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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.

Precursor mRNA (pre-mRNA) splicing proceeds by two consecutive transesterification reactions via a lariat–intron intermediate. Here we present the 3.8 Å cryo-electron microscopy structure of the spliceosome immediately after lariat formation. The 5′-splice site is cleaved but remains close to the catalytic Mg 2+ site in the U2/U6 small nuclear RNA (snRNA) triplex, and the 5′-phosphate of the intron nucleotide G(+1) is linked to the branch adenosine 2′OH. The 5′-exon is held between the Prp8 amino-terminal and linker domains, and base-pairs with U5 snRNA loop 1. Non-Watson–Crick interactions between the branch helix and 5′-splice site dock the branch adenosine into the active site, while intron nucleotides +3 to +6 base-pair with the U6 snRNA ACAG AGA sequence. Isy1 and the step-one factors Yju2 and Cwc25 stabilize docking of the branch helix. The intron downstream of the branch site emerges between the Prp8 reverse transcriptase and linker domains and extends towards the Prp16 helicase, suggesting a plausible mechanism of remodelling before exon ligation. Cryo-EM reveals the configuration of substrate pre-mRNA within the active spliceosome and suggests how remodelling occurs prior to exon ligation. Structure of the branched splicing complex The excision of introns from RNA is not a concerted process, but is rather an ordered one involving two transesterification reactions by the spliceosome. In the first step, the 5′-splice site is cleaved and the intron end is joined to make a lariat structure. Kiyoshi Nagai and colleagues have captured the Saccharomyces cerevisiae spliceosome stalled immediately after this first transesterification (branching) reaction by cryo-electron microscopy single-particle reconstruction at an overall resolution of 3.8 Å. The configuration of the RNA within the complex suggests that remodelling occurs before the second step, exon ligation.

Many protein-protein and protein-nucleic acid interactions have been experimentally characterized, whereas RNA-RNA interactions have generally only been predicted computationally. Here, we describe a high-throughput method to identify intramolecular and intermolecular RNA-RNA interactions experimentally by cross-linking, ligation, and sequencing of hybrids (CLASH). As validation, we identified 39 known target sites for box C/D modification-guide small nucleolar RNAs (snoRNAs) on the yeast pre-rRNA. Novel snoRNA-rRNA hybrids were recovered between snR4-5S and U14-25S. These are supported by native electrophoresis and consistent with previously unexplained data. The U3 snoRNA was found to be associated with sequences close to the 3' side of the central pseudoknot in 18S rRNA, supporting a role in formation of this structure. Applying CLASH to the yeast U2 spliceosomal snRNA led to a revised predicted secondary structure, featuring alternative folding of the 3' domain and long-range contacts between the 3' and 5' domains. CLASH should allow transcriptome-wide analyses of RNA-RNA interactions in many organisms.

Pseudouridine (Ψ) is one of the most abundant modifications in cellular RNA. However, its function remains elusive, mainly due to the lack of highly sensitive and accurate detection methods. Here, we introduced 2-bromoacrylamide-assisted cyclization sequencing (BACS), which enables Ψ-to-C transitions, for quantitative profiling of Ψ at single-base resolution. BACS allowed the precise identification of Ψ positions, especially in densely modified Ψ regions and consecutive uridine sequences. BACS detected all known Ψ sites in human rRNA and spliceosomal small nuclear RNAs and generated the quantitative Ψ map of human small nucleolar RNA and tRNA. Furthermore, BACS simultaneously detected adenosine-to-inosine editing sites and N 1 -methyladenosine. Depletion of pseudouridine synthases TRUB1, PUS7 and PUS1 elucidated their targets and sequence motifs. We further identified a highly abundant Ψ 114 site in Epstein–Barr virus-encoded small RNA EBER2. Surprisingly, applying BACS to a panel of RNA viruses demonstrated the absence of Ψ in their viral transcripts or genomes, shedding light on differences in pseudouridylation across virus families. This study introduces a chemical method, BACS, that generates Ψ-to-C mutation signatures, allowing for sequencing and quantification of Ψ at single-base resolution.

U4/U6.U5 tri-snRNP represents a substantial part of the spliceosome before activation. A cryo-electron microscopy structure of Saccharomyces cerevisiae U4/U6.U5 tri-snRNP at 3.7 Å resolution led to an essentially complete atomic model comprising 30 proteins plus U4/U6 and U5 small nuclear RNAs (snRNAs). The structure reveals striking interweaving interactions of the protein and RNA components, including extended polypeptides penetrating into subunit interfaces. The invariant ACAGAGA sequence of U6 snRNA, which base-pairs with the 5′-splice site during catalytic activation, forms a hairpin stabilized by Dib1 and Prp8 while the adjacent nucleotides interact with the exon binding loop 1 of U5 snRNA. Snu114 harbours GTP, but its putative catalytic histidine is held away from the γ-phosphate by hydrogen bonding to a tyrosine in the amino-terminal domain of Prp8. Mutation of this histidine to alanine has no detectable effect on yeast growth. The structure provides important new insights into the spliceosome activation process leading to the formation of the catalytic centre. A 3.7 Å resolution structure for the yeast U4/U6.U5 tri-snRNP, a complex involved in splicing, allows a better appreciation of the architecture of the tri-snRNP, and offers new functional insights into the activation of the spliceosome and the assembly of the catalytic core. Yeast U4/U6.U5 tri-snRNP structure Following up on their 5.9 Å cryo-electron microscopy structure published less than a year ago, Kiyoshi Nagai and colleagues have now achieved a resolution of 3.7 Å for the yeast U4/U6.U5 tri-snRNP, a complex involved in splicing of messenger RNA. The improved resolution allows a better appreciation of the architecture of the tri-snRNP, and offers new functional insights into the activation of the spliceosome and the assembly of the catalytic core.

The spliceosome, a ribonucleoprotein complex that includes proteins and small nuclear RNAs (snRNAs), catalyzes RNA splicing through intron excision and exon ligation to produce mature messenger RNAs, which, in turn serve as templates for protein translation. We identified four point mutations in the U4atac snRNA component of the minor spliceosome in patients with brain and bone malformations and unexplained postnatal death [microcephalic osteodysplastic primordial dwarfism type 1 (MOPD 1) or Taybi-Linder syndrome (TALS); Mendelian Inheritance in Man ID no. 210710]. Expression of a subgroup of genes, possibly linked to the disease phenotype, and minor intron splicing were affected in cell lines derived from TALS patients. Our findings demonstrate a crucial role of the minor spliceosome component U4atac snRNA in early human development and postnatal survival.

RNA molecules are synthesized in the cell nucleus, yet many have to be moved to the cytoplasm to be processed and/or to effect their function. Different classes of RNA are transported from the nucleus by different transport systems. Messenger RNAs (mRNAs) and uridine-rich small nuclear RNAs (U snRNAs) are transcribed by RNA polymerase II and are capped and bound by the cap-binding machinery in the nucleus but are exported by different protein complexes. The feature that distinguishes the two classes of RNA is their length: U snRNAs are short and mRNAs are long. Using an in vitro system and human tissue culture cells, McCloskey et al. (p. 1643 ) show that the length of the RNAs is measured by the heterogeneous nuclear ribonicleoprotein (hnRNP) C tetrameric protein complex. The hnRNP C cannot bind to the short U snRNAs, allowing the U snRNA-specific export adaptor protein, PHAX, to bind and mediate export. Longer mRNAs are bound by hnRNP C, which prevents the binding of PHAX, thus identifying these RNAs for export from the nucleus via the mRNA pathway. A nuclear protein measures the length of newly made RNAs and sorts them into distinct pathways for export. Specific RNA recognition is usually achieved by specific RNA sequences and/or structures. However, we show here a mechanism by which RNA polymerase II (Pol II) transcripts are classified according to their length. The heterotetramer of the heterogeneous nuclear ribonucleoprotein (hnRNP) C1/C2 measures the length of the transcripts like a molecular ruler, by selectively binding to the unstructured RNA regions longer than 200 to 300 nucleotides. Thus, the tetramer sorts the transcripts into two RNA categories, to be exported as either messenger RNA or uridine-rich small nuclear RNA (U snRNA), depending on whether or not they are longer than the threshold, respectively. Our findings reveal a new function of the C tetramer and highlight the biological importance of RNA recognition by the length.

Custom RNA base editing exploiting the human Adenosine Deaminase Acting on RNA (ADAR) enzyme may enable therapeutic gene editing without DNA damage or use of foreign proteins. ADAR’s adenosine-to-inosine (effectively A-to-G) deamination activity can be targeted to transcripts using an antisense guide RNA (gRNA), but efficacy is challenged by limits of in vivo delivery. Embedding gRNAs into a U7 small nuclear RNA (snRNA) framework greatly enhances RNA editing with endogenous ADAR, and a 750-plex single-cell mutagenesis screen further improved the framework. An optimized scaffold with a stronger synthetic U7 promoter enables 76% RNA editing in vitro from a single DNA construct per cell, and 75% editing in a Hurler syndrome mouse brain after one systemic AAV injection, surpassing circular gRNA approaches. The technology also improves published DMD exon-skipping designs 25-fold in differentiated myoblasts. Our engineered U7 framework represents a universal scaffold for ADAR-based RNA editing and other antisense RNA therapies. The human ADAR enzyme can deaminate additional transcripts for therapeutic A-to-G RNA base editing using an antisense guide RNA. Here, authors engineer a universal guide RNA scaffold from the U7 snRNA, boosting editing efficiency from minimal doses, especially in organs with limited AAV delivery.

Key Points We now understand that genomes in all three domains of life produce functional RNAs that do not encode proteins (non-coding (nc)RNAs), but do exert important influences on diverse cellular processes. Most ncRNAs function with essential partner proteins (that is, as non-coding ribonucleoproteins; ncRNPs) and use cognate antisense elements to interact with target molecules. The small nuclear (sn)RNAs and small nucleolar (sno)RNAs are founding members of the family of ncRNAs that helped to establish these common paradigms. Recent studies of the snRNPs and snoRNPs have revealed unexpectedly elaborate biogenesis pathways that will probably also be travelled by other ncRNPs. snRNAs and snoRNAs can be transcribed from independent promoters (similar to mRNAs) or can be encoded within intronic sequences. RNA function can require multiple partner proteins with roles that might include modulating RNA structure or securing an enzyme, as well as catalysing the reaction. Assembling a functional complex seems to involve a series of non-functional intermediate states that are matured by a series of cellular factors along a defined physical pathway in the cell. These transport and assembly steps might serve as control points for the regulation of the activity of a given ncRNP. Recent studies have revealed remarkable complexity in the biogenesis, trafficking and mechanisms of action of small nuclear and small nucleolar ribonucleoproteins (RNPs). The principles of RNP-complex formation can be used to understand the regulation and function of other non-coding RNPs. Recent advances have fuelled rapid growth in our appreciation of the tremendous number, diversity and biological importance of non-coding (nc)RNAs. Because ncRNAs typically function as ribonucleoprotein (RNP) complexes and not as naked RNAs, understanding their biogenesis is crucial to comprehending their regulation and function. The small nuclear and small nucleolar RNPs are two well studied classes of ncRNPs with elaborate assembly and trafficking pathways that provide paradigms for understanding the biogenesis of other ncRNPs.

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.