PRPF8-mediated dysregulation of hBrr2 helicase disrupts human spliceosome kinetics and 5´-splice-site selection causing tissue-specific defects.

The carboxy-terminus of the spliceosomal protein PRPF8, which regulates the RNA helicase Brr2, is a hotspot for mutations causing retinitis pigmentosa-type 13, with unclear role in human splicing and tissue-specificity mechanism. We used patient induced pluripotent stem cells-derived cells, carrying the heterozygous PRPF8 c.6926 A > C (p.H2309P) mutation to demonstrate retinal-specific endophenotypes comprising photoreceptor loss, apical-basal polarity and ciliary defects. Comprehensive molecular, transcriptomic, and proteomic analyses revealed a role of the PRPF8/Brr2 regulation in 5'-splice site (5'SS) selection by spliceosomes, for which disruption impaired alternative splicing and weak/suboptimal 5'SS selection, and enhanced cryptic splicing, predominantly in ciliary and retinal-specific transcripts. Altered splicing efficiency, nuclear speckles organisation, and PRPF8 interaction with U6 snRNA, caused accumulation of active spliceosomes and poly(A)+ mRNAs in unique splicing clusters located at the nuclear periphery of photoreceptors. Collectively these elucidate the role of PRPF8/Brr2 regulatory mechanisms in splicing and the molecular basis of retinal disease, informing therapeutic approaches.

[Targeting the spliceosome: A new therapeutic strategy to counteract chemotherapy resistance in lung cancer?] / Cibler le splicéosome : une nouvelle stratégie thérapeutique pour contrecarrer la résistance à la chimiothérapie dans les cancers du poumon ?

Lung cancer is the first cancer-related cause of death worldwide. This is in partially due to therapeutic resistance, which occurs in around 70% of patients, especially those receiving platinum salts, the gold-standard chemotherapy. The massive deregulation of alternative transcript splicing processes observed in many cancers has led to the development of a new class of pharmacological agents aimed at inhibiting the activity of the splicing machinery (spliceosome). The molecular mechanisms by which these inhibitors act remain largely unknown, as do the benefits of using them in combination with other therapies. In this context, our work is focused on an inhibitor of the SRPK1 kinase, a major regulator of the spliceosome.

The spliceosome-associated protein CWC15 promotes miRNA biogenesis in Arabidopsis.

MicroRNAs (miRNAs) play a key role in regulating gene expression and their biogenesis is precisely controlled through modulating the activity of microprocessor. Here, we report that CWC15, a spliceosome-associated protein, acts as a positive regulator of miRNA biogenesis. CWC15 binds the promoters of genes encoding miRNAs (MIRs), promotes their activity, and increases the occupancy of DNA-dependent RNA polymerases at MIR promoters, suggesting that CWC15 positively regulates the transcription of primary miRNA transcripts (pri-miRNAs). In addition, CWC15 interacts with Serrate (SE) and HYL1, two key components of microprocessor, and is required for efficient pri-miRNA processing and the HYL1-pri-miRNA interaction. Moreover, CWC15 interacts with the 20 S proteasome and PRP4KA, facilitating SE phosphorylation by PRP4KA, and subsequent non-functional SE degradation by the 20 S proteasome. These data reveal that CWC15 ensures optimal miRNA biogenesis by maintaining proper SE levels and by modulating pri-miRNA levels. Taken together, this study uncovers the role of a conserved splicing-related protein in miRNA biogenesis.

The splicing regulators RBM5 and RBM10 are subunits of the U2 snRNP engaged with intron branch sites on chromatin.

Understanding the mechanisms of pre-mRNA splicing is limited by the technical challenges to examining spliceosomes in vivo. Here, we report the isolation of RNP complexes derived from precatalytic A or B-like spliceosomes solubilized from the chromatin pellet of mammalian cell nuclei. We found that these complexes contain U2 snRNP proteins and a portion of the U2 snRNA bound with protected RNA fragments that precisely map to intronic branch sites across the transcriptome. These U2 complexes also contained the splicing regulators RBM5 and RBM10. We found RBM5 and RBM10 bound to nearly all branch site complexes and not simply those at regulated exons. The deletion of a conserved RBM5/RBM10 peptide sequence, including a zinc finger motif, disrupted U2 interaction and rendered the proteins inactive for the repression of many alternative exons. We propose a model where RBM5 and RBM10 regulate splicing as components of the U2 snRNP complex following branch site base pairing.

Tracing Allostery in the Spliceosome Ski2-like RNA Helicase Brr2.

RNA ATPases/helicases remodel substrate RNA-protein complexes in distinct ways. The different RNA ATPases/helicases, taking part in the spliceosome complex, reshape the RNA/RNA-protein contacts to enable premature-mRNA splicing. Among them, the bad response to refrigeration 2 (Brr2) helicase promotes U4/U6 small nuclear (sn)RNA unwinding via ATP-driven translocation of the U4 snRNA strand, thus playing a pivotal role during the activation, catalytic, and disassembly phases of splicing. The plastic Brr2 architecture consists of an enzymatically active N-terminal cassette (N-cassette) and a structurally similar but inactive C-terminal cassette (C-cassette). The C-cassette, along with other allosteric effectors and regulators, tightly and timely controls Brr2's function via an elusive mechanism. Here, microsecond-long molecular dynamics simulations, dynamical network theory, and community network analysis are combined to elucidate how allosteric effectors/regulators modulate the Brr2 function. We unexpectedly reveal that U4 snRNA itself acts as an allosteric regulator, amplifying the cross-talk of distal Brr2 domains and triggering a conformational reorganization of the protein. Our findings offer fundamental understanding into Brr2's mechanism of action and broaden our knowledge on the sophisticated regulatory mechanisms by which spliceosome ATPases/helicases control gene expression. This includes their allosteric regulation exerted by client RNA strands, a mechanism that may be broadly applicable to other RNA-dependent ATPases/helicases.

Xrp1 governs the stress response program to spliceosome dysfunction.

Co-transcriptional processing of nascent pre-mRNAs by the spliceosome is vital to regulating gene expression and maintaining genome integrity. Here, we show that the deficiency of functional U5 small nuclear ribonucleoprotein particles (snRNPs) in Drosophila imaginal cells causes extensive transcriptome remodeling and accumulation of highly mutagenic R-loops, triggering a robust stress response and cell cycle arrest. Despite compromised proliferative capacity, the U5 snRNP-deficient cells increased protein translation and cell size, causing intra-organ growth disbalance before being gradually eliminated via apoptosis. We identify the Xrp1-Irbp18 heterodimer as the primary driver of transcriptional and cellular stress program downstream of U5 snRNP malfunction. Knockdown of Xrp1 or Irbp18 in U5 snRNP-deficient cells attenuated JNK and p53 activity, restored normal cell cycle progression and growth, and inhibited cell death. Reducing Xrp1-Irbp18, however, did not rescue the splicing defects, highlighting the requirement of accurate splicing for cellular and tissue homeostasis. Our work provides novel insights into the crosstalk between splicing and the DNA damage response and defines the Xrp1-Irbp18 heterodimer as a critical sensor of spliceosome malfunction and mediator of the stress-induced cellular senescence program.

The removal of introns and the joining of exons into mature mRNA by the spliceosome is crucial in regulating gene expression, simultaneously safeguarding genome integrity and enhancing proteome diversity in multicellular organisms. Spliceosome dysfunction is thus associated with various diseases and organismal aging. Our study describes the cascade of events in response to spliceosome dysfunction. We identified two transcription factors as drivers of a stress response program triggered by spliceosome dysfunction, which dramatically remodel gene expression to protect tissue integrity and induce a senescent-like state in damaged cells prior to their inevitable elimination. Together, we highlight the indispensable role of spliceosomes in maintaining homeostasis and implicate spliceosome dysfunction in senescent cell accumulation associated with the pathomechanisms of spliceopathies and aging.

SNUPN deficiency causes a recessive muscular dystrophy due to RNA mis-splicing and ECM dysregulation.

SNURPORTIN-1, encoded by SNUPN, plays a central role in the nuclear import of spliceosomal small nuclear ribonucleoproteins. However, its physiological function remains unexplored. In this study, we investigate 18 children from 15 unrelated families who present with atypical muscular dystrophy and neurological defects. Nine hypomorphic SNUPN biallelic variants, predominantly clustered in the last coding exon, are ascertained to segregate with the disease. We demonstrate that mutant SPN1 failed to oligomerize leading to cytoplasmic aggregation in patients' primary fibroblasts and CRISPR/Cas9-mediated mutant cell lines. Additionally, mutant nuclei exhibit defective spliceosomal maturation and breakdown of Cajal bodies. Transcriptome analyses reveal splicing and mRNA expression dysregulation, particularly in sarcolemmal components, causing disruption of cytoskeletal organization in mutant cells and patient muscle tissues. Our findings establish SNUPN deficiency as the genetic etiology of a previously unrecognized subtype of muscular dystrophy and provide robust evidence of the role of SPN1 for muscle homeostasis.

USP4 regulates TUT1 ubiquitination status in concert with SART3.

The ubiquitin system plays pivotal roles in diverse cellular processes, including signal transduction, transcription and translation, organelle quality control, and protein degradation. Recent investigations have revealed the regulatory influence of ubiquitin systems on RNA metabolism. Previously, we reported that the deubiquitinating enzyme, ubiquitin specific peptidase 15 (USP15), promotes deubiquitination of terminal uridylyl transferase 1 (TUT1), a key regulator within the U4/U6 spliceosome, thereby instigating significant alterations in global RNA splicing [1]. In this study, we report that ubiquitin specific peptidase 4 (USP4), a homologous protein to USP15, also exerts control over the ubiquitination status of TUT1. Analogous to USP15, the expression of USP4 results in a reduction of TUT1 ubiquitination. Furthermore, squamous cell carcinoma antigen recognized by T-cells 3 (SART3) collaborates in enhancing the deubiquitinating activity of USP4 towards TUT1. A crucial revelation is that USP4 orchestrates the subnuclear relocation of TUT1 from the nucleolus to the nucleoplasm and facilitates the stability of U6 small nuclear RNA (snRNA). Notably, USP4 has a more profound effect on TUT1 redistribution compared to USP15. Our findings suggest that USP4 intricately modulates the ubiquitination status of TUT1, thereby exerting pronounced effects on the spliceosome functions.

Minor introns impact on hematopoietic malignancies.

In the intricate orchestration of the central dogma, pre-mRNA splicing plays a crucial role in the post-transcriptional process that transforms DNA into mature mRNA. Widely acknowledged as a pivotal RNA processing step, it significantly influences gene expression and alters the functionality of gene product proteins. Although U2-dependent spliceosomes efficiently manage the removal of over 99% of introns, a distinct subset of essential genes undergo splicing with a different intron type, denoted as minor introns, using U12-dependent spliceosomes. Mutations in spliceosome component genes are now recognized as prevalent genetic abnormalities in cancer patients, especially those with hematologic malignancies. Despite the relative rarity of minor introns, genes containing them are evolutionarily conserved and play crucial roles in functions such as the RAS-MAPK pathway. Disruptions in U12-type minor intron splicing caused by mutations in snRNA or its regulatory components significantly contribute to cancer progression. Notably, recurrent mutations associated with myelodysplastic syndrome (MDS) in the minor spliceosome component ZRSR2 underscore its significance. Examination of ZRSR2-mutated MDS cells has revealed that only a subset of minor spliceosome-dependent genes, such as LZTR1, consistently exhibit missplicing. Recent technological advancements have uncovered insights into minor introns, raising inquiries beyond current understanding. This review comprehensively explores the importance of minor intron regulation, the molecular implications of minor (U12-type) spliceosomal mutations and cis-regulatory regions, and the evolutionary progress of studies on minor, aiming to provide a sophisticated understanding of their intricate role in cancer biology.

THUMPD2 catalyzes the N2-methylation of U6 snRNA of the spliceosome catalytic center and regulates pre-mRNA splicing and retinal degeneration.

The mechanisms by which the relatively conserved spliceosome manages the enormously large number of splicing events that occur in humans (∼200 000 versus ∼300 in yeast) are poorly understood. Here, we show deposition of one RNA modification-N2-methylguanosine (m2G) on the G72 of U6 snRNA (the catalytic center of the spliceosome) promotes efficient pre-mRNA splicing activity in human cells. This modification was identified to be conserved among vertebrates. Further, THUMPD2 was demonstrated as the methyltransferase responsible for U6 m2G72 by explicitly recognizing the U6-specific sequences and structural elements. The knock-out of THUMPD2 eliminated U6 m2G72 and impaired the pre-mRNA splicing activity, resulting in thousands of changed alternative splicing events of endogenous pre-mRNAs in human cells. Notably, the aberrantly spliced pre-mRNA population elicited the nonsense-mediated mRNA decay pathway. We further show that THUMPD2 was associated with age-related macular degeneration and retinal function. Our study thus demonstrates how an RNA epigenetic modification of the major spliceosome regulates global pre-mRNA splicing and impacts physiology and disease.

Spliceosomal helicases DDX41/SACY-1 and PRP22/MOG-5 both contribute to proofreading against proximal 3' splice site usage.

RNA helicases drive necessary rearrangements and ensure fidelity during the pre-mRNA splicing cycle. DEAD-box helicase DDX41 has been linked to human disease and has recently been shown to interact with DEAH-box helicase PRP22 in the spliceosomal C* complex, yet its function in splicing remains unknown. Depletion of DDX41 homolog SACY-1 from somatic cells has been previously shown to lead to changes in alternative 3' splice site (3'ss) usage. Here, we show by transcriptomic analysis of published and novel data sets that SACY-1 perturbation causes a previously unreported pattern in alternative 3' splicing in introns with pairs of 3' splice sites ≤18 nt away from each other. We find that both SACY-1 depletion and the allele sacy-1(G533R) lead to a striking unidirectional increase in the usage of the proximal (upstream) 3'ss. We previously discovered a similar alternative splicing pattern between germline tissue and somatic tissue, in which there is a unidirectional increase in proximal 3'ss usage in the germline for ∼200 events; many of the somatic SACY-1 alternative 3' splicing events overlap with these developmentally regulated events. We generated targeted mutant alleles of the Caenorhabditis elegans homolog of PRP22, mog-5, in the region of MOG-5 that is predicted to interact with SACY-1 based on the human C* structure. These viable alleles, and a mimic of the myelodysplastic syndrome-associated allele DDX41(R525H), all promote the usage of proximal alternative adjacent 3' splice sites. We show that PRP22/MOG-5 and DDX41/SACY-1 have overlapping roles in proofreading the 3'ss.

RIP-PEN-seq identifies a class of kink-turn RNAs as splicing regulators.

A kink-turn (K-turn) is a three-dimensional RNA structure that exists in all three primary phylogenetic domains. In this study, we developed the RIP-PEN-seq method to identify the full-length sequences of RNAs bound by the K-turn binding protein 15.5K and discovered a previously uncharacterized class of RNAs with backward K-turn motifs (bktRNAs) in humans and mice. All bktRNAs share two consensus sequence motifs at their fixed terminal position and have complex folding properties, expression and evolution patterns. We found that a highly conserved bktRNA1 guides the methyltransferase fibrillarin to install RNA methylation of U12 small nuclear RNA in humans. Depletion of bktRNA1 causes global splicing dysregulation of U12-type introns by impairing the recruitment of ZCRB1 to the minor spliceosome. Most bktRNAs regulate the splicing of local introns by interacting with the 15.5K protein. Taken together, our findings characterize a class of small RNAs and uncover another layer of gene expression regulation that involves crosstalk among bktRNAs, RNA splicing and RNA methylation.

Structural basis of branching during RNA splicing.

Branching is a critical step in RNA splicing that is essential for 5' splice site selection. Recent spliceosome structures have led to competing models for the recognition of the invariant adenosine at the branch point. However, there are no structures of any splicing complex with the adenosine nucleophile docked in the active site and positioned to attack the 5' splice site. Thus we lack a mechanistic understanding of adenosine selection and splice site recognition during RNA splicing. Here we present a cryo-electron microscopy structure of a group II intron that reveals that active site dynamics are coupled to the formation of a base triple within the branch-site helix that positions the 2'-OH of the adenosine for nucleophilic attack on the 5' scissile phosphate. This structure, complemented with biochemistry and comparative analyses to splicing complexes, supports a base triple model of adenosine recognition for branching within group II introns and the evolutionarily related spliceosome.

JANUS, a spliceosome-associated protein, promotes miRNA biogenesis in Arabidopsis.

MicroRNAs (miRNAs) are important regulators of genes expression. Their levels are precisely controlled through modulating the activity of the microprocesser complex (MC). Here, we report that JANUS, a homology of the conserved U2 snRNP assembly factor in yeast and human, is required for miRNA accumulation. JANUS associates with MC components Dicer-like 1 (DCL1) and SERRATE (SE) and directly binds the stem-loop of pri-miRNAs. In a hypomorphic janus mutant, the activity of DCL1, the numbers of MC, and the interaction of primary miRNA transcript (pri-miRNAs) with MC are reduced. These data suggest that JANUS promotes the assembly and activity of MC through its interaction with MC and/or pri-miRNAs. In addition, JANUS modulates the transcription of some pri-miRNAs as it binds the promoter of pri-miRNAs and facilitates Pol II occupancy of at their promoters. Moreover, global splicing defects are detected in janus. Taken together, our study reveals a novel role of a conserved splicing factor in miRNA biogenesis.

Spliceosome component PHD finger 5A is essential for early B lymphopoiesis.

The spliceosome, a multi-megadalton ribonucleoprotein complex, is essential for pre-mRNA splicing in the nucleus and ensuring genomic stability. Its precise and dynamic assembly is pivotal for its function. Spliceosome malfunctions can lead to developmental abnormalities and potentially contribute to tumorigenesis. The specific role of the spliceosome in B cell development is poorly understood. Here, we reveal that the spliceosomal U2 snRNP component PHD finger protein 5A (Phf5a) is vital for early B cell development. Loss of Phf5a results in pronounced defects in B cell development, causing an arrest at the transition from pre-pro-B to early pro-B cell stage in the bone marrow of mutant mice. Phf5a-deficient B cells exhibit impaired immunoglobulin heavy (IgH) chain expression due to defective V-to-DJ gene rearrangement. Mechanistically, our findings suggest that Phf5a facilitates IgH gene rearrangement by regulating the activity of recombination-activating gene endonuclease and influencing chromatin interactions at the Igh locus.

Global analysis of binding sites of U2AF1 and ZRSR2 reveals RNA elements required for mutually exclusive splicing by the U2- and U12-type spliceosome.

Recurring mutations in genes encoding 3' splice-site recognition proteins, U2AF1 and ZRSR2 are associated with human cancers. Here, we determined binding sites of the proteins to reveal that U2-type and U12-type splice sites are recognized by U2AF1 and ZRSR2, respectively. However, some sites are spliced by both the U2-type and U12-type spliceosomes, indicating that well-conserved consensus motifs in some U12-type introns could be recognized by the U2-type spliceosome. Nucleotides flanking splice sites of U12-type introns are different from those flanking U2-type introns. Remarkably, the AG dinucleotide at the positions -1 and -2 of 5' splice sites of U12-type introns with GT-AG termini is not present. AG next to 5' splice site introduced by a single nucleotide substitution at the -2 position could convert a U12-type splice site to a U2-type site. The class switch of introns by a single mutation and the bias against G at the -1 position of U12-type 5' splice site support the notion that the identities of nucleotides in exonic regions adjacent to splice sites are fine-tuned to avoid recognition by the U2-type spliceosome. These findings may shed light on the mechanism of selectivity in U12-type intron splicing and the mutations that affect splicing.

AI-assisted proofreading of RNA splicing.

RNA helicases orchestrate proofreading mechanisms that facilitate accurate intron removal from pre-mRNAs. How these activities are recruited to spliceosome/pre-mRNA complexes remains poorly understood. In this issue of Genes & Development, Zhang and colleagues (pp. 968-983) combine biochemical experiments with AI-based structure prediction methods to generate a model for the interaction between SF3B1, a core splicing factor essential for the recognition of the intron branchpoint, and SUGP1, a protein that bridges SF3B1 with the helicase DHX15. Interaction with SF3B1 exposes the G-patch domain of SUGP1, facilitating binding to and activation of DHX15. The model can explain the activation of cryptic 3' splice sites induced by mutations in SF3B1 or SUGP1 frequently found in cancer.

SAP30BP interacts with RBM17/SPF45 to promote splicing in a subset of human short introns.

Human pre-mRNA splicing requires the removal of introns with highly variable lengths, from tens to over a million nucleotides. Therefore, mechanisms of intron recognition and splicing are likely not universal. Recently, we reported that splicing in a subset of human short introns with truncated polypyrimidine tracts depends on RBM17 (SPF45), instead of the canonical splicing factor U2 auxiliary factor (U2AF) heterodimer. Here, we demonstrate that SAP30BP, a factor previously implicated in transcriptional control, is an essential splicing cofactor for RBM17. In vitro binding and nuclear magnetic resonance analyses demonstrate that a U2AF-homology motif (UHM) in RBM17 binds directly to a newly identified UHM-ligand motif in SAP30BP. We show that this RBM17-SAP30BP interaction is required to specifically recruit RBM17 to phosphorylated SF3B1 (SF3b155), a U2 small nuclear ribonucleoprotein (U2 snRNP) component in active spliceosomes. We propose a mechanism for splicing in a subset of short introns, in which SAP30BP guides RBM17 in the assembly of active spliceosomes.

Exploring the highly reduced spliceosome of Pseudoloma neurophilia.

Spliceosomal introns evolved early in eukaryogenesis, originating from self-splicing group II introns that invaded the proto-eukaryotic genome1. Elements of these ribozymes, now called snRNAs (U1, U2, U4, U5, U6), were co-opted to excise these invasive elements. Prior to eukaryotic diversification, the spliceosome is predicted to have accumulated hundreds of proteins2. This early complexification has obscured our understanding of spliceosomal evolution. Reduced systems with few introns and tiny spliceosomes give insights into the plasticity of the splicing reaction and provide an opportunity to study the evolution of the spliceosome3,4. Microsporidia are intracellular parasites possessing extremely reduced genomes that have lost many, and in some instances all, introns5. In the purportedly intron-lacking genome of the microsporidian Pseudoloma neurophilia6, we identified two introns that are spliced at high levels. Furthermore, with only 14 predicted proteins, the P. neurophilia spliceosome could be the smallest known. Intriguingly, the few proteins retained are divergent compared to canonical orthologs. Even the central spliceosomal protein Prp8, which originated from the proteinaceous component of group II introns, is extremely divergent. This is unusual given that Prp8 is highly conserved across eukaryotes, including other microsporidia. All five P. neurophilia snRNAs are present, and all but U2 have diverged extensively, likely resulting from the loss of interacting proteins. Despite this divergence, U1 and U2 are predicted to pair with intron sequences more extensively than previously described. The P. neurophilia spliceosome is retained to splice a mere two introns and, with few proteins and reliance on RNA-RNA interactions, could function in a manner more reminiscent of presumed ancestral splicing.