RESUMO
The spliceosome executes pre-mRNA splicing through four sequential stages: assembly, activation, catalysis, and disassembly. Activation of the spliceosome, namely remodeling of the pre-catalytic spliceosome (B complex) into the activated spliceosome (Bact complex) and the catalytically activated spliceosome (B* complex), involves major flux of protein components and structural rearrangements. Relying on a splicing inhibitor, we have captured six intermediate states between the B and B* complexes: pre-Bact, Bact-I, Bact-II, Bact-III, Bact-IV, and post-Bact. Their cryo-EM structures, together with an improved structure of the catalytic step I spliceosome (C complex), reveal how the catalytic center matures around the internal stem loop of U6 snRNA, how the branch site approaches 5'-splice site, how the RNA helicase PRP2 rearranges to bind pre-mRNA, and how U2 snRNP undergoes remarkable movement to facilitate activation. We identify a previously unrecognized key role of PRP2 in spliceosome activation. Our study recapitulates a molecular choreography of the human spliceosome during its catalytic activation.
Assuntos
Microscopia Crioeletrônica , Precursores de RNA , Splicing de RNA , RNA Nuclear Pequeno , Spliceossomos , Spliceossomos/metabolismo , Humanos , Precursores de RNA/metabolismo , Precursores de RNA/genética , RNA Nuclear Pequeno/metabolismo , Ribonucleoproteína Nuclear Pequena U2/metabolismo , Ribonucleoproteína Nuclear Pequena U2/genética , Modelos Moleculares , RNA Helicases DEAD-box/metabolismo , RNA Helicases DEAD-box/genética , Domínio CatalíticoRESUMO
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.
Assuntos
Ribonucleoproteína Nuclear Pequena U2 , Spliceossomos , Animais , Spliceossomos/genética , Spliceossomos/metabolismo , Ribonucleoproteína Nuclear Pequena U2/genética , Ribonucleoproteína Nuclear Pequena U2/metabolismo , Íntrons/genética , Cromatina/genética , Cromatina/metabolismo , Splicing de RNA , Precursores de RNA/metabolismo , Mamíferos/metabolismoRESUMO
The revised 4th edition of the World Health Organization (WHO4R) classification lists myelodysplastic syndromes with ring sideroblasts (MDS-RS) as a separate entity with single lineage (MDS-RS-SLD) or multilineage (MDS-RS-MLD) dysplasia. The more recent International Consensus Classification (ICC) distinguishes between MDS with SF3B1 mutation (MDS-SF3B1) and MDS-RS without SF3B1 mutation; the latter is instead included under the category of MDS not otherwise specified. The current study includes 170 Mayo Clinic patients with WHO4R-defined MDS-RS, including MDS-RS-SLD (N=83) and MDS-RSMLD (N=87); a subset of 145 patients were also evaluable for the presence of SF3B1 and other mutations, including 126 with (87%) and 19 (13%) without SF3B1 mutation. Median overall survival for all 170 patients was 6.6 years with 5- and 10-year survival rates of 59% and 25%, respectively. A significant difference in overall survival was apparent between MDS-RS-MLD and MDS-RS-SLD (P<0.01) but not between MDS-RS with and without SF3B1 mutation (P=0.36). Multivariable analysis confirmed the independent prognostic contribution of MLD (hazard ratio=1.8, 95% confidence interval: 1.1-2.8; P=0.01) and also identified age (P<0.01), transfusion need at diagnosis (P<0.01), and abnormal karyotype (P<0.01), as additional risk factors; the impact from SF3B1 or other mutations was not significant. Leukemia-free survival was independently affected by abnormal karyotype (P<0.01), RUNX1 (P=0.02) and IDH1 (P=0.01) mutations, but not by MLD or SF3B1 mutation. Exclusion of patients not meeting ICC-criteria for MDS-SF3B1 did not change the observations on overall survival. MLD-based, as opposed to SF3B1 mutation-based, disease classification for MDS-RS might be prognostically more relevant.
Assuntos
Anemia Sideroblástica , Mutação , Síndromes Mielodisplásicas , Fosfoproteínas , Fatores de Processamento de RNA , Humanos , Fatores de Processamento de RNA/genética , Masculino , Feminino , Idoso , Pessoa de Meia-Idade , Prognóstico , Idoso de 80 Anos ou mais , Adulto , Fosfoproteínas/genética , Anemia Sideroblástica/genética , Anemia Sideroblástica/diagnóstico , Anemia Sideroblástica/mortalidade , Anemia Sideroblástica/patologia , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/mortalidade , Síndromes Mielodisplásicas/diagnóstico , Síndromes Mielodisplásicas/patologia , Ribonucleoproteína Nuclear Pequena U2/genética , Linhagem da Célula , Adulto JovemRESUMO
Japanese encephalitis virus (JEV) is a zoonotic pathogen belonging to the Flavivirus genus, causing viral encephalitis in humans and reproductive failure in swine. The 3' untranslated region (3'UTR) of JEV contains highly conservative secondary structures required for viral translation, RNA synthesis, and pathogenicity. Identification of host factors interacting with JEV 3'UTR is crucial for elucidating the underlying mechanism of flavivirus replication and pathogenesis. In this study, U2 snRNP auxiliary factor 2 (U2AF2) was identified as a novel cellular protein that interacts with the JEV genomic 3'UTR (the SL-I, SL-II, SL-III, and DB region) via its 1 to 148 amino acids. JEV infection or JEV 3' UTR on its own triggered the nuclear-localized U2AF2 redistributed to the cytoplasm and colocalized with viral replication complex. U2AF2 also interacts with JEV NS3 and NS5 protein, the downregulation of U2AF2 nearly abolished the formation of flavivirus replication vesicles. The production of JEV protein, RNA, and viral titers were all increased by U2AF2 overexpression and decreased by knockdown. U2AF2 also functioned as a pro-viral factor for Zika virus (ZIKV) and West Nile virus (WNV), but not for vesicular stomatitis virus (VSV). Mechanically, U2AF2 facilitated the synthesis of both positive- and negative-strand flavivirus RNA without affecting viral attachment, internalization or release process. Collectively, our work paves the way for developing U2AF2 as a potential flavivirus therapeutic target.
Assuntos
Vírus da Encefalite Japonesa (Espécie) , Flavivirus , Doenças dos Suínos , Infecção por Zika virus , Zika virus , Humanos , Animais , Suínos , Flavivirus/genética , Regiões 3' não Traduzidas , Ribonucleoproteína Nuclear Pequena U2/genética , Infecção por Zika virus/genética , Infecção por Zika virus/veterinária , Replicação Viral/genética , Linhagem Celular , Zika virus/genética , Zika virus/metabolismo , Vírus da Encefalite Japonesa (Espécie)/genética , RNA Viral/genética , RNA Viral/metabolismo , Fator de Processamento U2AF/genética , Doenças dos Suínos/genéticaRESUMO
Selection of the pre-mRNA branch site (BS) by the U2 small nuclear ribonucleoprotein (snRNP) is crucial to prespliceosome (A complex) assembly. The RNA helicase PRP5 proofreads BS selection but the underlying mechanism remains unclear. Here we report the atomic structures of two sequential complexes leading to prespliceosome assembly: human 17S U2 snRNP and a cross-exon pre-A complex. PRP5 is anchored on 17S U2 snRNP mainly through occupation of the RNA path of SF3B1 by an acidic loop of PRP5; the helicase domain of PRP5 associates with U2 snRNA; the BS-interacting stem-loop (BSL) of U2 snRNA is shielded by TAT-SF1, unable to engage the BS. In the pre-A complex, an initial U2-BS duplex is formed; the translocated helicase domain of PRP5 stays with U2 snRNA and the acidic loop still occupies the RNA path. The pre-A conformation is specifically stabilized by the splicing factors SF1, DNAJC8 and SF3A2. Cancer-derived mutations in SF3B1 damage its association with PRP5, compromising BS proofreading. Together, these findings reveal key insights into prespliceosome assembly and BS selection or proofreading by PRP5.
Assuntos
Modelos Moleculares , Fatores de Processamento de RNA , Spliceossomos , Humanos , Spliceossomos/metabolismo , Spliceossomos/química , Fatores de Processamento de RNA/metabolismo , Fatores de Processamento de RNA/química , Ribonucleoproteína Nuclear Pequena U2/metabolismo , Ribonucleoproteína Nuclear Pequena U2/química , Ribonucleoproteína Nuclear Pequena U2/genética , Microscopia Crioeletrônica , Splicing de RNA , Precursores de RNA/metabolismo , Conformação de Ácido Nucleico , RNA Nuclear Pequeno/metabolismo , RNA Nuclear Pequeno/química , FosfoproteínasRESUMO
Intron branchpoint (BP) recognition by the U2 snRNP is a critical step of splicing, vulnerable to recurrent cancer mutations and bacterial natural product inhibitors. The BP binds a conserved pocket in the SF3B1 (human) or Hsh155 (yeast) U2 snRNP protein. Amino acids that line this pocket affect the binding of splicing inhibitors like Pladienolide-B (Plad-B), such that organisms differ in their sensitivity. To study the mechanism of splicing inhibitor action in a simplified system, we modified the naturally Plad-B resistant yeast Saccharomyces cerevisiae by changing 14 amino acids in the Hsh155 BP pocket to those from human. This humanized yeast grows normally, and splicing is largely unaffected by the mutation. Splicing is inhibited within minutes after the addition of Plad-B, and different introns appear inhibited to different extents. Intron-specific inhibition differences are also observed during cotranscriptional splicing in Plad-B using single-molecule intron tracking to minimize gene-specific transcription and decay rates that cloud estimates of inhibition by standard RNA-seq. Comparison of Plad-B intron sensitivities to those of the structurally distinct inhibitor Thailanstatin-A reveals intron-specific differences in sensitivity to different compounds. This work exposes a complex relationship between the binding of different members of this class of inhibitors to the spliceosome and intron-specific rates of BP recognition and catalysis. Introns with variant BP sequences seem particularly sensitive, echoing observations from mammalian cells, where monitoring individual introns is complicated by multi-intron gene architecture and alternative splicing. The compact yeast system may hasten the characterization of splicing inhibitors, accelerating improvements in selectivity and therapeutic efficacy.
Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Humanos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Íntrons/genética , Ribonucleoproteína Nuclear Pequena U2/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Splicing de RNA , Spliceossomos/genética , Aminoácidos/genética , Precursores de RNA/genéticaRESUMO
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.
Assuntos
Splicing de RNA , Spliceossomos , Humanos , Íntrons/genética , Fator de Processamento U2AF/genética , Fator de Processamento U2AF/metabolismo , Splicing de RNA/genética , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/metabolismo , Spliceossomos/metabolismo , Ribonucleoproteína Nuclear Pequena U2/genética , Fatores de Transcrição/metabolismo , Precursores de RNA/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismoRESUMO
The conserved SR-like protein Npl3 promotes splicing of diverse pre-mRNAs. However, the RNA sequence(s) recognized by the RNA Recognition Motifs (RRM1 & RRM2) of Npl3 during the splicing reaction remain elusive. Here, we developed a split-iCRAC approach in yeast to uncover the consensus sequence bound to each RRM. High-resolution NMR structures show that RRM2 recognizes a 5´-GNGG-3´ motif leading to an unusual mille-feuille topology. These structures also reveal how RRM1 preferentially interacts with a CC-dinucleotide upstream of this motif, and how the inter-RRM linker and the region C-terminal to RRM2 contribute to cooperative RNA-binding. Structure-guided functional studies show that Npl3 genetically interacts with U2 snRNP specific factors and we provide evidence that Npl3 melts U2 snRNA stem-loop I, a prerequisite for U2/U6 duplex formation within the catalytic center of the Bact spliceosomal complex. Thus, our findings suggest an unanticipated RNA chaperoning role for Npl3 during spliceosome active site formation.
Assuntos
Splicing de RNA , RNA , Conformação de Ácido Nucleico , Ribonucleoproteína Nuclear Pequena U2/metabolismo , RNA/metabolismo , RNA Nuclear Pequeno/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Spliceossomos/metabolismoRESUMO
The pre-spliceosomal complex involves interactions between U1 and U2 snRNPs, where a ubiquitin-like domain (ULD) of SF3A1, a component of U2 snRNP, binds to the stem-loop 4 (SL4; the UUCG tetraloop) of U1 snRNA in U1 snRNP. Here, we reported the 1.80 Å crystal structure of human SF3A1 ULD (ULDSF3A1) complexed with SL4. The structural part of ULDSF3A1 (res. 704-785) adopts a typical ß-grasp fold with a topology of ß1-ß2-α1-310a-ß3-ß4-310b-ß5, closely resembling that of ubiquitin, except for the length and structure of the ß1/ß2 loop. A patch on the surface formed by three ULDSF3A1-specific residues, Lys756 (ß3), Phe763 (ß4) and Lys765 (following ß4), contacts the canonical UUCG tetraloop structure. In contrast, the directly following C-terminal tail composed of 786KERGGRKK793 was essentially stretched. The main or side chains of all the residues interacted with the major groove of the stem helix; the RGG residues adopted a peculiar conformation for RNA recognition. These findings were confirmed by mutational studies using bio-layer interferometry. Collectively, a unique combination of the ß-grasp fold and the C-terminal tail constituting ULDSF3A1 is required for the SL4-specific binding. This interaction mode also suggests that putative post-translational modifications, including ubiquitination in ULDSF3A1, directly inhibit SL4 binding.
Assuntos
Ribonucleoproteína Nuclear Pequena U2 , Ubiquitina , Humanos , Ribonucleoproteína Nuclear Pequena U2/genética , Ubiquitina/metabolismo , RNA Nuclear Pequeno/genética , RNA Nuclear Pequeno/metabolismo , RNA , Fatores de Processamento de RNARESUMO
The formation of mature mRNA requires cutting introns and splicing exons. The occurrence of splicing involves the participation of the spliceosome. Common spliceosomes mainly include five snRNPs: U1, U2, U4/U6, and U5. SF3a2, an essential component of spliceosome U2 snRNP, participates in splicing a series of genes. There is no definition of SF3a2 in plants. The paper elaborated on SF3a2s from a series of plants through protein sequence similarity. We constructed the evolutionary relationship of SF3a2s in plants. Moreover, we analyzed the similarities and differences in gene structure, protein structure, the cis-element of the promoter, and expression pattern; we predicted their interacting proteins and constructed their collinearity. We have preliminarily analyzed SF3a2s in plants and clarified the evolutionary relationship between different species; these studies can better serve for in-depth research on the members of the spliceosome in plants.
Assuntos
Ribonucleoproteínas Nucleares Pequenas , Spliceossomos , Spliceossomos/metabolismo , Filogenia , Ribonucleoproteínas Nucleares Pequenas/genética , Splicing de RNA/genética , Ribonucleoproteína Nuclear Pequena U2/química , Ribonucleoproteína Nuclear Pequena U2/genética , Ribonucleoproteína Nuclear Pequena U2/metabolismo , RNA Mensageiro/metabolismoRESUMO
Splicing factor 3B subunit 1 (SF3B1) is the largest component of SF3b protein complex which is involved in the pre-mRNA splicing mechanism. Somatic mutations of SF3B1 were shown to be associated with aberrant splicing, producing abnormal transcripts that drive cancer development and/or prognosis. In this study, we focus on the relationship between SF3B1 and four types of cancer, namely myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), and chronic lymphocytic leukemia (CLL) and breast cancer (BC). For this purpose, we identified from the Pubmed library only articles which mentioned SF3B1 in connection with the investigated types of cancer for the period 2007 to 2018 to reveal how the connection has developed over time. We left out all published articles which mentioned SF3B1 in other contexts. We retrieved the target articles and investigated the association between SF3B1 and the mentioned four types of cancer. For this we utilized some of the publicly available databases to retrieve gene/variant/disease information related to SF3B1. We used the outcome to derive and analyze a variety of complex networks that reflect the correlation between the considered diseases and variants associated with SF3B1. The results achieved based on the analyzed articles and reported in this article illustrated that SF3B1 is associated with hematologic malignancies, such as MDS, AML, and CLL more than BC. We found that different gene networks may be required for investigating the impact of mutant splicing factors on cancer development based on the target cancer type. Additionally, based on the literature analyzed in this study, we highlighted and summarized what other researchers have reported as the set of genes and cellular pathways that are affected by aberrant splicing in cancerous cells.
Assuntos
Neoplasias da Mama , Leucemia Linfocítica Crônica de Células B , Leucemia Mieloide Aguda , Síndromes Mielodisplásicas , Humanos , Feminino , Ribonucleoproteína Nuclear Pequena U2/genética , Leucemia Linfocítica Crônica de Células B/genética , Mutação , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/metabolismo , Splicing de RNA , Síndromes Mielodisplásicas/genética , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Fatores de Transcrição/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismoRESUMO
Three RNA helicases - DDX42, DDX46 and DHX15 - are found to be associated with human U2 snRNP, but their roles and mechanisms in U2 snRNP and spliceosome assembly are insufficiently understood. Here we report the cryo-electron microscopy (cryo-EM) structures of the DDX42-SF3b complex and a putative assembly precursor of 17S U2 snRNP that contains DDX42 (DDX42-U2 complex). DDX42 is anchored on SF3B1 through N-terminal sequences, with its N-plug occupying the RNA path of SF3B1. The binding mode of DDX42 to SF3B1 is in striking analogy to that of DDX46. In the DDX42-U2 complex, the N-terminus of DDX42 remains anchored on SF3B1, but the helicase domain has been displaced by U2 snRNA and TAT-SF1. Through in vitro assays, we show DDX42 and DDX46 are mutually exclusive in terms of binding to SF3b. Cancer-driving mutations of SF3B1 target the residues in the RNA path that directly interact with DDX42 and DDX46. These findings reveal the distinct roles of DDX42 and DDX46 in assembly of 17S U2 snRNP and provide insights into the mechanisms of SF3B1 cancer mutations.
Assuntos
Neoplasias , Spliceossomos , Humanos , Spliceossomos/metabolismo , Ribonucleoproteína Nuclear Pequena U2/metabolismo , RNA Helicases/genética , RNA Helicases/metabolismo , Microscopia Crioeletrônica , Ligação Proteica , RNA Nuclear Pequeno/metabolismo , Neoplasias/metabolismo , Splicing de RNA , Precursores de RNA/metabolismo , Fatores de Processamento de RNA/metabolismo , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismoRESUMO
SART3 is a multifunctional protein that acts in several steps of gene expression, including assembly and recycling of the spliceosomal U4/U6 small nuclear ribonucleoprotein particle (snRNP). In this work, we provide evidence that SART3 associates via its N-terminal HAT domain with the 12S U2 snRNP. Further analysis showed that SART3 associates with the post-splicing complex containing U2 and U5 snRNP components. In addition, we observed an interaction between SART3 and the RNA helicase DHX15, which disassembles post-splicing complexes. Based on our data, we propose a model that SART3 associates via its N-terminal HAT domain with the post-splicing complex, where it interacts with U6 snRNA to protect it and to initiate U6 snRNA recycling before a next round of splicing.
Assuntos
Splicing de RNA , Spliceossomos , Splicing de RNA/genética , Spliceossomos/genética , Spliceossomos/metabolismo , RNA Nuclear Pequeno/genética , RNA Nuclear Pequeno/metabolismo , Ribonucleoproteína Nuclear Pequena U4-U6/genética , Ribonucleoproteína Nuclear Pequena U4-U6/metabolismo , Ribonucleoproteína Nuclear Pequena U5/genética , Ribonucleoproteína Nuclear Pequena U5/metabolismo , Ribonucleoproteína Nuclear Pequena U2/genética , Ribonucleoproteína Nuclear Pequena U2/metabolismo , Ribonucleoproteínas Nucleares Pequenas/genética , Ribonucleoproteínas Nucleares Pequenas/metabolismoRESUMO
DDX46, a member of DEAD-box (DDX) proteins, is associated with various cancers, while its involvement in the pathogenesis of breast cancer hasn't been reported so far. The study demonstrated the overexpression of DDX46 in human breast cancer cells and tissue samples, and correlated with high histological grade and lymph node metastasis. Downregulation of DDX46 in the breast cancer cell lines inhibited their proliferation and invasiveness in vitro. Furthermore, the growth of MDA-MB-231 xenografts was suppressed in nude mice by DDX46 knockingdown. Taken together, our findings suggest that DDX46 is an oncogenic factor in human breast cancer, and a potential therapeutic target.
Assuntos
Neoplasias da Mama , Proliferação de Células , RNA Helicases DEAD-box , Animais , Feminino , Humanos , Camundongos , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Regulação Neoplásica da Expressão Gênica , Células MCF-7 , Camundongos Nus , Invasividade Neoplásica/genética , Ribonucleoproteína Nuclear Pequena U2/genética , Ribonucleoproteína Nuclear Pequena U2/metabolismoRESUMO
BACKGROUND: Alternative splicing (AS) is an important channel for gene expression regulation and protein diversification, in addition to a major reason for the considerable differences in the number of genes and proteins in eukaryotes. In plants, U2 small nuclear ribonucleoprotein Bâ³ (U2Bâ³), a component of splicing complex U2 snRNP, plays an important role in AS. Currently, few studies have investigated plant U2Bâ³, and its mechanism remains unclear. RESULT: Phylogenetic analysis, including gene and protein structures, revealed that U2Bâ³ is highly conserved in plants and typically contains two RNA recognition motifs. Subcellular localisation showed that OsU2Bâ³ is located in the nucleus and cytoplasm, indicating that it has broad functions throughout the cell. Elemental analysis of the promoter region showed that it responded to numerous external stimuli, including hormones, stress, and light. Subsequent qPCR experiments examining response to stress (cold, salt, drought, and heavy metal cadmium) corroborated the findings. The prediction results of protein-protein interactions showed that its function is largely through a single pathway, mainly through interaction with snRNP proteins. CONCLUSION: U2Bâ³ is highly conserved in the plant kingdom, functions in the nucleus and cytoplasm, and participates in a wide range of processes in plant growth and development.
Assuntos
Ribonucleoproteína Nuclear Pequena U2 , Spliceossomos , Proteínas Centrais de snRNP/genética , Ribonucleoproteína Nuclear Pequena U2/química , Ribonucleoproteína Nuclear Pequena U2/genética , Ribonucleoproteína Nuclear Pequena U2/metabolismo , Filogenia , Sequência de Aminoácidos , RNA Nuclear Pequeno/genética , Splicing de RNARESUMO
SURP domains are exclusively found in splicing-related proteins in all eukaryotes. SF3A1, a component of the U2 snRNP, has two tandem SURP domains, SURP1, and SURP2. SURP2 is permanently associated with a specific short region of SF3A3 within the SF3A protein complex whereas, SURP1 binds to the splicing factor SF1 for recruitment of U2 snRNP to the early spliceosomal complex, from which SF1 is dissociated during complex conversion. Here, we determined the solution structure of the complex of SURP1 and the human SF1 fragment using nuclear magnetic resonance (NMR) methods. SURP1 adopts the canonical topology of α1-α2-310 -α3, in which α1 and α2 are connected by a single glycine residue in a particular backbone conformation, allowing the two α-helices to be fixed at an acute angle. A hydrophobic patch, which is part of the characteristic surface formed by α1 and α2, specifically contacts a hydrophobic cluster on a 16-residue α-helix of the SF1 fragment. Furthermore, whereas only hydrophobic interactions occurred between SURP2 and the SF3A3 fragment, several salt bridges and hydrogen bonds were found between the residues of SURP1 and the SF1 fragment. This finding was confirmed through mutational studies using bio-layer interferometry. The study also revealed that the dissociation constant between SURP1 and the SF1 fragment peptide was approximately 20 µM, indicating a weak or transient interaction. Collectively, these results indicate that the interplay between U2 snRNP and SF1 involves a transient interaction of SURP1, and this transient interaction appears to be common to most SURP domains, except for SURP2.
Assuntos
Fatores de Processamento de RNA , Ribonucleoproteína Nuclear Pequena U2 , Spliceossomos , Glicina , Humanos , Ligação Proteica , Splicing de RNA , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/metabolismo , Ribonucleoproteína Nuclear Pequena U2/genética , Ribonucleoproteína Nuclear Pequena U2/metabolismo , Spliceossomos/genética , Spliceossomos/metabolismoRESUMO
Intron diversity facilitates regulated gene expression and alternative splicing. Spliceosomes excise introns after recognizing their splicing signals: the 5'-splice site (5'ss), branchpoint (BP) and 3'-splice site (3'ss). The latter two signals are recognized by U2 small nuclear ribonucleoprotein (snRNP) and its accessory factors (U2AFs), but longer spacings between them result in weaker splicing. Here, we show that excision of introns with a BP-distant 3'ss (e.g. rap1 intron 2) requires the ubiquitin-fold-activated splicing regulator Sde2 in Schizosaccharomyces pombe. By monitoring splicing-specific ura4 reporters in a collection of S. pombe mutants, Cay1 and Tls1 were identified as additional regulators of this process. The role of Sde2, Cay1 and Tls1 was further confirmed by increasing BP-3'ss spacings in a canonical tho5 intron. We also examined BP-distant exons spliced independently of these factors and observed that RNA secondary structures possibly bridged the gap between the two signals. These proteins may guide the 3'ss towards the spliceosome's catalytic centre by folding the RNA between the BP and 3'ss. Orthologues of Sde2, Cay1 and Tls1, although missing in the intron-poor Saccharomyces cerevisiae, are present in intron-rich eukaryotes, including humans. This type of intron-specific pre-mRNA splicing appears to have evolved for regulated gene expression and alternative splicing of key heterochromatin factors.
Assuntos
Precursores de RNA , Schizosaccharomyces , Processamento Alternativo , Proteínas de Transporte , Proteínas de Ligação a DNA/genética , Éxons , Heterocromatina , Humanos , Íntrons/genética , Proteínas Nucleares/metabolismo , Precursores de RNA/metabolismo , Sítios de Splice de RNA , Splicing de RNA , Ribonucleoproteína Nuclear Pequena U2/genética , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe , Complexo Shelterina , Proteínas de Ligação a Telômeros , Ubiquitina/genética , Ubiquitina/metabolismoRESUMO
RNA splicing, the process of intron removal from pre-mRNA, is essential for the regulation of gene expression. It is controlled by the spliceosome, a megadalton RNA-protein complex that assembles de novo on each pre-mRNA intron through an ordered assembly of intermediate complexes1,2. Spliceosome activation is a major control step that requires substantial protein and RNA rearrangements leading to a catalytically active complex1-5. Splicing factor 3B subunit 1 (SF3B1) protein-a subunit of the U2 small nuclear ribonucleoprotein6-is phosphorylated during spliceosome activation7-10, but the kinase that is responsible has not been identified. Here we show that cyclin-dependent kinase 11 (CDK11) associates with SF3B1 and phosphorylates threonine residues at its N terminus during spliceosome activation. The phosphorylation is important for the association between SF3B1 and U5 and U6 snRNAs in the activated spliceosome, termed the Bact complex, and the phosphorylation can be blocked by OTS964, a potent and selective inhibitor of CDK11. Inhibition of CDK11 prevents spliceosomal transition from the precatalytic complex B to the activated complex Bact and leads to widespread intron retention and accumulation of non-functional spliceosomes on pre-mRNAs and chromatin. We demonstrate a central role of CDK11 in spliceosome assembly and splicing regulation and characterize OTS964 as a highly selective CDK11 inhibitor that suppresses spliceosome activation and splicing.
Assuntos
Quinases Ciclina-Dependentes , Fosfoproteínas , Precursores de RNA , Splicing de RNA , Ribonucleoproteína Nuclear Pequena U2 , Spliceossomos , Cromatina/metabolismo , Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/metabolismo , Ativação Enzimática/efeitos dos fármacos , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Fosforilação , Quinolonas/farmacologia , Precursores de RNA/genética , Precursores de RNA/metabolismo , Splicing de RNA/efeitos dos fármacos , Ribonucleoproteína Nuclear Pequena U2/química , Ribonucleoproteína Nuclear Pequena U2/metabolismo , Spliceossomos/efeitos dos fármacos , Spliceossomos/metabolismo , Treonina/metabolismoRESUMO
DExH/D-box helicases are essential nucleic acid and ribonucleoprotein remodelers involved in all aspects of nucleic acid metabolism including replication, gene expression and post-transcriptional modifications. In parallel to their importance in basic cellular functions, DExH/D-box helicases play multiple roles in viral life cycles, with some of them highjacked by viruses or negatively regulating innate immune activation. However, other DExH/D-box helicases have recurrently been highlighted as direct antiviral effectors or as positive regulators of innate immune activation. Innate immunity relies on the ability of Pathogen Recognition Receptors to recognize viral signatures and trigger the production of interferons (IFNs) and pro-inflammatory cytokines. Secreted IFNs interact with their receptors to establish antiviral cellular reprogramming via expression regulation of the interferon-stimulated genes (ISGs). Several DExH/D-box helicases have been reported to act as viral sensors (DDX3, DDX41, DHX9, DDX1/DDX21/DHX36 complex), and others to play roles in innate immune activation (DDX60, DDX60L, DDX23). In contrast, the DDX39A, DDX46, DDX5 and DDX24 helicases act as negative regulators and impede IFN production upon viral infection. Beyond their role in viral sensing, the ISGs DDX60 and DDX60L act as viral inhibitors. Interestingly, the constitutively expressed DEAD-box helicases DDX56, DDX17, DDX42 intrinsically restrict viral replication. Hence, DExH/D-box helicases appear to form a multilayer network of primary and secondary factors involved in both intrinsic and innate antiviral immunity. In this review, we highlight recent findings on the extent of antiviral defences played by helicases and emphasize the need to better understand their immune functions as well as their complex interplay.