RESUMO
Early spliceosome assembly can occur through an intron-defined pathway, whereby U1 and U2 small nuclear ribonucleoprotein particles (snRNPs) assemble across the intron1. Alternatively, it can occur through an exon-defined pathway2-5, whereby U2 binds the branch site located upstream of the defined exon and U1 snRNP interacts with the 5' splice site located directly downstream of it. The U4/U6.U5 tri-snRNP subsequently binds to produce a cross-intron (CI) or cross-exon (CE) pre-B complex, which is then converted to the spliceosomal B complex6,7. Exon definition promotes the splicing of upstream introns2,8,9 and plays a key part in alternative splicing regulation10-16. However, the three-dimensional structure of exon-defined spliceosomal complexes and the molecular mechanism of the conversion from a CE-organized to a CI-organized spliceosome, a pre-requisite for splicing catalysis, remain poorly understood. Here cryo-electron microscopy analyses of human CE pre-B complex and B-like complexes reveal extensive structural similarities with their CI counterparts. The results indicate that the CE and CI spliceosome assembly pathways converge already at the pre-B stage. Add-back experiments using purified CE pre-B complexes, coupled with cryo-electron microscopy, elucidate the order of the extensive remodelling events that accompany the formation of B complexes and B-like complexes. The molecular triggers and roles of B-specific proteins in these rearrangements are also identified. We show that CE pre-B complexes can productively bind in trans to a U1 snRNP-bound 5' splice site. Together, our studies provide new mechanistic insights into the CE to CI switch during spliceosome assembly and its effect on pre-mRNA splice site pairing at this stage.
Assuntos
Éxons , Íntrons , Splicing de RNA , Spliceossomos , Humanos , Processamento Alternativo , Microscopia Crioeletrônica , Éxons/genética , Íntrons/genética , Modelos Moleculares , Sítios de Splice de RNA/genética , Splicing de RNA/genética , Spliceossomos/metabolismo , Spliceossomos/química , Spliceossomos/ultraestrutura , Ribonucleoproteínas Nucleares Pequenas/química , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Ribonucleoproteínas Nucleares Pequenas/ultraestruturaRESUMO
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.
Assuntos
Sítios de Splice de RNA , Retinose Pigmentar , Spliceossomos , Humanos , Spliceossomos/genética , Spliceossomos/metabolismo , Proteômica , Splicing de RNA/genética , Processamento Alternativo/genética , RNA Nuclear Pequeno/genética , RNA Nuclear Pequeno/metabolismo , RNA Mensageiro/metabolismo , Mutação , DNA Helicases/metabolismo , Proteínas de Ligação a RNA/metabolismoRESUMO
The B complex is a key intermediate stage of spliceosome assembly. To improve the structural resolution of monomeric, human spliceosomal B (hB) complexes and thereby generate a more comprehensive hB molecular model, we determined the cryo-EM structure of B complex dimers formed in the presence of ATP γ S. The enhanced resolution of these complexes allows a finer molecular dissection of how the 5' splice site (5'ss) is recognized in hB, and new insights into molecular interactions of FBP21, SNU23 and PRP38 with the U6/5'ss helix and with each other. It also reveals that SMU1 and RED are present as a heterotetrameric complex and are located at the interface of the B dimer protomers. We further show that MFAP1 and UBL5 form a 5' exon binding channel in hB, and elucidate the molecular contacts stabilizing the 5' exon at this stage. Our studies thus yield more accurate models of protein and RNA components of hB complexes. They further allow the localization of additional proteins and protein domains (such as SF3B6, BUD31 and TCERG1) whose position was not previously known, thereby uncovering new functions for B-specific and other hB proteins during pre-mRNA splicing.
Assuntos
Splicing de RNA , Spliceossomos , Humanos , Spliceossomos/genética , Microscopia Crioeletrônica , Sítios de Splice de RNA , Éxons , Precursores de RNA/genética , Precursores de RNA/metabolismo , Fatores de Elongação da Transcrição/genética , Proteínas Nucleares/metabolismoRESUMO
Alternative precursor messenger RNA splicing is instrumental in expanding the proteome of higher eukaryotes, and changes in 3' splice site (3'ss) usage contribute to human disease. We demonstrate by small interfering RNA-mediated knockdowns, followed by RNA sequencing, that many proteins first recruited to human C* spliceosomes, which catalyze step 2 of splicing, regulate alternative splicing, including the selection of alternatively spliced NAGNAG 3'ss. Cryo-electron microscopy and protein cross-linking reveal the molecular architecture of these proteins in C* spliceosomes, providing mechanistic and structural insights into how they influence 3'ss usage. They further elucidate the path of the 3' region of the intron, allowing a structure-based model for how the C* spliceosome potentially scans for the proximal 3'ss. By combining biochemical and structural approaches with genome-wide functional analyses, our studies reveal widespread regulation of alternative 3'ss usage after step 1 of splicing and the likely mechanisms whereby C* proteins influence NAGNAG 3'ss choices.
Assuntos
Sítios de Splice de RNA , Spliceossomos , Humanos , Microscopia Crioeletrônica , Processamento Alternativo , ÍntronsRESUMO
INTRODUCTION: Mutations in pre-mRNA processing factor 31 (PRPF31), a core protein of the spliceosomal tri-snRNP complex, cause autosomal-dominant retinitis pigmentosa (adRP). It has remained an enigma why mutations in ubiquitously expressed tri-snRNP proteins result in retina-specific disorders, and so far, the underlying mechanism of splicing factors-related RP is poorly understood. METHODS: We used the induced pluripotent stem cell (iPSC) technology to generate retinal organoids and RPE models from four patients with severe and very severe PRPF31-adRP, unaffected individuals and a CRISPR/Cas9 isogenic control. RESULTS: To fully assess the impacts of PRPF31 mutations, quantitative proteomics analyses of retinal organoids and RPE cells were carried out showing RNA splicing, autophagy and lysosome, unfolded protein response (UPR) and visual cycle-related pathways to be significantly affected. Strikingly, the patient-derived RPE and retinal cells were characterised by the presence of large amounts of cytoplasmic aggregates containing the mutant PRPF31 and misfolded, ubiquitin-conjugated proteins including key visual cycle and other RP-linked tri-snRNP proteins, which accumulated progressively with time. The mutant PRPF31 variant was not incorporated into splicing complexes, but reduction of PRPF31 wild-type levels led to tri-snRNP assembly defects in Cajal bodies of PRPF31 patient retinal cells, altered morphology of nuclear speckles and reduced formation of active spliceosomes giving rise to global splicing dysregulation. Moreover, the impaired waste disposal mechanisms further exacerbated aggregate formation, and targeting these by activating the autophagy pathway using Rapamycin reduced cytoplasmic aggregates, leading to improved cell survival. CONCLUSIONS: Our data demonstrate that it is the progressive aggregate accumulation that overburdens the waste disposal machinery rather than direct PRPF31-initiated mis-splicing, and thus relieving the RPE cells from insoluble cytoplasmic aggregates presents a novel therapeutic strategy that can be combined with gene therapy studies to fully restore RPE and retinal cell function in PRPF31-adRP patients.
Assuntos
Autofagia , Proteínas do Olho , Células-Tronco Pluripotentes Induzidas , Agregados Proteicos , Retinose Pigmentar , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/metabolismo , Retinose Pigmentar/genética , Retinose Pigmentar/metabolismo , Ribonucleoproteínas Nucleares PequenasRESUMO
During the splicing of introns from precursor messenger RNAs (pre-mRNAs), the U2 small nuclear ribonucleoprotein (snRNP) must undergo stable integration into the spliceosomal A complex-a poorly understood, multistep process that is facilitated by the DEAD-box helicase Prp5 (refs. 1-4). During this process, the U2 small nuclear RNA (snRNA) forms an RNA duplex with the pre-mRNA branch site (the U2-BS helix), which is proofread by Prp5 at this stage through an unclear mechanism5. Here, by deleting the branch-site adenosine (BS-A) or mutating the branch-site sequence of an actin pre-mRNA, we stall the assembly of spliceosomes in extracts from the yeast Saccharomyces cerevisiae directly before the A complex is formed. We then determine the three-dimensional structure of this newly identified assembly intermediate by cryo-electron microscopy. Our structure indicates that the U2-BS helix has formed in this pre-A complex, but is not yet clamped by the HEAT domain of the Hsh155 protein (Hsh155HEAT), which exhibits an open conformation. The structure further reveals a large-scale remodelling/repositioning of the U1 and U2 snRNPs during the formation of the A complex that is required to allow subsequent binding of the U4/U6.U5 tri-snRNP, but that this repositioning is blocked in the pre-A complex by the presence of Prp5. Our data suggest that binding of Hsh155HEAT to the bulged BS-A of the U2-BS helix triggers closure of Hsh155HEAT, which in turn destabilizes Prp5 binding. Thus, Prp5 proofreads the branch site indirectly, hindering spliceosome assembly if branch-site mutations prevent the remodelling of Hsh155HEAT. Our data provide structural insights into how a spliceosomal helicase enhances the fidelity of pre-mRNA splicing.
Assuntos
RNA Helicases DEAD-box/química , RNA Helicases DEAD-box/metabolismo , Precursores de RNA/química , Precursores de RNA/genética , Splicing de RNA , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae , Spliceossomos/enzimologia , Actinas/genética , Adenosina/metabolismo , Sítios de Ligação , Microscopia Crioeletrônica , RNA Helicases DEAD-box/ultraestrutura , Modelos Moleculares , Mutação , Domínios Proteicos , Precursores de RNA/metabolismo , Precursores de RNA/ultraestrutura , Splicing de RNA/genética , Ribonucleoproteína Nuclear Pequena U1/metabolismo , Ribonucleoproteína Nuclear Pequena U2/química , Ribonucleoproteína Nuclear Pequena U2/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Spliceossomos/química , Spliceossomos/metabolismoRESUMO
Splicing is catalyzed by the spliceosome, a compositionally dynamic complex assembled stepwise on pre-mRNA. We reveal links between splicing machinery components and the intrinsically disordered ciliopathy protein SANS. Pathogenic mutations in SANS/USH1G lead to Usher syndrome-the most common cause of deaf-blindness. Previously, SANS was shown to function only in the cytosol and primary cilia. Here, we have uncovered molecular links between SANS and pre-mRNA splicing catalyzed by the spliceosome in the nucleus. We show that SANS is found in Cajal bodies and nuclear speckles, where it interacts with components of spliceosomal sub-complexes such as SF3B1 and the large splicing cofactor SON but also with PRPFs and snRNAs related to the tri-snRNP complex. SANS is required for the transfer of tri-snRNPs between Cajal bodies and nuclear speckles for spliceosome assembly and may also participate in snRNP recycling back to Cajal bodies. SANS depletion alters the kinetics of spliceosome assembly, leading to accumulation of complex A. SANS deficiency and USH1G pathogenic mutations affects splicing of genes related to cell proliferation and human Usher syndrome. Thus, we provide the first evidence that splicing dysregulation may participate in the pathophysiology of Usher syndrome.
Assuntos
Processamento Alternativo/genética , Proteínas do Tecido Nervoso/metabolismo , Precursores de RNA/metabolismo , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Spliceossomos/metabolismo , Síndromes de Usher/metabolismo , Núcleo Celular/metabolismo , Proliferação de Células/genética , Corpos Enovelados/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas do Olho/metabolismo , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Espectrometria de Massas , Microscopia Eletrônica de Transmissão , Antígenos de Histocompatibilidade Menor/metabolismo , Proteínas do Tecido Nervoso/genética , Fosfoproteínas/metabolismo , Proteômica , Precursores de RNA/genética , Fatores de Processamento de RNA/metabolismo , RNA Nuclear Pequeno/genética , RNA Nuclear Pequeno/metabolismo , Ribonucleoproteína Nuclear Pequena U4-U6/metabolismo , Ribonucleoproteínas Nucleares Pequenas/genética , Spliceossomos/genética , Fatores de Transcrição/metabolismo , Síndromes de Usher/genéticaRESUMO
To initiate cotranscriptional splicing, RNA polymerase II (Pol II) recruits the U1 small nuclear ribonucleoprotein particle (U1 snRNP) to nascent precursor messenger RNA (pre-mRNA). Here, we report the cryo-electron microscopy structure of a mammalian transcribing Pol II-U1 snRNP complex. The structure reveals that Pol II and U1 snRNP interact directly. This interaction positions the pre-mRNA 5' splice site near the RNA exit site of Pol II. Extension of pre-mRNA retains the 5' splice site, leading to the formation of a "growing intron loop." Loop formation may facilitate scanning of nascent pre-mRNA for the 3' splice site, functional pairing of distant intron ends, and prespliceosome assembly. Our results provide a starting point for a mechanistic analysis of cotranscriptional spliceosome assembly and the biogenesis of mRNA isoforms by alternative splicing.
Assuntos
Processamento Alternativo , RNA Polimerase II/química , RNA Mensageiro/biossíntese , Ribonucleoproteína Nuclear Pequena U1/química , Spliceossomos/química , Animais , Microscopia Crioeletrônica , Humanos , Íntrons , Conformação de Ácido Nucleico , Ligação Proteica , Domínios Proteicos , Precursores de RNA/química , RNA Mensageiro/química , Spliceossomos/metabolismo , Sus scrofa , Transcrição GênicaRESUMO
Interactions between U2AF homology motifs (UHMs) and U2AF ligand motifs (ULMs) play a crucial role in early spliceosome assembly in eukaryotic gene regulation. UHM-ULM interactions mediate heterodimerization of the constitutive splicing factors U2AF65 and U2AF35 and between other splicing factors that regulate spliceosome assembly at the 3' splice site, where UHM domains of alternative splicing factors, such as SPF45 and PUF60, contribute to alternative splicing regulation. Here, we performed high-throughput screening using fluorescence polarization assays with hit validation by NMR and identified phenothiazines as general inhibitors of UHM-ULM interactions. NMR studies show that these compounds occupy the tryptophan binding pocket of UHM domains. Co-crystal structures of the inhibitors with the PUF60 UHM domain and medicinal chemistry provide structure-activity-relationships and reveal functional groups important for binding. These inhibitors inhibit early spliceosome assembly on pre-mRNA substrates in vitro. Our data show that spliceosome assembly can be inhibited by targeting UHM-ULM interactions by small molecules, thus extending the toolkit of splicing modulators for structural and biochemical studies of the spliceosome and splicing regulation.
Assuntos
Fenotiazinas/química , Fenotiazinas/farmacologia , Spliceossomos/efeitos dos fármacos , Spliceossomos/metabolismo , Processamento Alternativo , Humanos , Ligação Proteica/efeitos dos fármacos , Domínios Proteicos , Precursores de RNA/genética , Precursores de RNA/metabolismo , Fatores de Processamento de RNA/química , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Spliceossomos/genética , Fator de Processamento U2AF/química , Fator de Processamento U2AF/genética , Fator de Processamento U2AF/metabolismoRESUMO
Spliceosome activation involves extensive protein and RNA rearrangements that lead to formation of a catalytically active U2/U6 RNA structure. At present, little is known about the assembly pathway of the latter and the mechanism whereby proteins aid its proper folding. Here, we report the cryo-electron microscopy structures of two human, activated spliceosome precursors (that is, pre-Bact complexes) at core resolutions of 3.9 and 4.2 angstroms. These structures elucidate the order of the numerous protein exchanges that occur during activation, the mutually exclusive interactions that ensure the correct order of ribonucleoprotein rearrangements needed to form the U2/U6 catalytic RNA, and the stepwise folding pathway of the latter. Structural comparisons with mature Bact complexes reveal the molecular mechanism whereby a conformational change in the scaffold protein PRP8 facilitates final three-dimensional folding of the U2/U6 catalytic RNA.
Assuntos
Dobramento de RNA , Splicing de RNA , RNA Nuclear Pequeno/química , Proteínas de Ligação a RNA/química , Spliceossomos/química , Domínio Catalítico , Microscopia Crioeletrônica , Humanos , Conformação Proteica , RNA Catalítico/química , RNA Nuclear Pequeno/genética , Spliceossomos/genéticaRESUMO
Human spliceosomes contain numerous proteins absent in yeast, whose functions remain largely unknown. Here we report a 3D cryo-EM structure of the human spliceosomal C complex at 3.4 Å core resolution and 4.5-5.7 Å at its periphery, and aided by protein crosslinking we determine its molecular architecture. Our structure provides additional insights into the spliceosome's architecture between the catalytic steps of splicing, and how proteins aid formation of the spliceosome's catalytically active RNP (ribonucleoprotein) conformation. It reveals the spatial organization of the metazoan-specific proteins PPWD1, WDR70, FRG1, and CIR1 in human C complexes, indicating they stabilize functionally important protein domains and RNA structures rearranged/repositioned during the Bact to C transition. Structural comparisons with human Bact, C∗, and P complexes reveal an intricate cascade of RNP rearrangements during splicing catalysis, with intermediate RNP conformations not found in yeast, and additionally elucidate the structural basis for the sequential recruitment of metazoan-specific spliceosomal proteins.
Assuntos
Fatores de Processamento de RNA/química , Fatores de Processamento de RNA/metabolismo , Spliceossomos/metabolismo , Animais , Catálise , Células HeLa , Humanos , Íntrons/genética , Modelos Moleculares , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/ultraestrutura , Ligação Proteica , Estabilidade Proteica , RNA/química , RNA/metabolismo , Ribonucleoproteínas/metabolismo , Saccharomyces cerevisiae/metabolismo , Especificidade da Espécie , Fatores de TempoRESUMO
The U2 small nuclear ribonucleoprotein (snRNP) has an essential role in the selection of the precursor mRNA branch-site adenosine, the nucleophile for the first step of splicing1. Stable addition of U2 during early spliceosome formation requires the DEAD-box ATPase PRP52-7. Yeast U2 small nuclear RNA (snRNA) nucleotides that form base pairs with the branch site are initially sequestered in a branchpoint-interacting stem-loop (BSL)8, but whether the human U2 snRNA folds in a similar manner is unknown. The U2 SF3B1 protein, a common mutational target in haematopoietic cancers9, contains a HEAT domain (SF3B1HEAT) with an open conformation in isolated SF3b10, but a closed conformation in spliceosomes11, which is required for stable interaction between U2 and the branch site. Here we report a 3D cryo-electron microscopy structure of the human 17S U2 snRNP at a core resolution of 4.1 Å and combine it with protein crosslinking data to determine the molecular architecture of this snRNP. Our structure reveals that SF3B1HEAT interacts with PRP5 and TAT-SF1, and maintains its open conformation in U2 snRNP, and that U2 snRNA forms a BSL that is sandwiched between PRP5, TAT-SF1 and SF3B1HEAT. Thus, substantial remodelling of the BSL and displacement of BSL-interacting proteins must occur to allow formation of the U2-branch-site helix. Our studies provide a structural explanation of why TAT-SF1 must be displaced before the stable addition of U2 to the spliceosome, and identify RNP rearrangements facilitated by PRP5 that are required for stable interaction between U2 and the branch site.
Assuntos
Microscopia Crioeletrônica , Ribonucleoproteína Nuclear Pequena U2/química , Ribonucleoproteína Nuclear Pequena U2/ultraestrutura , Sequência de Bases , RNA Helicases DEAD-box/química , RNA Helicases DEAD-box/metabolismo , Células HeLa , Humanos , Modelos Moleculares , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Ligação Proteica , Conformação Proteica , Fatores de Processamento de RNA/química , Fatores de Processamento de RNA/metabolismo , Ribonucleoproteína Nuclear Pequena U2/genética , Ribonucleoproteína Nuclear Pequena U2/metabolismo , Transativadores/química , Transativadores/metabolismoRESUMO
Deregulated expression of MYC induces a dependence on the NUAK1 kinase, but the molecular mechanisms underlying this dependence have not been fully clarified. Here, we show that NUAK1 is a predominantly nuclear protein that associates with a network of nuclear protein phosphatase 1 (PP1) interactors and that PNUTS, a nuclear regulatory subunit of PP1, is phosphorylated by NUAK1. Both NUAK1 and PNUTS associate with the splicing machinery. Inhibition of NUAK1 abolishes chromatin association of PNUTS, reduces spliceosome activity, and suppresses nascent RNA synthesis. Activation of MYC does not bypass the requirement for NUAK1 for spliceosome activity but significantly attenuates transcription inhibition. Consequently, NUAK1 inhibition in MYC-transformed cells induces global accumulation of RNAPII both at the pause site and at the first exon-intron boundary but does not increase mRNA synthesis. We suggest that NUAK1 inhibition in the presence of deregulated MYC traps non-productive RNAPII because of the absence of correctly assembled spliceosomes.
Assuntos
Núcleo Celular/metabolismo , Cromatina/metabolismo , Proteínas Quinases/metabolismo , Proteína Fosfatase 1/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Repressoras/metabolismo , Spliceossomos/metabolismo , Transcrição Gênica , Animais , Núcleo Celular/genética , Cromatina/genética , Regulação da Expressão Gênica , Células HeLa , Humanos , Camundongos , Células NIH 3T3 , Fosforilação , Proteínas Quinases/genética , Proteína Fosfatase 1/genética , Proteína Fosfatase 1/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Splicing de RNA , Proteínas Repressoras/genética , Spliceossomos/genéticaRESUMO
Human pre-catalytic spliceosomes contain several proteins that associate transiently just prior to spliceosome activation and are absent in yeast, suggesting that this critical step is more complex in higher eukaryotes. We demonstrate via RNAi coupled with RNA-Seq that two of these human-specific proteins, Smu1 and RED, function both as alternative splicing regulators and as general splicing factors and are required predominantly for efficient splicing of short introns. In vitro splicing assays reveal that Smu1 and RED promote spliceosome activation, and are essential for this step when the distance between the pre-mRNA's 5' splice site (SS) and branch site (BS) is sufficiently short. This Smu1-RED requirement can be bypassed when the 5' and 3' regions of short introns are physically separated. Our observations suggest that Smu1 and RED relieve physical constraints arising from a short 5'SS-BS distance, thereby enabling spliceosomes to overcome structural challenges associated with the splicing of short introns.
Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Citocinas/metabolismo , Íntrons , Splicing de RNA , Spliceossomos/metabolismo , Proteínas Cromossômicas não Histona/genética , Citocinas/genética , Humanos , Precursores de RNA/genética , Precursores de RNA/metabolismo , Sítios de Splice de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Spliceossomos/genéticaRESUMO
A precise genetic diagnosis is the single most important step for families with genetic disorders to enable personalized and preventative medicine. In addition to genetic variants in coding regions (exons) that can change a protein sequence, abnormal pre-mRNA splicing can be devastating for the encoded protein, inducing a frameshift or in-frame deletion/insertion of multiple residues. Non-coding variants that disrupt splicing are extremely challenging to identify. Stemming from an initial clinical discovery in two index Australian families, we define 25 families with genetic disorders caused by a class of pathogenic non-coding splice variant due to intronic deletions. These pathogenic intronic deletions spare all consensus splice motifs, though they critically shorten the minimal distance between the 5' splice-site (5'SS) and branchpoint. The mechanistic basis for abnormal splicing is due to biophysical constraint precluding U1/U2 spliceosome assembly, which stalls in A-complexes (that bridge the 5'SS and branchpoint). Substitution of deleted nucleotides with non-specific sequences restores spliceosome assembly and normal splicing, arguing against loss of an intronic element as the primary causal basis. Incremental lengthening of 5'SS-branchpoint length in our index EMD case subject defines 45-47 nt as the critical elongation enabling (inefficient) spliceosome assembly for EMD intron 5. The 5'SS-branchpoint space constraint mechanism, not currently factored by genomic informatics pipelines, is relevant to diagnosis and precision medicine across the breadth of Mendelian disorders and cancer genomics.
Assuntos
Íntrons , Splicing de RNA , Spliceossomos , Adolescente , Adulto , Fenômenos Biofísicos , Criança , Feminino , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , LinhagemRESUMO
The spliceosome is a highly complex, dynamic ribonucleoprotein molecular machine that undergoes numerous structural and compositional rearrangements that lead to the formation of its active site. Recent advances in cyroelectron microscopy (cryo-EM) have provided a plethora of near-atomic structural information about the inner workings of the spliceosome. Aided by previous biochemical, structural, and functional studies, cryo-EM has confirmed or provided a structural basis for most of the prevailing models of spliceosome function, but at the same time allowed novel insights into splicing catalysis and the intriguing dynamics of the spliceosome. The mechanism of pre-mRNA splicing is highly conserved between humans and yeast, but the compositional dynamics and ribonucleoprotein (RNP) remodeling of the human spliceosome are more complex. Here, we summarize recent advances in our understanding of the molecular architecture of the human spliceosome, highlighting differences between the human and yeast splicing machineries.
Assuntos
Conformação de Ácido Nucleico , Precursores de RNA/metabolismo , Splicing de RNA , RNA Mensageiro/metabolismo , Catálise , Microscopia Crioeletrônica , Células Eucarióticas , Mutação , Neoplasias/metabolismo , Conformação Proteica , Precursores de RNA/química , RNA Mensageiro/química , Ribonucleoproteínas Nucleares Pequenas/química , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Saccharomyces cerevisiae/metabolismo , Spliceossomos/metabolismoRESUMO
The active 3D conformation of the spliceosome's catalytic U2/U6 RNA core is stabilised by a network of secondary and tertiary RNA interactions, but also depends on spliceosomal proteins for its formation. To determine the contribution towards splicing of specific RNA secondary and tertiary interactions in the U2/U6 RNA core, we introduced mutations in critical U6 nucleotides and tested their effect on splicing using a yeast in vitro U6 depletion/complementation system. Elimination of selected RNA tertiary interactions involving the U6 catalytic triad, or deletions of the bases of U6-U80 or U6-A59, had moderate to no effect on splicing, showing that the affected secondary and tertiary interactions are not required for splicing catalysis. However, removal of the base of U6-G60 of the catalytic triad completely blocked splicing, without affecting assembly of the activated spliceosome or its subsequent conversion into a B*-like complex. Our data suggest that the catalytic configuration of the RNA core that allows catalytic metal M1 binding can be maintained by Protein-RNA contacts. However, RNA stacking interactions in the U2/U6 RNA core are required for productive coordination of metal M2. The functional conformation of the U2/U6 RNA core is thus highly buffered, with overlapping contributions from RNA-RNA and Protein-RNA interactions.
Assuntos
Conformação de Ácido Nucleico , Splicing de RNA , RNA Fúngico/química , RNA Nuclear Pequeno/química , Spliceossomos/genética , Sítios de Ligação , Catálise , Domínio Catalítico , Microscopia Crioeletrônica , Cristalografia por Raios X , Humanos , Ligação de Hidrogênio , Metais/química , Mutação , Precursores de RNA/química , RNA Mensageiro/metabolismo , Saccharomyces cerevisiaeRESUMO
Mutations in pre-mRNA processing factors (PRPFs) cause autosomal-dominant retinitis pigmentosa (RP), but it is unclear why mutations in ubiquitously expressed genes cause non-syndromic retinal disease. Here, we generate transcriptome profiles from RP11 (PRPF31-mutated) patient-derived retinal organoids and retinal pigment epithelium (RPE), as well as Prpf31+/- mouse tissues, which revealed that disrupted alternative splicing occurred for specific splicing programmes. Mis-splicing of genes encoding pre-mRNA splicing proteins was limited to patient-specific retinal cells and Prpf31+/- mouse retinae and RPE. Mis-splicing of genes implicated in ciliogenesis and cellular adhesion was associated with severe RPE defects that include disrupted apical - basal polarity, reduced trans-epithelial resistance and phagocytic capacity, and decreased cilia length and incidence. Disrupted cilia morphology also occurred in patient-derived photoreceptors, associated with progressive degeneration and cellular stress. In situ gene editing of a pathogenic mutation rescued protein expression and key cellular phenotypes in RPE and photoreceptors, providing proof of concept for future therapeutic strategies.
Assuntos
Proteínas do Olho/metabolismo , Retinose Pigmentar/etiologia , Retinose Pigmentar/metabolismo , Processamento Alternativo/genética , Processamento Alternativo/fisiologia , Animais , Adesão Celular/genética , Adesão Celular/fisiologia , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Cílios/genética , Cílios/metabolismo , Cílios/fisiologia , Proteínas do Olho/genética , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Mutação/genética , Organoides/citologia , Organoides/metabolismo , Splicing de RNA/genética , Splicing de RNA/fisiologia , Retina/citologia , Retina/metabolismo , Retinose Pigmentar/genéticaRESUMO
SF3B is a multi-protein complex essential for branch site (BS) recognition and selection during pre-mRNA splicing. Several splicing modulators with antitumor activity bind SF3B and thereby modulate splicing. Here we report the crystal structure of a human SF3B core in complex with pladienolide B (PB), a macrocyclic splicing modulator and potent inhibitor of tumor cell proliferation. PB stalls SF3B in an open conformation by acting like a wedge within a hinge, modulating SF3B's transition to the closed conformation needed to form the BS adenosine-binding pocket and stably accommodate the BS/U2 duplex. This work explains the structural basis for the splicing modulation activity of PB and related compounds, and reveals key interactions between SF3B and a common pharmacophore, providing a framework for future structure-based drug design.