Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 4.340
Filtrar
1.
Nat Commun ; 10(1): 3639, 2019 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-31409787

RESUMO

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 , Processamento 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/genética
2.
Genes Dev ; 33(17-18): 1208-1220, 2019 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-31416967

RESUMO

The PIWI-interacting RNA (piRNA) pathway is a conserved small RNA-based immune system that protects animal germ cell genomes from the harmful effects of transposon mobilization. In Drosophila ovaries, most piRNAs originate from dual-strand clusters, which generate piRNAs from both genomic strands. Dual-strand clusters use noncanonical transcription mechanisms. Although transcribed by RNA polymerase II, cluster transcripts lack splicing signatures and poly(A) tails. mRNA processing is important for general mRNA export mediated by nuclear export factor 1 (Nxf1). Although UAP56, a component of the transcription and export complex, has been implicated in piRNA precursor export, it remains unknown how dual-strand cluster transcripts are specifically targeted for piRNA biogenesis by export from the nucleus to cytoplasmic processing centers. Here we report that dual-strand cluster transcript export requires CG13741/Bootlegger and the Drosophila nuclear export factor family protein Nxf3. Bootlegger is specifically recruited to piRNA clusters and in turn brings Nxf3. We found that Nxf3 specifically binds to piRNA precursors and is essential for their export to piRNA biogenesis sites, a process that is critical for germline transposon silencing. Our data shed light on how dual-strand clusters compensate for a lack of canonical features of mature mRNAs to be specifically exported via Nxf3, ensuring proper piRNA production.


Assuntos
Transporte Ativo do Núcleo Celular/genética , Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Precursores de RNA/metabolismo , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Elementos de DNA Transponíveis/genética , Drosophila/genética , Proteínas de Drosophila/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Transporte Nucleocitoplasmático/genética , Proteínas de Ligação a RNA/genética
3.
PLoS Pathog ; 15(6): e1007884, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31206552

RESUMO

In contrast to human cells, very few HSV-1 genes are known to be spliced, although the same pre-mRNA processing machinery is shared. Here, through global analysis of splice junctions in cells infected with HSV-1 and an HSV-1 mutant virus with deletion of infectious cell culture protein 27 (ICP27), one of two viral immediate early (IE) genes essential for viral replication, we identify hundreds of novel alternative splice junctions mapping to both previously known HSV-1 spliced genes and previously unknown spliced genes, the majority of which alter the coding potential of viral genes. Quantitative and qualitative splicing efficiency analysis of these novel alternatively spliced genes based on RNA-Seq and RT-PCR reveals that splicing at these novel splice sites is efficient only when ICP27 is absent; while in wildtype HSV-1 infected cells, the splicing of these novel splice junctions is largely silenced in a gene/sequence specific manner, suggesting that ICP27 not only promotes accumulation of ICP27 targeted transcripts but also ensures correctness of the functional coding sequences through inhibition of alternative splicing. Furthermore, ICP27 toggles expression of ICP34.5, the major viral neurovirulence factor, through inhibition of splicing and activation of a proximal polyadenylation signal (PAS) in the newly identified intron, revealing a novel regulatory mechanism for expression of a viral gene. Thus, through the viral IE protein ICP27, HSV-1 co-opts both splicing and polyadenylation machinery to achieve optimal viral gene expression during lytic infection. On the other hand, during latent infection when ICP27 is absent, HSV-1 likely takes advantages of host splicing machinery to restrict expression of randomly activated antigenic viral genes to achieve immune evasion.


Assuntos
Regulação Viral da Expressão Gênica , Herpesvirus Humano 1/fisiologia , Proteínas Imediatamente Precoces/biossíntese , Poliadenilação , Precursores de RNA/metabolismo , Processamento de RNA , RNA Viral/metabolismo , Latência Viral/fisiologia , Células HEK293 , Humanos , Proteínas Imediatamente Precoces/genética , Precursores de RNA/genética , RNA Viral/genética , Proteínas Virais/biossíntese , Proteínas Virais/genética
4.
Nucleic Acids Res ; 47(11): 5867-5879, 2019 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-30949712

RESUMO

In the yeast U1 snRNP the Prp39/Prp42 heterodimer is essential for early steps of spliceosome assembly. In metazoans no Prp42 ortholog exists, raising the question how the heterodimer is functionally substituted. Here we present the crystal structure of murine PRPF39, which forms a homodimer. Structure-guided point mutations disrupt dimer formation and inhibit splicing, manifesting the homodimer as functional unit. PRPF39 expression is controlled by NMD-inducing alternative splicing in mice and human, suggesting a role in adapting splicing efficiency to cell type specific requirements. A phylogenetic analysis reveals coevolution of shortened U1 snRNA and the absence of Prp42, which correlates with overall splicing complexity in different fungi. While current models correlate the diversity of spliceosomal proteins with splicing complexity, our study highlights a contrary case. We find that organisms with higher splicing complexity have substituted the Prp39/Prp42 heterodimer with a PRPF39 homodimer.


Assuntos
Proteínas Nucleares/fisiologia , Proteínas de Ligação a RNA/fisiologia , Ribonucleoproteína Nuclear Pequena U1/química , Proteínas de Saccharomyces cerevisiae/química , Processamento Alternativo , Animais , Linfócitos T CD8-Positivos/citologia , Dimerização , Células HEK293 , Humanos , Camundongos , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Fases de Leitura Aberta , Filogenia , Mutação Puntual , Precursores de RNA/metabolismo , Processamento de RNA , Fatores de Processamento de RNA/genética , RNA Nuclear Pequeno/metabolismo , Proteínas de Ligação a RNA/química , Ribonucleoproteína Nuclear Pequena U1/metabolismo , Saccharomyces cerevisiae/genética , Spliceossomos/metabolismo
5.
Nucleic Acids Res ; 47(11): 5906-5921, 2019 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-31006803

RESUMO

In most bacteria, ribosomal RNA is transcribed as a single polycistronic precursor that is first processed by RNase III. This double-stranded specific RNase cleaves two large stems flanking the 23S and 16S rRNA mature sequences, liberating three 16S, 23S and 5S rRNA precursors, which are further processed by other ribonucleases. Here, we investigate the rRNA maturation pathway of the human gastric pathogen Helicobacter pylori. This bacterium has an unusual arrangement of its rRNA genes, the 16S rRNA gene being separated from a 23S-5S rRNA cluster. We show that RNase III also initiates processing in this organism, by cleaving two typical stem structures encompassing 16S and 23S rRNAs and an atypical stem-loop located upstream of the 5S rRNA. Deletion of RNase III leads to the accumulation of a large 23S-5S precursor that is found in polysomes, suggesting that it can function in translation. Finally, we characterize a cis-encoded antisense RNA overlapping the leader of the 23S-5S rRNA precursor. We present evidence that this antisense RNA interacts with this precursor, forming an intermolecular complex that is cleaved by RNase III. This pairing induces additional specific cleavages of the rRNA precursor coupled with a rapid degradation of the antisense RNA.


Assuntos
Helicobacter pylori/genética , Precursores de RNA/metabolismo , RNA Ribossômico 16S/genética , RNA Ribossômico 23S/metabolismo , RNA Ribossômico 5S/metabolismo , Ribonuclease III/metabolismo , Escherichia coli/metabolismo , Humanos , Conformação de Ácido Nucleico , Oligonucleotídeos/genética , Polirribossomos/metabolismo , RNA Antissenso , RNA Bacteriano/metabolismo , RNA Ribossômico/metabolismo , Gastropatias/microbiologia
6.
Nucleic Acids Res ; 47(10): 5293-5306, 2019 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-30916337

RESUMO

Nonsense-mediated decay (NMD) is a eukaryotic mRNA surveillance system that selectively degrades transcripts with premature termination codons (PTC). Many RNA-binding proteins (RBP) regulate their expression levels by a negative feedback loop, in which RBP binds its own pre-mRNA and causes alternative splicing to introduce a PTC. We present a bioinformatic analysis integrating three data sources, eCLIP assays for a large RBP panel, shRNA inactivation of NMD pathway, and shRNA-depletion of RBPs followed by RNA-seq, to identify novel such autoregulatory feedback loops. We show that RBPs frequently bind their own pre-mRNAs, their exons respond prominently to NMD pathway disruption, and that the responding exons are enriched with nearby eCLIP peaks. We confirm previously proposed models of autoregulation in SRSF7 and U2AF1 genes and present two novel models, in which (i) SFPQ binds its mRNA and promotes switching to an alternative distal 3'-UTR that is targeted by NMD, and (ii) RPS3 binding activates a poison 5'-splice site in its pre-mRNA that leads to a frame shift and degradation by NMD. We also suggest specific splicing events that could be implicated in autoregulatory feedback loops in RBM39, HNRNPM, and U2AF2 genes. The results are available through a UCSC Genome Browser track hub.


Assuntos
Códon sem Sentido , Degradação do RNAm Mediada por Códon sem Sentido , Processamento de RNA , RNA Interferente Pequeno/metabolismo , Transcriptoma , Regiões 3' não Traduzidas , Processamento Alternativo , Biologia Computacional , Éxons , Mutação da Fase de Leitura , Ribonucleoproteínas Nucleares Heterogêneas Grupo M/metabolismo , Humanos , Proteínas Nucleares/metabolismo , Precursores de RNA/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fatores de Processamento de Serina-Arginina/metabolismo , Spliceossomos , Fator de Processamento U2AF/metabolismo
7.
Cell ; 177(2): 339-351.e13, 2019 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-30879786

RESUMO

Pre-mRNA splicing is executed by the spliceosome. Structural characterization of the catalytically activated complex (B∗) is pivotal for understanding the branching reaction. In this study, we assembled the B∗ complexes on two different pre-mRNAs from Saccharomyces cerevisiae and determined the cryo-EM structures of four distinct B∗ complexes at overall resolutions of 2.9-3.8 Å. The duplex between U2 small nuclear RNA (snRNA) and the branch point sequence (BPS) is discretely away from the 5'-splice site (5'SS) in the three B∗ complexes that are devoid of the step I splicing factors Yju2 and Cwc25. Recruitment of Yju2 into the active site brings the U2/BPS duplex into the vicinity of 5'SS, with the BPS nucleophile positioned 4 Å away from the catalytic metal M2. This analysis reveals the functional mechanism of Yju2 and Cwc25 in branching. These structures on different pre-mRNAs reveal substrate-specific conformations of the spliceosome in a major functional state.


Assuntos
Spliceossomos/fisiologia , Spliceossomos/ultraestrutura , Domínio Catalítico/fisiologia , Microscopia Crioeletrônica/métodos , Éxons , Íntrons , Proteínas Nucleares/metabolismo , Precursores de RNA/metabolismo , Sítios de Splice de RNA/genética , Processamento de RNA/fisiologia , Fatores de Processamento de RNA/metabolismo , RNA Nuclear Pequeno/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Spliceossomos/metabolismo
8.
Proc Natl Acad Sci U S A ; 116(14): 6868-6877, 2019 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-30886105

RESUMO

Arginine methylation is a ubiquitous posttranslational modification that regulates critical cellular processes including signal transduction and pre-mRNA splicing. Here, we report that the tumor-suppressor PTEN is methylated by protein arginine methyltransferase 6 (PRMT6). Mass-spectrometry analysis reveals that PTEN is dimethylated at arginine 159 (R159). We found that PTEN is mutated at R159 in cancers, and the PTEN mutant R159K loses its capability to inhibit the PI3K-AKT cascade. Furthermore, PRMT6 is physically associated with PTEN, promotes asymmetrical dimethylation of PTEN, and regulates the PI3K-AKT cascade through PTEN R159 methylation. In addition, using transcriptome analyses, we found that PTEN R159 methylation is involved in modulation of pre-mRNA alternative splicing. Our results demonstrate that PTEN is functionally regulated by arginine methylation. We propose that PTEN arginine methylation modulates pre-mRNA alternative splicing and influences diverse physiologic processes.


Assuntos
Processamento Alternativo , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Precursores de RNA/metabolismo , RNA Neoplásico/metabolismo , Transdução de Sinais , Linhagem Celular Tumoral , Células HEK293 , Humanos , Metilação , Proteínas de Neoplasias/genética , Neoplasias/genética , Proteínas Nucleares/genética , PTEN Fosfo-Hidrolase/genética , Fosfatidilinositol 3-Quinases/genética , Proteína-Arginina N-Metiltransferases/genética , Proteínas Proto-Oncogênicas c-akt/genética , Precursores de RNA/genética , RNA Neoplásico/genética
9.
Wiley Interdiscip Rev RNA ; 10(4): e1529, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30848101

RESUMO

Processing and maturation of precursor RNA species is coupled to RNA polymerase II transcription. Co-transcriptional RNA processing helps to ensure efficient and proper capping, splicing, and 3' end processing of different RNA species to help ensure quality control of the transcriptome. Many improperly processed transcripts are not exported from the nucleus, are restricted to the site of transcription, and are in some cases degraded, which helps to limit any possibility of aberrant RNA causing harm to cellular health. These critical quality control pathways are regulated by the highly dynamic protein-protein interaction network at the site of transcription. Recent work has further revealed the extent to which the processes of transcription and RNA processing and quality control are integrated, and how critically their coupling relies upon the dynamic protein interactions that take place co-transcriptionally. This review focuses specifically on the intricate balance between 3' end processing and RNA decay during transcription termination. This article is categorized under: RNA Turnover and Surveillance > Turnover/Surveillance Mechanisms RNA Processing > 3' End Processing RNA Processing > Splicing Mechanisms RNA Processing > Capping and 5' End Modifications.


Assuntos
RNA Polimerase II/metabolismo , Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA , RNA Mensageiro/metabolismo , Transcrição Genética , Mapas de Interação de Proteínas
10.
BMC Evol Biol ; 19(1): 55, 2019 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-30764768

RESUMO

BACKGROUND: Micro RNAs (miRNAs), a class of small non-coding RNAs, have been implicated in various aspects of plant development. miR394 is required for shoot apical meristem organization, stem cell maintenance and abiotic stress responses in Arabidopsis, where it functions by negatively regulating the transcript level of target LEAF CURLING RESPONSIVENESS (LCR), which is an F-box protein-coding gene. The evolutionary conservation of stem cell regulatory miR394-LCR module among plants remains elusive. RESULTS: Our study has identified 79 miR394 and 43 target sequences across 40 plant species using various homology based search tools and databases, and analysed their co-evolution pattern. We customised an annotation workflow which computationally validates 20 novel miR394s from 14 plant species. Independent phylogenetic trees were reconstructed with precursor MIR394s, mature miR394s, and their target sequences along with complementary miR394 binding sites. The phylogeny revealed that mature sequences of miR394s as well as their targets belonging to the F-box protein encoding gene families, were highly conserved. Though, miR394-3p were complementary to miR394s/miR394-5p, they clustered separately. CONCLUSION: The existence and separate clustering of miR394-3p and miR394s/miR394-5p indicate their independent regulation. The phylogeny also suggests that miR394s had evolved at the beginning of gymnosperm-angiosperm divergence. Despite strong conservation, some level of sequence variation in miR394s and the complementary binding sites of their targets suggests possible functional diversification of miR394-LCR mediated stem cell regulation in plants.


Assuntos
Evolução Molecular , MicroRNAs/genética , Plantas/genética , RNA de Plantas/genética , Arabidopsis/genética , Sequência de Bases , Sequência Conservada/genética , Regulação da Expressão Gênica de Plantas , Genoma de Planta , MicroRNAs/metabolismo , Anotação de Sequência Molecular , Família Multigênica , Filogenia , Precursores de RNA/genética , Precursores de RNA/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Plantas/metabolismo , Especificidade da Espécie , Estresse Fisiológico/genética
11.
J Antibiot (Tokyo) ; 72(4): 225-236, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30737453

RESUMO

In bacteria, RNase III cleaves the initial long primary ribosomal RNA transcripts/precursors (pre-rRNAs), thereby releasing the pre-16S and pre-23S rRNAs for maturation. This cleavage is specified by the double-stranded secondary structures flanking the mature rRNAs, and not necessarily by the nucleotide sequences. Inhibition of this cleavage would lead to a build-up of pre-rRNA molecules. Doxycycline has earlier been shown to bind synthetic double-stranded RNAs and inhibit their cleavage by RNase III. Since bacterial rRNA processing is primarily dependent on RNase III cleavage (which is inhibited by doxycycline), doxycycline could therefore inhibit the normal processing of bacterial rRNA. In this study, the effect of doxycycline on bacterial rRNA processing was investigated by analyzing the amounts of various rRNAs in growing Escherichia coli cells treated with doxycycline. The results showed a doxycycline dose-dependent decrease in mature 16S and 23S rRNAs, concurrent with an accumulation of the initial rRNA transcripts and long precursors. Morphologically, treated cells were elongated at low drug concentrations, while nucleoid degeneration indicative of cell death occurred at higher drug concentrations. These observations suggest that doxycycline inhibits the cleavage and processing of bacterial rRNA transcripts/precursors, leading to impaired formation of mature rRNAs, and the consequent inhibition of protein synthesis for which the tetracycline group of antibiotics are renowned. Since rRNA structure and processing pathway is conserved among bacterial species, this mechanism may account for the broad spectrum of antibiotic activity and selective microbial protein synthesis inhibition of doxycycline and the tetracyclines.


Assuntos
Antibacterianos/farmacologia , Doxiciclina/farmacologia , Escherichia coli/efeitos dos fármacos , Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA/efeitos dos fármacos , RNA Ribossômico 16S/metabolismo , RNA Ribossômico 23S/metabolismo
12.
mBio ; 10(1)2019 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-30755517

RESUMO

Herpes simplex virus 1 (HSV-1) switches between two infection programs, productive ("lytic") and latent infection. Some HSV-1 microRNAs (miRNAs) have been hypothesized to help control this switch, and yet little is known about regulation of their expression. Using Northern blot analyses, we found that, despite inherent differences in biogenesis efficiency among six HSV-1 miRNAs, all six exhibited high pre-miRNA/miRNA ratios during lytic infection of different cell lines and, when detectable, in acutely infected mouse trigeminal ganglia. In contrast, considerably lower ratios were observed in latently infected ganglia and in cells transduced with lentiviral vectors expressing the miRNAs, suggesting that HSV-1 lytic infection blocks miRNA biogenesis. This phenomenon is not specific to viral miRNAs, as a host miRNA expressed from recombinant HSV-1 also exhibited high pre-miRNA/miRNA ratios late during lytic infection. The levels of most of the mature miRNAs remained stable during infection in the presence of actinomycin D, indicating that the high ratios are due to inefficient pre-miRNA conversion to miRNA. Cellular fractionation experiments showed that late (but not early) during infection, pre-miRNAs were enriched in the nucleus and depleted in the cytoplasm, indicating that nuclear export was blocked. A mutation eliminating ICP27 expression or addition of acyclovir reduced pre-miRNA/miRNA ratios, but mutations drastically reducing Us11 expression did not. Thus, HSV-1 lytic infection inhibits miRNA biogenesis at the step of nuclear export and does so in an ICP27- and viral DNA synthesis-dependent manner. This mechanism may benefit the virus by reducing expression of repressive miRNAs during lytic infection while permitting elevated expression during latency.IMPORTANCE Various mechanisms have been identified by which viruses target host small RNA biogenesis pathways to achieve optimal infection outcomes. Herpes simplex virus 1 (HSV-1) is a ubiquitous human pathogen whose successful persistence in the host entails both productive ("lytic") and latent infection. Although many HSV-1 miRNAs have been discovered and some are thought to help control the lytic/latent switch, little is known about regulation of their biogenesis. By characterizing expression of both pre-miRNAs and mature miRNAs under various conditions, this study revealed striking differences in miRNA biogenesis between lytic and latent infection and uncovered a regulatory mechanism that blocks pre-miRNA nuclear export and is dependent on viral protein ICP27 and viral DNA synthesis. This mechanism represents a new virus-host interaction that could limit the repressive effects of HSV-1 miRNAs hypothesized to promote latency and may shed light on the regulation of miRNA nuclear export, which has been relatively unexplored.


Assuntos
Transporte Ativo do Núcleo Celular , Herpes Simples/virologia , Herpesvirus Humano 1/crescimento & desenvolvimento , Interações Hospedeiro-Patógeno , MicroRNAs/metabolismo , Precursores de RNA/metabolismo , Animais , Northern Blotting , Linhagem Celular , Modelos Animais de Doenças , Regulação da Expressão Gênica , Herpes Simples/patologia , Humanos , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/metabolismo , Camundongos , Mutação , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Gânglio Trigeminal/patologia , Gânglio Trigeminal/virologia , Proteínas Virais/genética , Proteínas Virais/metabolismo , Latência Viral
13.
Nucleic Acids Res ; 47(8): 4181-4197, 2019 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-30767021

RESUMO

Src associated in mitosis (SAM68) plays major roles in regulating RNA processing events, such as alternative splicing and mRNA translation, implicated in several developmental processes. It was previously shown that SAM68 regulates the alternative splicing of the mechanistic target of rapamycin (mTor), but the mechanism regulating this process remains elusive. Here, we report that SAM68 interacts with U1 small nuclear ribonucleoprotein (U1 snRNP) to promote splicing at the 5' splice site in intron 5 of mTor. We also show that this direct interaction is mediated through U1A, a core-component of U1snRNP. SAM68 was found to bind the RRM1 domain of U1A through its C-terminal tyrosine rich region (YY domain). Deletion of the U1A-SAM68 interaction domain or mutation in SAM68-binding sites in intron 5 of mTor abrogates U1A recruitment and 5' splice site recognition by the U1 snRNP, leading to premature intron 5 termination and polyadenylation. Taken together, our results provide the first mechanistic study by which SAM68 modulates alternative splicing decision, by affecting U1 snRNP recruitment at 5' splice sites.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Precursores de RNA/genética , Processamento de RNA , Proteínas de Ligação a RNA/genética , RNA/genética , Ribonucleoproteína Nuclear Pequena U1/genética , Serina-Treonina Quinases TOR/genética , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Linhagem Celular , Éxons , Fibroblastos/citologia , Fibroblastos/metabolismo , Deleção de Genes , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Íntrons , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Transporte Proteico , RNA/metabolismo , Precursores de RNA/metabolismo , Ribonucleoproteína Nuclear Pequena U1/química , Ribonucleoproteína Nuclear Pequena U1/metabolismo , Serina-Treonina Quinases TOR/metabolismo
14.
Cell Chem Biol ; 26(3): 443-448.e3, 2019 03 21.
Artigo em Inglês | MEDLINE | ID: mdl-30639260

RESUMO

The spliceosome mediates precursor mRNA splicing in eukaryotes, including the model organism Saccharomyces cerevisiae (yeast). Despite decades of study, no chemical inhibitors of yeast splicing in vivo are available. We have developed a system to efficiently inhibit splicing and block proliferation in living yeast cells using compounds that target the human spliceosome protein SF3B1. Potent inhibition is observed in yeast expressing a chimeric protein containing portions of human SF3B1. However, only a single point mutation in the yeast homolog of SF3B1 is needed for selective inhibition of splicing by pladienolide B, herboxidiene, or meayamycin in liquid culture. Mutations that enable inhibition also improve splicing of branch sites containing mismatches between the intron and small nuclear RNA-suggesting a link between inhibitor sensitivity and usage of weak branch sites in humans. This approach provides powerful new tools for manipulating splicing in live yeast and studies of spliceosome inhibitors.


Assuntos
Precursores de RNA/metabolismo , Ribonucleoproteína Nuclear Pequena U2/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Bibliotecas de Moléculas Pequenas/química , Sequência de Aminoácidos , Compostos de Epóxi/química , Compostos de Epóxi/farmacologia , Álcoois Graxos/química , Álcoois Graxos/farmacologia , Humanos , Macrolídeos/química , Macrolídeos/farmacologia , Mutagênese , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Piranos/química , Piranos/farmacologia , Precursores de RNA/antagonistas & inibidores , Processamento de RNA/efeitos dos fármacos , Fatores de Processamento de RNA/química , Fatores de Processamento de RNA/metabolismo , Ribonucleoproteína Nuclear Pequena U2/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Bibliotecas de Moléculas Pequenas/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia
15.
RNA Biol ; 16(2): 155-159, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30596342

RESUMO

The family of heterogeneous ribonucleoproteins (hnRNPs) have multiple functions in RNA metabolism. In recent years, several hnRNPs have also been shown to be essential for the maintenance of transcriptome integrity, by preventing intronic cryptic splicing signals from mis-splicing of many endogeneous pre-mRNA transcripts. Here we discuss the possibility for a general role of this family of proteins and their expansion in transcriptome protection.


Assuntos
Regulação da Expressão Gênica , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Família Multigênica , Transcrição Genética , Transcriptoma , Animais , Sítios de Ligação , Ribonucleoproteínas Nucleares Heterogêneas/genética , Humanos , Motivos de Nucleotídeos , Ligação Proteica , Precursores de RNA/genética , Precursores de RNA/metabolismo , Processamento de RNA
16.
BMC Res Notes ; 12(1): 33, 2019 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-30658689

RESUMO

OBJECTIVE: The pre-mRNA of the Epstein-Barr virus LMP2 (latent membrane protein 2) has a region of unusual RNA structure that partially spans two consecutive exons and the entire intervening intron; suggesting RNA folding might affect splicing-particularly via interactions with human regulatory proteins. To better understand the roles of protein associations with this structured intronic region, we undertook a combined bioinformatics (motif searching) and experimental analysis (biotin pulldowns and RNA immunoprecipitations) of protein binding. RESULT: Characterization of the ribonucleoprotein composition of this region revealed several human proteins as interactors: regulatory proteins hnRNP A1 (heterogeneous nuclear ribonucleoprotein A1), hnRNP U, HuR (human antigen R), and PSF (polypyrimidine tract-binding protein-associated splicing factor), as well as, unexpectedly, the cytoskeletal protein actin. Treatment of EBV-positive cells with drugs that alter actin polymerization specifically showed marked effects on splicing in this region. This suggests a potentially novel role for nuclear actin in regulation of viral RNA splicing.


Assuntos
Herpesvirus Humano 4/metabolismo , Precursores de RNA/metabolismo , RNA Viral/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas da Matriz Viral/metabolismo , Actinas/metabolismo , Núcleo Celular/metabolismo , Proteína Semelhante a ELAV 1/metabolismo , Herpesvirus Humano 4/genética , Ribonucleoproteína Nuclear Heterogênea A1/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo U/metabolismo , Humanos , Íntrons/genética , Fator de Processamento Associado a PTB/metabolismo , Ligação Proteica , Precursores de RNA/genética , RNA Viral/genética , Proteínas da Matriz Viral/genética
17.
BMC Genomics ; 20(1): 90, 2019 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-30691391

RESUMO

BACKGROUND: As important female reproductive tissues, the rice (Oryza sativa L.) ovule and female gametophyte is significant in terms of their fertility. Long noncoding RNAs (lncRNAs) play important and wide-ranging roles in the growth and development of plants and have become a major research focus in recent years. Therefore, we explored the characterization and expression change of lncRNAs during ovule development and female gametophytic abortion. RESULTS: In our study, whole-transcriptome strand-specific RNA sequencing (ssRNA-seq) was performed in the ovules of a high-frequency female-sterile rice line (fsv1) and a wild-type rice line (Gui99) at the megaspore mother cell meiosis stage (stage 1), functional megaspore mitosis stage (stage 2) and female gametophyte mature stage (stage 3). By comparing two rice lines, we identified 152, 233, and 197 differentially expressed lncRNAs at the three ovule developmental stages. Functional analysis of the coherent target genes of these differentially expressed lncRNAs indicated that many lncRNAs participate in multiple pathways such as hormone and cellular metabolism and signal transduction. Moreover, there were many differentially expressed lncRNAs acting as the precursors of some miRNAs that are involved in the development of ovules and female gametophytes. In addition, we have found that lncRNAs can act as decoys, competing with mRNAs for binding to miRNAs to maintain the normal expression of genes related to ovule and female gametophyte development. CONCLUSION: These results provide important clues for elucidating the female gametophyte abortion mechanism in rice. This study also expands our understanding about the biological functions of lncRNAs and the annotation of the rice genome.


Assuntos
Regulação da Expressão Gênica de Plantas , Células Germinativas Vegetais/metabolismo , Oryza/genética , Óvulo Vegetal/genética , RNA Longo não Codificante/metabolismo , Perfilação da Expressão Gênica , Genoma de Planta , Sequenciamento de Nucleotídeos em Larga Escala , MicroRNAs/química , MicroRNAs/metabolismo , Oryza/crescimento & desenvolvimento , Oryza/metabolismo , Óvulo Vegetal/crescimento & desenvolvimento , Proteínas de Plantas/genética , Precursores de RNA/química , Precursores de RNA/metabolismo , RNA Longo não Codificante/química , RNA Longo não Codificante/fisiologia , RNA Mensageiro/metabolismo , Análise de Sequência de RNA
18.
Development ; 146(2)2019 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-30696714

RESUMO

The scarcity of embryonic/foetal material as a resource for direct study means that there is still limited understanding of human retina development. Here, we present an integrated transcriptome analysis combined with immunohistochemistry in human eye and retinal samples from 4 to 19 post-conception weeks. This analysis reveals three developmental windows with specific gene expression patterns that informed the sequential emergence of retinal cell types and enabled identification of stage-specific cellular and biological processes, and transcriptional regulators. Each stage is characterised by a specific set of alternatively spliced transcripts that code for proteins involved in the formation of the photoreceptor connecting cilium, pre-mRNA splicing and epigenetic modifiers. Importantly, our data show that the transition from foetal to adult retina is characterised by a large increase in the percentage of mutually exclusive exons that code for proteins involved in photoreceptor maintenance. The circular RNA population is also defined and shown to increase during retinal development. Collectively, these data increase our understanding of human retinal development and the pre-mRNA splicing process, and help to identify new candidate disease genes.


Assuntos
Perfilação da Expressão Gênica , Retina/embriologia , Retina/metabolismo , Processamento Alternativo/genética , Animais , Biomarcadores/metabolismo , Cílios/metabolismo , Feto/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Organogênese/genética , Células Fotorreceptoras de Vertebrados/citologia , Células Fotorreceptoras de Vertebrados/metabolismo , Análise de Componente Principal , RNA/genética , RNA/metabolismo , Precursores de RNA/genética , Precursores de RNA/metabolismo , Retina/citologia , Retina/ultraestrutura , Transcriptoma/genética
19.
RNA ; 25(4): 465-471, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30670483

RESUMO

The eukaryotic ribosome is assembled through a complex process involving more than 200 factors. As preribosomal RNA is transcribed, assembly factors bind the nascent pre-rRNA and guide its correct folding, modification, and cleavage. While these early events in the assembly of the small ribosomal subunit have been relatively well characterized, assembly of the large subunit precursors, or pre-60S, is less well understood. Recent structures of nucleolar intermediates of large subunit assembly have shed light on the role of many early large subunit assembly factors, but how these particles emerge is still unknown. Here, we use the expression and purification of truncated pre-rRNAs to examine the initial assembly of pre-60S particles. Using this approach, we can recapitulate the early recruitment of large subunit assembly factors mainly to the domains I, II, and VI of the assembling 25S rRNA.


Assuntos
Biogênese de Organelas , Precursores de RNA/genética , RNA Ribossômico/genética , Proteínas Ribossômicas/genética , Subunidades Ribossômicas Maiores de Eucariotos/genética , Saccharomyces cerevisiae/genética , Aptâmeros de Nucleotídeos/síntese química , Aptâmeros de Nucleotídeos/metabolismo , Clonagem Molecular , Plasmídeos/química , Plasmídeos/metabolismo , Precursores de RNA/metabolismo , RNA Ribossômico/metabolismo , Proteínas Ribossômicas/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos/ultraestrutura , Saccharomyces cerevisiae/metabolismo , Coloração e Rotulagem/métodos
20.
Cell ; 176(3): 535-548.e24, 2019 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-30661751

RESUMO

The splicing of pre-mRNAs into mature transcripts is remarkable for its precision, but the mechanisms by which the cellular machinery achieves such specificity are incompletely understood. Here, we describe a deep neural network that accurately predicts splice junctions from an arbitrary pre-mRNA transcript sequence, enabling precise prediction of noncoding genetic variants that cause cryptic splicing. Synonymous and intronic mutations with predicted splice-altering consequence validate at a high rate on RNA-seq and are strongly deleterious in the human population. De novo mutations with predicted splice-altering consequence are significantly enriched in patients with autism and intellectual disability compared to healthy controls and validate against RNA-seq in 21 out of 28 of these patients. We estimate that 9%-11% of pathogenic mutations in patients with rare genetic disorders are caused by this previously underappreciated class of disease variation.


Assuntos
Previsões/métodos , Precursores de RNA/genética , Processamento de RNA/genética , Algoritmos , Processamento Alternativo/genética , Transtorno Autístico/genética , Aprendizado Profundo , Éxons/genética , Humanos , Deficiência Intelectual/genética , Íntrons/genética , Precursores de RNA/metabolismo , Sítios de Splice de RNA/genética , Sítios de Splice de RNA/fisiologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA