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1.
Nat Commun ; 12(1): 4910, 2021 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-34389706

RESUMO

Human pre-mRNA introns vary in size from under fifty to over a million nucleotides. We searched for essential factors involved in the splicing of human short introns by screening siRNAs against 154 human nuclear proteins. The splicing activity was assayed with a model HNRNPH1 pre-mRNA containing short 56-nucleotide intron. We identify a known alternative splicing regulator SPF45 (RBM17) as a constitutive splicing factor that is required to splice out this 56-nt intron. Whole-transcriptome sequencing of SPF45-deficient cells reveals that SPF45 is essential in the efficient splicing of many short introns. To initiate the spliceosome assembly on a short intron with the truncated poly-pyrimidine tract, the U2AF-homology motif (UHM) of SPF45 competes out that of U2AF65 (U2AF2) for binding to the UHM-ligand motif (ULM) of the U2 snRNP protein SF3b155 (SF3B1). We propose that splicing in a distinct subset of human short introns depends on SPF45 but not U2AF heterodimer.


Assuntos
Íntrons/genética , Fatores de Processamento de RNA/metabolismo , Splicing de RNA , Fator de Processamento U2AF/metabolismo , Sequência de Bases , Sítios de Ligação/genética , Humanos , Modelos Genéticos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Ligação Proteica , Precursores de RNA/genética , Precursores de RNA/metabolismo , Fatores de Processamento de RNA/genética , Ribonucleoproteína Nuclear Pequena U2/genética , Ribonucleoproteína Nuclear Pequena U2/metabolismo , Spliceossomos/genética , Spliceossomos/metabolismo , Fator de Processamento U2AF/genética
2.
Nature ; 596(7871): 296-300, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34349264

RESUMO

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/metabolismo
3.
Int J Mol Sci ; 22(13)2021 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-34209806

RESUMO

Pre-mRNA splicing is a key process in the regulation of gene expression. In the fission yeast Schizosaccharomyces pombe, Nrl1 regulates splicing and expression of several genes and non-coding RNAs, and also suppresses the accumulation of R-loops. Here, we report analysis of interactions between Nrl1 and selected RNA-processing proteins and regulation of Nrl1 function by phosphorylation. Bacterial two-hybrid system (BACTH) assays revealed that the N-terminal region of Nrl1 is important for the interaction with ATP-dependent RNA helicase Mtl1 while the C-terminal region of Nrl1 is important for interactions with spliceosome components Ctr1, Ntr2, and Syf3. Consistent with this result, tandem affinity purification showed that Mtl1, but not Ctr1, Ntr2, or Syf3, co-purifies with the N-terminal region of Nrl1. Interestingly, mass-spectrometry analysis revealed that in addition to previously identified phosphorylation sites, Nrl1 is also phosphorylated on serines 86 and 112, and that Nrl1-TAP co-purifies with Cka1, the catalytic subunit of casein kinase 2. In vitro assay showed that Cka1 can phosphorylate bacterially expressed Nrl1 fragments. An analysis of non-phosphorylatable nrl1 mutants revealed defects in gene expression and splicing consistent with the notion that phosphorylation is an important regulator of Nrl1 function. Taken together, our results provide insights into two mechanisms that are involved in the regulation of the spliceosome-associated factor Nrl1, namely domain-specific interactions between Nrl1 and RNA-processing proteins and post-translational modification of Nrl1 by phosphorylation.


Assuntos
Proteínas de Ligação a RNA/metabolismo , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/metabolismo , Caseína Quinase II/metabolismo , Fosforilação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas , Processamento Pós-Transcricional do RNA , Splicing de RNA , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/fisiologia , Spliceossomos/metabolismo , Técnicas do Sistema de Duplo-Híbrido
4.
Int J Mol Sci ; 22(11)2021 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-34199764

RESUMO

Pre-mRNA splicing is an essential step in gene expression and is catalyzed by two machineries in eukaryotes: the major (U2 type) and minor (U12 type) spliceosomes. While the majority of introns in humans are U2 type, less than 0.4% are U12 type, also known as minor introns (mi-INTs), and require a specialized spliceosome composed of U11, U12, U4atac, U5, and U6atac snRNPs. The high evolutionary conservation and apparent splicing inefficiency of U12 introns have set them apart from their major counterparts and led to speculations on the purpose for their existence. However, recent studies challenged the simple concept of mi-INTs splicing inefficiency due to low abundance of their spliceosome and confirmed their regulatory role in alternative splicing, significantly impacting the expression of their host genes. Additionally, a growing list of minor spliceosome-associated diseases with tissue-specific pathologies affirmed the importance of minor splicing as a key regulatory pathway, which when deregulated could lead to tissue-specific pathologies due to specific alterations in the expression of some minor-intron-containing genes. Consequently, uncovering how mi-INTs splicing is regulated in a tissue-specific manner would allow for better understanding of disease pathogenesis and pave the way for novel therapies, which we highlight in this review.


Assuntos
Doença/genética , Íntrons/genética , Splicing de RNA/genética , Animais , Evolução Molecular , Humanos , Especificidade de Órgãos/genética , Spliceossomos/metabolismo
5.
RNA ; 27(10): 1186-1203, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34234030

RESUMO

Human pre-mRNA splicing is primarily catalyzed by the major spliceosome, comprising five small nuclear ribonucleoprotein complexes, U1, U2, U4, U5, and U6 snRNPs, each of which contains the corresponding U-rich snRNA. These snRNAs are encoded by large gene families exhibiting significant sequence variation, but it remains unknown if most human snRNA genes are untranscribed pseudogenes or produce variant snRNAs with the potential to differentially influence splicing. Since gene duplication and variation are powerful mechanisms of evolutionary adaptation, we sought to address this knowledge gap by systematically profiling human U1, U2, U4, and U5 snRNA variant gene transcripts. We identified 55 transcripts that are detectably expressed in human cells, 38 of which incorporate into snRNPs and spliceosomes in 293T cells. All U1 snRNA variants are more than 1000-fold less abundant in spliceosomes than the canonical U1, whereas at least 1% of spliceosomes contain a variant of U2 or U4. In contrast, eight U5 snRNA sequence variants occupy spliceosomes at levels of 1% to 46%. Furthermore, snRNA variants display distinct expression patterns across five human cell lines and adult and fetal tissues. Different RNA degradation rates contribute to the diverse steady state levels of snRNA variants. Our findings suggest that variant spliceosomes containing noncanonical snRNAs may contribute to different tissue- and cell-type-specific alternative splicing patterns.


Assuntos
Splicing de RNA , RNA Mensageiro/genética , RNA Nuclear Pequeno/genética , Spliceossomos/genética , Adulto , Pareamento de Bases , Sequência de Bases , Fracionamento Celular/métodos , Éxons , Feto , Células HEK293 , Humanos , Íntrons , Anotação de Sequência Molecular , Conformação de Ácido Nucleico , Especificidade de Órgãos , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/química , RNA Mensageiro/metabolismo , RNA Nuclear Pequeno/química , RNA Nuclear Pequeno/metabolismo , Spliceossomos/química , Spliceossomos/metabolismo
6.
Nat Commun ; 12(1): 4491, 2021 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-34301950

RESUMO

Intron selection during the formation of prespliceosomes is a critical event in pre-mRNA splicing. Chemical modulation of intron selection has emerged as a route for cancer therapy. Splicing modulators alter the splicing patterns in cells by binding to the U2 snRNP (small nuclear ribonucleoprotein)-a complex chaperoning the selection of branch and 3' splice sites. Here we report crystal structures of the SF3B module of the U2 snRNP in complex with spliceostatin and sudemycin FR901464 analogs, and the cryo-electron microscopy structure of a cross-exon prespliceosome-like complex arrested with spliceostatin A. The structures reveal how modulators inactivate the branch site in a sequence-dependent manner and stall an E-to-A prespliceosome intermediate by covalent coupling to a nucleophilic zinc finger belonging to the SF3B subunit PHF5A. These findings support a mechanism of intron recognition by the U2 snRNP as a toehold-mediated strand invasion and advance an unanticipated drug targeting concept.


Assuntos
DNA/genética , Íntrons/genética , Piranos/metabolismo , Ribonucleoproteína Nuclear Pequena U2/metabolismo , Compostos de Espiro/metabolismo , Spliceossomos/metabolismo , Microscopia Crioeletrônica , Cristalografia por Raios X , DNA/química , DNA/metabolismo , Humanos , Lactonas/química , Lactonas/metabolismo , Modelos Moleculares , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica , Piranos/química , Pironas/química , Pironas/metabolismo , Ribonucleoproteína Nuclear Pequena U2/química , Compostos de Espiro/química , Spliceossomos/ultraestrutura
7.
J Biol Chem ; 297(1): 100882, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34144037

RESUMO

Alteration of RNA splicing is a hallmark of cellular senescence, which is associated with age-related disease and cancer development. However, the roles of splicing factors in cellular senescence are not fully understood. In this study, we identified the splicing factor PRPF19 as a critical regulator of cellular senescence in normal human diploid fibroblasts. PRPF19 was downregulated during replicative senescence, and PRPF19 knockdown prematurely induced senescence-like cell cycle arrest through the p53-p21 pathway. RNA-sequencing analysis revealed that PRPF19 knockdown caused a switch of the MDM4 splicing isoform from stable full-length MDM4-FL to unstable MDM4-S lacking exon 6. We also found that PRPF19 regulates MDM4 splicing by promoting the physical interaction of other splicing factors, PRPF3 and PRPF8, which are key components of the core spliceosome, U4/U6.U5 tri-snRNP. Given that MDM4 is a major negative regulator of p53, our findings imply that PRPF19 downregulation inhibits MDM4-mediated p53 inactivation, resulting in induction of cellular senescence. Thus, PRPF19 plays an important role in the induction of p53-dependent cellular senescence.


Assuntos
Processamento Alternativo , Proteínas de Ciclo Celular/genética , Senescência Celular , Enzimas Reparadoras do DNA/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas/genética , Fatores de Processamento de RNA/metabolismo , Proteínas de Ciclo Celular/metabolismo , Enzimas Reparadoras do DNA/genética , Células HEK293 , Humanos , Proteínas Nucleares/genética , Ligação Proteica , Proteínas Proto-Oncogênicas/metabolismo , Fatores de Processamento de RNA/genética , Spliceossomos/metabolismo , Proteína Supressora de Tumor p53/metabolismo
8.
Nat Commun ; 12(1): 3646, 2021 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-34131137

RESUMO

U5 snRNP is a complex particle essential for RNA splicing. U5 snRNPs undergo intricate biogenesis that ensures that only a fully mature particle assembles into a splicing competent U4/U6•U5 tri-snRNP and enters the splicing reaction. During splicing, U5 snRNP is substantially rearranged and leaves as a U5/PRPF19 post-splicing particle, which requires re-generation before the next round of splicing. Here, we show that a previously uncharacterized protein TSSC4 is a component of U5 snRNP that promotes tri-snRNP formation. We provide evidence that TSSC4 associates with U5 snRNP chaperones, U5 snRNP and the U5/PRPF19 particle. Specifically, TSSC4 interacts with U5-specific proteins PRPF8, EFTUD2 and SNRNP200. We also identified TSSC4 domains critical for the interaction with U5 snRNP and the PRPF19 complex, as well as for TSSC4 function in tri-snRNP assembly. TSSC4 emerges as a specific chaperone that acts in U5 snRNP de novo biogenesis as well as post-splicing recycling.


Assuntos
Ribonucleoproteína Nuclear Pequena U5/química , Ribonucleoproteína Nuclear Pequena U5/metabolismo , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Spliceossomos/metabolismo , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Regulação para Baixo , Células HeLa , Humanos , Proteínas Nucleares/metabolismo , Fatores de Alongamento de Peptídeos , Domínios Proteicos , Domínios e Motivos de Interação entre Proteínas , Splicing de RNA , Fatores de Processamento de RNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes de Fusão , Ribonucleoproteínas Nucleares Pequenas/química , Fatores de Transcrição , Proteínas Supressoras de Tumor/genética
9.
Int J Mol Sci ; 22(11)2021 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-34073089

RESUMO

Sox proteins are known as crucial transcription factors for many developmental processes and for a wide range of common diseases. They were believed to specifically bind and bend DNA with other transcription factors and elicit transcriptional activation or repression activities in the early stage of transcription. However, their functions are not limited to transcription initiation. It has been showed that Sox proteins are involved in the regulation of alternative splicing regulatory networks and translational control. In this review, we discuss the current knowledge on how Sox transcription factors such as Sox2, Sry, Sox6, and Sox9 allow the coordination of co-transcriptional splicing and also the mechanism of SOX4-mediated translational control in the context of RNA polymerase III.


Assuntos
Fatores de Transcrição SOX , Processamento Alternativo , Animais , Humanos , Biossíntese de Proteínas , RNA Polimerase III , Splicing de RNA , Fatores de Transcrição SOX/genética , Fatores de Transcrição SOX/fisiologia , Spliceossomos/metabolismo , Ativação Transcricional
10.
Mol Cell ; 81(11): 2275-2277, 2021 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-34087179

RESUMO

Wan et al. (2021) establish a powerful new platform to measure the dynamics of transcription and splicing of endogenous genes in single cells in real time. Combining real-time measurements with multiple deep-sequencing tools reveals an unexpectedly high amount of spliceosome activity, prompting a reconsideration of current models of how introns are removed from pre-mRNA.


Assuntos
RNA , Spliceossomos , Íntrons/genética , RNA/metabolismo , Precursores de RNA/genética , Precursores de RNA/metabolismo , Splicing de RNA , Spliceossomos/genética , Spliceossomos/metabolismo
11.
Int J Mol Sci ; 22(10)2021 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-34065983

RESUMO

Dysregulation of messenger RNA (mRNA) processing-in particular mRNA splicing-is a hallmark of cancer. Compared to normal cells, cancer cells frequently present aberrant mRNA splicing, which promotes cancer progression and treatment resistance. This hallmark provides opportunities for developing new targeted cancer treatments. Splicing of precursor mRNA into mature mRNA is executed by a dynamic complex of proteins and small RNAs called the spliceosome. Spliceosomes are part of the supraspliceosome, a macromolecular structure where all co-transcriptional mRNA processing activities in the cell nucleus are coordinated. Here we review the biology of the mRNA splicing machinery in the context of other mRNA processing activities in the supraspliceosome and present current knowledge of its dysregulation in lung cancer. In addition, we review investigations to discover therapeutic targets in the spliceosome and give an overview of inhibitors and modulators of the mRNA splicing process identified so far. Together, this provides insight into the value of targeting the spliceosome as a possible new treatment for lung cancer.


Assuntos
Neoplasias Pulmonares/genética , Splicing de RNA , Spliceossomos/metabolismo , Processamento Alternativo , Núcleo Celular/metabolismo , Progressão da Doença , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/metabolismo
12.
Commun Biol ; 4(1): 529, 2021 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-33953336

RESUMO

The SF3B complex, a multiprotein component of the U2 snRNP of the spliceosome, plays a crucial role in recognizing branch point sequence and facilitates spliceosome assembly and activation. Several chemicals that bind SF3B1 and PHF5A subunits of the SF3B complex inhibit splicing. We recently generated a splicing inhibitor-resistant SF3B1 mutant named SF3B1 GEX1A RESISTANT 4 (SGR4) using CRISPR-mediated directed evolution, whereas splicing inhibitor-resistant mutant of PHF5A (Overexpression-PHF5A GEX1A Resistance, OGR) was generated by expressing an engineered version PHF5A-Y36C. Global analysis of splicing in wild type and these two mutants revealed the role of SF3B1 and PHF5A in splicing regulation. This analysis uncovered a set of genes whose intron retention is regulated by both proteins. Further analysis of these retained introns revealed that they are shorter, have a higher GC content, and contain shorter and weaker polypyrimidine tracts. Furthermore, splicing inhibition increased seedlings sensitivity to salt stress, consistent with emerging roles of splicing regulation in stress responses. In summary, we uncovered the functions of two members of the plant branch point recognition complex. The novel strategies described here should be broadly applicable in elucidating functions of splicing regulators, especially in studying the functions of redundant paralogs in plants.


Assuntos
Regulação da Expressão Gênica de Plantas , Oryza/metabolismo , Proteínas de Plantas/metabolismo , Fatores de Processamento de RNA/metabolismo , Splicing de RNA , Proteínas de Ligação a RNA/metabolismo , Spliceossomos/metabolismo , Oryza/genética , Oryza/crescimento & desenvolvimento , Proteínas de Plantas/genética , Fatores de Processamento de RNA/genética , Proteínas de Ligação a RNA/genética , Spliceossomos/genética
13.
Nat Commun ; 12(1): 3082, 2021 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-34035302

RESUMO

Splicing, a key step in the eukaryotic gene-expression pathway, converts precursor messenger RNA (pre-mRNA) into mRNA by excising introns and ligating exons. This task is accomplished by the spliceosome, a macromolecular machine that must undergo sequential conformational changes to establish its active site. Each of these major changes requires a dedicated DExD/H-box ATPase, but how these enzymes are activated remain obscure. Here we show that Prp28, a yeast DEAD-box ATPase, transiently interacts with the conserved 5' splice-site (5'SS) GU dinucleotide and makes splicing-dependent contacts with the U1 snRNP protein U1C, and U4/U6.U5 tri-snRNP proteins, Prp8, Brr2, and Snu114. We further show that Prp28's ATPase activity is potentiated by the phosphorylated Npl3, but not the unphosphorylated Npl3, thus suggesting a strategy for regulating DExD/H-box ATPases. We propose that Npl3 is a functional counterpart of the metazoan-specific Prp28 N-terminal region, which can be phosphorylated and serves as an anchor to human spliceosome.


Assuntos
RNA Helicases DEAD-box/metabolismo , Proteínas Nucleares/metabolismo , Splicing de RNA , Proteínas de Ligação a RNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Spliceossomos/metabolismo , Trifosfato de Adenosina/metabolismo , RNA Helicases DEAD-box/genética , Humanos , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Mutação , Proteínas Nucleares/genética , Fosforilação , Ligação Proteica , RNA Helicases/genética , RNA Helicases/metabolismo , Precursores de RNA/genética , Precursores de RNA/metabolismo , Proteínas de Ligação a RNA/genética , Ribonuclease H/metabolismo , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Spliceossomos/genética
14.
Science ; 372(6543)2021 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-33986153

RESUMO

Aberrant alternative splicing is a hallmark of cancer, yet the underlying regulatory programs that control this process remain largely unknown. Here, we report a systematic effort to decipher the RNA structural code that shapes pathological splicing during breast cancer metastasis. We discovered a previously unknown structural splicing enhancer that is enriched near cassette exons with increased inclusion in highly metastatic cells. We show that the spliceosomal protein small nuclear ribonucleoprotein polypeptide A' (SNRPA1) interacts with these enhancers to promote cassette exon inclusion. This interaction enhances metastatic lung colonization and cancer cell invasion, in part through SNRPA1-mediated regulation of PLEC alternative splicing, which can be counteracted by splicing modulating morpholinos. Our findings establish a noncanonical regulatory role for SNRPA1 as a prometastatic splicing enhancer in breast cancer.


Assuntos
Processamento Alternativo , Neoplasias da Mama/patologia , Metástase Neoplásica/genética , RNA/genética , RNA/metabolismo , Ribonucleoproteína Nuclear Pequena U2/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Algoritmos , Animais , Sítios de Ligação , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Progressão da Doença , Éxons , Técnicas de Silenciamento de Genes , Humanos , Neoplasias Pulmonares/secundário , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Invasividade Neoplásica , Transplante de Neoplasias , Conformação de Ácido Nucleico , Plectina/genética , Ligação Proteica , Interferência de RNA , RNA Nuclear Pequeno/química , RNA Nuclear Pequeno/metabolismo , RNA-Seq , Ribonucleoproteína Nuclear Pequena U2/genética , Software , Spliceossomos/metabolismo , Proteínas Supressoras de Tumor/genética
15.
Neuro Oncol ; 23(10): 1693-1708, 2021 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-34042961

RESUMO

BACKGROUND: The glioblastoma (GBM) mesenchymal (MES) phenotype, induced by NF-κB activation, is characterized by aggressive tumor progression and poor clinical outcomes. Our previous analysis indicated that MES GBM has a unique alternative splicing (AS) pattern; however, the underlying mechanism remains obscure. We aimed to reveal how splicing regulation contributes to MES phenotype promotion in GBM. METHODS: We screened novel candidate splicing factors that participate in NF-κB activation and MES phenotype promotion in GBM. In vitro and in vivo assays were used to explore the function of RSRP1 in MES GBM. RESULTS: Here, we identified that arginine/serine-rich protein 1 (RSRP1) promotes the MES phenotype by facilitating GBM cell invasion and apoptosis resistance. Proteomic, transcriptomic, and functional analyses confirmed that RSRP1 regulates AS in MES GBM through mediating spliceosome assembly. One RSRP1-regulated AS event resulted in skipping PARP6 exon 18 to form truncated, oncogenic PARP6-s. This isoform was unable to effectively suppress NF-κB. Cotreatment of cultured GBM cells and GBM tumor-bearing mice with spliceosome and NF-κB inhibitors exerted a synergistic effect on MES GBM growth. CONCLUSION: We identified a novel mechanism through which RSRP1-dependent splicing promotes the GBM MES phenotype. Targeting AS via RSRP1-related spliceosomal factors might constitute a promising treatment for GBM.


Assuntos
Neoplasias Encefálicas , Glioblastoma , Proteínas de Neoplasias/genética , Animais , Neoplasias Encefálicas/genética , Linhagem Celular Tumoral , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Glioblastoma/genética , Camundongos , NF-kappa B/genética , NF-kappa B/metabolismo , Fenótipo , Proteômica , Spliceossomos/genética , Spliceossomos/metabolismo
16.
Nucleic Acids Res ; 49(10): 5845-5866, 2021 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-34023904

RESUMO

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/genética
17.
Nat Commun ; 12(1): 3153, 2021 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-34039990

RESUMO

RNA splicing, transcription and the DNA damage response are intriguingly linked in mammals but the underlying mechanisms remain poorly understood. Using an in vivo biotinylation tagging approach in mice, we show that the splicing factor XAB2 interacts with the core spliceosome and that it binds to spliceosomal U4 and U6 snRNAs and pre-mRNAs in developing livers. XAB2 depletion leads to aberrant intron retention, R-loop formation and DNA damage in cells. Studies in illudin S-treated cells and Csbm/m developing livers reveal that transcription-blocking DNA lesions trigger the release of XAB2 from all RNA targets tested. Immunoprecipitation studies reveal that XAB2 interacts with ERCC1-XPF and XPG endonucleases outside nucleotide excision repair and that the trimeric protein complex binds RNA:DNA hybrids under conditions that favor the formation of R-loops. Thus, XAB2 functionally links the spliceosomal response to DNA damage with R-loop processing with important ramifications for transcription-coupled DNA repair disorders.


Assuntos
Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Endonucleases/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Processamento de RNA/metabolismo , Fatores de Transcrição/metabolismo , Animais , Linhagem Celular , Dano ao DNA/efeitos dos fármacos , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Introdução de Genes , Técnicas de Silenciamento de Genes , Fígado/crescimento & desenvolvimento , Fígado/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Células-Tronco Embrionárias Murinas , Sesquiterpenos Policíclicos/farmacologia , Estruturas R-Loop/genética , Precursores de RNA/genética , Precursores de RNA/metabolismo , Fatores de Processamento de RNA/genética , RNA Nuclear Pequeno , RNA-Seq , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Spliceossomos/metabolismo , Transcrição Genética
18.
J Phys Chem B ; 125(15): 3815-3823, 2021 04 22.
Artigo em Inglês | MEDLINE | ID: mdl-33826329

RESUMO

While extensive studies have been carried out to determine protein-RNA binding affinities, mechanisms, and dynamics in vitro, such studies do not take into consideration the effect of the many weak nonspecific interactions in a cell filled with potential binding partners. Here we experimentally tested the role of the cellular environment on affinity and binding dynamics between a protein and RNA in living U-2 OS cells. Our model system is the spliceosomal protein U1A and its binding partner SL2 of the U1 snRNA. The binding equilibrium was perturbed by a laser-induced temperature jump and monitored by Förster resonance energy transfer. The apparent binding affinity in live cells was reduced by up to 2 orders of magnitude compared to in vitro. The measured in-cell dissociation rate coefficients were up to 2 orders of magnitude larger, whereas no change in the measured association rate coefficient was observed. The latter is not what would be anticipated due to macromolecular crowding or nonspecific sticking of the uncomplexed U1A and SL2 in the cell. A quantitative model fits our experimental results, with the major cellular effect being that U1A and SL2 sticking to cellular components are capable of binding, just not as strongly as the free complex. This observation suggests that high binding affinities measured or designed in vitro are necessary for proper binding in vivo, where competition with many nonspecific interactions exists, especially for strongly interacting species with high charge or large hydrophobic surface areas.


Assuntos
RNA Nuclear Pequeno , Ribonucleoproteína Nuclear Pequena U1 , Sítios de Ligação , Transtornos Dissociativos , Humanos , Ligação Proteica , RNA/metabolismo , RNA Nuclear Pequeno/metabolismo , Ribonucleoproteína Nuclear Pequena U1/metabolismo , Spliceossomos/metabolismo
19.
Molecules ; 26(8)2021 Apr 14.
Artigo em Inglês | MEDLINE | ID: mdl-33919699

RESUMO

RNA splicing is an essential step in producing mature messenger RNA (mRNA) and other RNA species. Harnessing RNA splicing modifiers as a new pharmacological modality is promising for the treatment of diseases caused by aberrant splicing. This drug modality can be used for infectious diseases by disrupting the splicing of essential pathogenic genes. Several antisense oligonucleotide splicing modifiers were approved by the U.S. Food and Drug Administration (FDA) for the treatment of spinal muscular atrophy (SMA) and Duchenne muscular dystrophy (DMD). Recently, a small-molecule splicing modifier, risdiplam, was also approved for the treatment of SMA, highlighting small molecules as important warheads in the arsenal for regulating RNA splicing. The cellular targets of these approved drugs are all mRNA precursors (pre-mRNAs) in human cells. The development of novel RNA-targeting splicing modifiers can not only expand the scope of drug targets to include many previously considered "undruggable" genes but also enrich the chemical-genetic toolbox for basic biomedical research. In this review, we summarized known splicing modifiers, screening methods for novel splicing modifiers, and the chemical space occupied by the small-molecule splicing modifiers.


Assuntos
Desenvolvimento de Medicamentos , Avaliação Pré-Clínica de Medicamentos , Splicing de RNA/genética , Animais , Sequência de Bases , Doença/genética , Humanos , Bibliotecas de Moléculas Pequenas/farmacologia , Spliceossomos/metabolismo
20.
Int J Mol Sci ; 22(8)2021 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-33920773

RESUMO

Rice false smut is a fungal disease distributed worldwide and caused by Ustilaginoidea virens. In this study, we identified a putative ester cyclase (named as UvEC1) as being significantly upregulated during U. virens infection. UvEC1 contained a SnoaL-like polyketide cyclase domain, but the functions of ketone cyclases such as SnoaL in plant fungal pathogens remain unclear. Deletion of UvEC1 caused defects in vegetative growth and conidiation. UvEC1 was also required for response to hyperosmotic and oxidative stresses and for maintenance of cell wall integrity. Importantly, ΔUvEC1 mutants exhibited reduced virulence. We performed a tandem mass tag (TMT)-based quantitative proteomic analysis to identify differentially accumulating proteins (DAPs) between the ΔUvEC1-1 mutant and the wild-type isolate HWD-2. Proteomics data revealed that UvEC1 has a variety of effects on metabolism, protein localization, catalytic activity, binding, toxin biosynthesis and the spliceosome. Taken together, our findings suggest that UvEC1 is critical for the development and virulence of U. virens.


Assuntos
Proteínas Fúngicas/metabolismo , Hypocreales/metabolismo , Hypocreales/patogenicidade , Isomerases/metabolismo , Oryza/microbiologia , Doenças das Plantas/microbiologia , Proteômica , Sequência de Aminoácidos , Proteínas Fúngicas/química , Deleção de Genes , Genoma Fúngico , Hypocreales/genética , Hypocreales/crescimento & desenvolvimento , Isomerases/química , Micotoxinas/genética , Micotoxinas/metabolismo , Proteoma/metabolismo , Spliceossomos/metabolismo , Esporos Fúngicos/metabolismo , Estresse Fisiológico , Frações Subcelulares/metabolismo
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