA mutations occur in the 5′ splice-site binding region, and snRNA-mutant tumours have significantly disrupted RNA splicing and an excess of 5′ cryptic splicing events. Alternative splicing mediated by mutant U1 snRNA inactivates tumour-suppressor genes ( PTCH1 ) and activates oncogenes ( GLI2 and CCND2 ), and represents a target for therapy. These U1 snRNA mutations provide an example of highly recurrent and tissue-specific mutations of a non-protein-coding gene in cancer. Highly recurrent hotspot r.3A>G mutations are identified in U1 splicesomal small nuclear RNAs in about 50% of Sonic hedgehog medulloblastomas, which result in disrupted RNA splicing and the activation of oncogenes.

The synthesis of pre-mRNA by RNA polymerase II (Pol II) involves the formation of a transcription initiation complex, and a transition to an elongation complex 1 – 4 . The large subunit of Pol II contains an intrinsically disordered C-terminal domain that is phosphorylated by cyclin-dependent kinases during the transition from initiation to elongation, thus influencing the interaction of the C-terminal domain with different components of the initiation or the RNA-splicing apparatus 5 , 6 . Recent observations suggest that this model provides only a partial picture of the effects of phosphorylation of the C-terminal domain 7 – 12 . Both the transcription-initiation machinery and the splicing machinery can form phase-separated condensates that contain large numbers of component molecules: hundreds of molecules of Pol II and mediator are concentrated in condensates at super-enhancers 7 , 8 , and large numbers of splicing factors are concentrated in nuclear speckles, some of which occur at highly active transcription sites 9 – 12 . Here we investigate whether the phosphorylation of the Pol II C-terminal domain regulates the incorporation of Pol II into phase-separated condensates that are associated with transcription initiation and splicing. We find that the hypophosphorylated C-terminal domain of Pol II is incorporated into mediator condensates and that phosphorylation by regulatory cyclin-dependent kinases reduces this incorporation. We also find that the hyperphosphorylated C-terminal domain is preferentially incorporated into condensates that are formed by splicing factors. These results suggest that phosphorylation of the Pol II C-terminal domain drives an exchange from condensates that are involved in transcription initiation to those that are involved in RNA processing, and implicates phosphorylation as a mechanism that regulates condensate preference. RNA polymerase II with a hypophosphorylated C-terminal domain preferentially incorporates into mediator condensates, and with a hyperphosphorylated C-terminal domain into splicing-factor condensates, revealing phosphorylation as a regulatory mechanism in condensate preference.

Spliceosome rearrangements facilitated by RNA helicase PRP16 before catalytic step two of splicing are poorly understood. Here we report a 3D cryo-electron microscopy structure of the human spliceosomal C complex stalled directly after PRP16 action (C*). The architecture of the catalytic U2–U6 ribonucleoprotein (RNP) core of the human C* spliceosome is very similar to that of the yeast pre-Prp16 C complex. However, in C* the branched intron region is separated from the catalytic centre by approximately 20 Å, and its position close to the U6 small nuclear RNA ACAGA box is stabilized by interactions with the PRP8 RNase H-like and PRP17 WD40 domains. RNA helicase PRP22 is located about 100 Å from the catalytic centre, suggesting that it destabilizes the spliced mRNA after step two from a distance. Comparison of the structure of the yeast C and human C* complexes reveals numerous RNP rearrangements that are likely to be facilitated by PRP16, including a large-scale movement of the U2 small nuclear RNP. The cryo-EM structure of the splicing intermediate known as the C* complex from human. Structure of the spliceosomal C* complex Recent years have seen substantial progress in understanding the structure of various intermediates of the splicing process. Two groups, led by Reinhard Lührmann and Kiyoshi Nagai, now describe the cryo-electron microscopy structures (from human and yeast cells, respectively) of the splicing intermediate known as the C* complex. The notable feature observed in this complex, relative to the preceding catalytic intermediate (the C complex), is a remodelling that positions the branch-site adenosine and the branched intron out of the catalytic core, opening up space for the 3′ exon to dock in preparation for exon ligation.

Patterns of splicing are frequently altered in cancer, and genes that encode splicing regulatory factors are often mutated. Thus, recent strategies have emerged to target splicing alterations in cancer, which are reviewed here. Recent studies have highlighted that splicing patterns are frequently altered in cancer and that mutations in genes encoding spliceosomal proteins, as well as mutations affecting the splicing of key cancer-associated genes, are enriched in cancer. In parallel, there is also accumulating evidence that several molecular subtypes of cancer are highly dependent on splicing function for cell survival. These findings have resulted in a growing interest in targeting splicing catalysis, splicing regulatory proteins, and/or specific key altered splicing events in the treatment of cancer. Here we present strategies that exist and that are in development to target altered dependency on the spliceosome, as well as aberrant splicing, in cancer. These include drugs to target global splicing in cancer subtypes that are preferentially dependent on wild-type splicing for survival, methods to alter post-translational modifications of splicing-regulating proteins, and strategies to modulate pathologic splicing events and protein–RNA interactions in cancer.

The cryo-electron microscopy structure of a yeast spliceosome stalled before mature RNA formation provides insight into the mechanism of exon ligation. Structure of the spliceosomal C* complex Recent years have seen substantial progress in understanding the structure of various intermediates of the splicing process. Two groups, led by Reinhard Lührmann and Kiyoshi Nagai, now describe the cryo-electron microscopy structures (from human and yeast cells, respectively) of the splicing intermediate known as the C* complex. The notable feature observed in this complex, relative to the preceding catalytic intermediate (the C complex), is a remodelling that positions the branch-site adenosine and the branched intron out of the catalytic core, opening up space for the 3′ exon to dock in preparation for exon ligation. The spliceosome excises introns from pre-mRNAs in two sequential transesterifications—branching and exon ligation 1 —catalysed at a single catalytic metal site in U6 small nuclear RNA (snRNA) 2 , 3 . Recently reported structures of the spliceosomal C complex 4 , 5 with the cleaved 5′ exon and lariat–3′-exon bound to the catalytic centre revealed that branching-specific factors such as Cwc25 lock the branch helix into position for nucleophilic attack of the branch adenosine at the 5′ splice site. Furthermore, the ATPase Prp16 is positioned to bind and translocate the intron downstream of the branch point to destabilize branching-specific factors and release the branch helix from the active site 4 . Here we present, at 3.8 Å resolution, the cryo-electron microscopy structure of a Saccharomyces cerevisiae spliceosome stalled after Prp16-mediated remodelling but before exon ligation. While the U6 snRNA catalytic core remains firmly held in the active site cavity of Prp8 by proteins common to both steps, the branch helix has rotated by 75° compared to the C complex and is stabilized in a new position by Prp17, Cef1 and the reoriented Prp8 RNase H-like domain. This rotation of the branch helix removes the branch adenosine from the catalytic core, creates a space for 3′ exon docking, and restructures the pairing of the 5′ splice site with the U6 snRNA ACAGAGA region. Slu7 and Prp18, which promote exon ligation, bind together to the Prp8 RNase H-like domain. The ATPase Prp22, bound to Prp8 in place of Prp16, could interact with the 3′ exon, suggesting a possible basis for mRNA release after exon ligation 6 , 7 . Together with the structure of the C complex 4 , our structure of the C* complex reveals the two major conformations of the spliceosome during the catalytic stages of splicing.

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.

Many protein-coding genes in higher eukaryotes can produce circular RNAs (circRNAs) through back-splicing of exons. CircRNAs differ from mRNAs in their production, structure and turnover and thereby have unique cellular functions and potential biomedical applications. In this Review, I discuss recent progress in our understanding of the biogenesis of circRNAs and the regulation of their abundance and of their biological functions, including in transcription and splicing, sequestering or scaffolding of macromolecules to interfere with microRNA activities or signalling pathways, and serving as templates for translation. I further discuss the emerging roles of circRNAs in regulating immune responses and cell proliferation, and the possibilities of applying circRNA technologies in biomedical research.Circular RNAs, which are produced through back-splicing of exons, are emerging as key regulators of immune responses and cell proliferation. Recent studies have shed new light on the biogenesis and functions of circular RNAs, which include the modulation of transcription and splicing, and interference with microRNAs and other cellular signalling pathways.

Intron removal requires assembly of the spliceosome on precursor mRNA (pre-mRNA) and extensive remodelling to form the spliceosome’s catalytic centre. Here we report the cryo-electron microscopy structure of the yeast Saccharomyces cerevisiae pre-catalytic B complex spliceosome at near-atomic resolution. The mobile U2 small nuclear ribonucleoprotein particle (snRNP) associates with U4/U6.U5 tri-snRNP through the U2/U6 helix II and an interface between U4/U6 di-snRNP and the U2 snRNP SF3b-containing domain, which also transiently contacts the helicase Brr2. The 3′ region of the U2 snRNP is flexibly attached to the SF3b-containing domain and protrudes over the concave surface of tri-snRNP, where the U1 snRNP may reside before its release from the pre-mRNA 5′ splice site. The U6 ACAGAGA sequence forms a hairpin that weakly tethers the 5′ splice site. The B complex proteins Prp38, Snu23 and Spp381 bind the Prp8 N-terminal domain and stabilize U6 ACAGAGA stem–pre-mRNA and Brr2–U4 small nuclear RNA interactions. These results provide important insights into the events leading to active site formation. The cryo-electron microscopy structure of the yeast spliceosome in a pre-catalytic state provides insights into the molecular events leading to formation of the spliceosome active site. Visualization of a poised spliceosome Protein-coding regions of DNA can be interrupted by non-coding regions, or introns. A large multisubunit complex, the spliceosome, is used to excise introns from the messenger RNA before it is translated into protein. Formation of an active spliceosome complex on an intron requires stepwise assembly of subcomplexes, followed by their rearrangement and the loss of some factors. Kiyoshi Nagai and colleagues have solved the structure of the B complex spliceosome, poised in a pre-catalytic state. The detection of several factors that were not visualized in previous spliceosome structures provides new insights regarding the process by which the complex is activated.

Previous studies demonstrated that splicing factor mutations are recurrent events in hematopoietic malignancies with both clinical and functional implications. However, their aberrant splicing patterns in acute myeloid leukemia remain largely unexplored. In this study, we characterized mutations in SRSF2, U2AF1, and SF3B1, the most commonly mutated splicing factors. In our clinical analysis of 2678 patients, splicing factor mutations showed inferior relapse-free and overall survival, however, these mutations did not represent independent prognostic markers. RNA-sequencing of 246 and independent validation in 177 patients revealed an isoform expression profile which is highly characteristic for each individual mutation, with several isoforms showing a strong dysregulation. By establishing a custom differential splice junction usage pipeline, we accurately detected aberrant splicing in splicing factor mutated samples. A large proportion of differentially used junctions were novel, including several junctions in leukemia-associated genes. In SRSF2(P95H) mutants, we further explored the possibility of a cascading effect through the dysregulation of the splicing pathway. Furthermore, we observed a validated impact on overall survival for two junctions overused in SRSF2(P95H) mutants. We conclude that splicing factor mutations do not represent independent prognostic markers. However, they do have genome-wide consequences on gene splicing leading to dysregulated isoform expression of several genes.

R-loops are stable nucleic acid structures that have important physiological functions, but which also pose a significant threat to genomic stability. Increased R-loops cause replication stress and chromosome fragility and have been associated with diseases such as neurodegeneration and cancer. Although excessive R-loops are a feature of cells that are defective in RNA processing, what causes them to form is unclear. Here, we demonstrate that DHX9 (RNA helicase A) promotes the formation of pathological and non-pathological R-loops. In the absence of splicing factors, formation of R-loops correlates with the prolonged association of DHX9 with RNA Polymerase II (RNA Pol II). This leads to the production of DNA–RNA hybrid, which traps RNA Pol II on chromatin with the potential to block DNA replication. Our data provide a molecular mechanism for the formation of R-loops that is relevant to neurodegenerative diseases and cancers in which deregulated RNA processing is a feature. Unresolved R-loops can represent a threat to genome stability. Here the authors reveal that DHX9 helicase can promote R-loop formation in the absence of splicing factors SFPQ and SF3B3.