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1.
Proc Natl Acad Sci U S A ; 118(39)2021 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-34548404

RESUMO

Homozygous mutation of the RNA kinase CLP1 (cleavage factor polyribonucleotide kinase subunit 1) causes pontocerebellar hypoplasia type 10 (PCH10), a pediatric neurodegenerative disease. CLP1 is associated with the transfer RNA (tRNA) splicing endonuclease complex and the cleavage and polyadenylation machinery, but its function remains unclear. We generated two mouse models of PCH10: one homozygous for the disease-associated Clp1 mutation, R140H, and one heterozygous for this mutation and a null allele. Both models exhibit loss of lower motor neurons and neurons of the deep cerebellar nuclei. To explore whether Clp1 mutation impacts tRNA splicing, we profiled the products of intron-containing tRNA genes. While mature tRNAs were expressed at normal levels in mutant mice, numerous other products of intron-containing tRNA genes were dysregulated, with pre-tRNAs, introns, and certain tRNA fragments up-regulated, and other fragments down-regulated. However, the spatiotemporal patterns of dysregulation do not correlate with pathogenicity for most altered tRNA products. To elucidate the effect of Clp1 mutation on precursor messenger RNA (pre-mRNA) cleavage, we analyzed poly(A) site (PAS) usage and gene expression in Clp1 R140H/- spinal cord. PAS usage was shifted from proximal to distal sites in the mutant mouse, particularly in short and closely spaced genes. Many such genes were also expressed at lower levels in the Clp1 R140H/- mouse, possibly as a result of impaired transcript maturation. These findings are consistent with the hypothesis that select genes are particularly dependent upon CLP1 for proper pre-mRNA cleavage, suggesting that impaired mRNA 3' processing may contribute to pathogenesis in PCH10.


Assuntos
Doenças Cerebelares/patologia , Doenças Neurodegenerativas/patologia , Poliadenilação , Processamento Pós-Transcricional do RNA , RNA Mensageiro/metabolismo , RNA de Transferência/metabolismo , Proteínas de Ligação a RNA/fisiologia , Fatores de Transcrição/fisiologia , Animais , Doenças Cerebelares/genética , Doenças Cerebelares/metabolismo , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismo , Precursores de RNA/genética , Precursores de RNA/metabolismo , RNA Mensageiro/genética , RNA de Transferência/genética
2.
Science ; 373(6558): 984-991, 2021 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-34446600

RESUMO

Protein kinase activity must be precisely regulated, but how a cell governs hyperactive kinases remains unclear. In this study, we generated a constitutively active mitogen-activated protein kinase DYF-5 (DYF-5CA) in Caenorhabditis elegans that disrupted sensory cilia. Genetic suppressor screens identified that mutations of ADR-2, an RNA adenosine deaminase, rescued ciliary phenotypes of dyf-5CA We found that dyf-5CA animals abnormally transcribed antisense RNAs that pair with dyf-5CA messenger RNA (mRNA) to form double-stranded RNA, recruiting ADR-2 to edit the region ectopically. RNA editing impaired dyf-5CA mRNA splicing, and the resultant intron retentions blocked DYF-5CA protein translation and activated nonsense-mediated dyf-5CA mRNA decay. The kinase RNA editing requires kinase hyperactivity. The similar RNA editing-dependent feedback regulation restricted the other ciliary kinases NEKL-4/NEK10 and DYF-18/CCRK, which suggests a widespread mechanism that underlies kinase regulation.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/enzimologia , Cílios/metabolismo , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Edição de RNA , Adenosina Desaminase/genética , Adenosina Desaminase/metabolismo , Animais , Caenorhabditis elegans/genética , Núcleo Celular/metabolismo , Cílios/enzimologia , Ativação Enzimática , Fenótipo , Biossíntese de Proteínas , Proteínas Serina-Treonina Quinases/metabolismo , Precursores de RNA/genética , Precursores de RNA/metabolismo , Splicing de RNA , Estabilidade de RNA , RNA Antissenso/genética , RNA Antissenso/metabolismo , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/metabolismo , RNA de Helmintos/genética , RNA de Helmintos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais , Transcrição Genética
3.
Nat Commun ; 12(1): 4696, 2021 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-34349113

RESUMO

Productive ribosomal RNA (rRNA) compaction during ribosome assembly necessitates establishing correct tertiary contacts between distant secondary structure elements. Here, we quantify the response of the yeast proteome to low temperature (LT), a condition where aberrant mis-paired RNA folding intermediates accumulate. We show that, at LT, yeast cells globally boost production of their ribosome assembly machinery. We find that the LT-induced assembly factor, Puf6, binds to the nascent catalytic RNA-rich subunit interface within the 60S pre-ribosome, at a site that eventually loads the nuclear export apparatus. Ensemble Förster resonance energy transfer studies show that Puf6 mimics the role of Mg2+ to usher a unique long-range tertiary contact to compact rRNA. At LT, puf6 mutants accumulate 60S pre-ribosomes in the nucleus, thus unveiling Puf6-mediated rRNA compaction as a critical temperature-regulated rescue mechanism that counters rRNA misfolding to prime export competence.


Assuntos
Núcleo Celular/metabolismo , Proteínas de Ligação a RNA/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transporte Ativo do Núcleo Celular , Temperatura Baixa , GTP Fosfo-Hidrolases/metabolismo , Mutação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteoma/metabolismo , Dobramento de RNA , Precursores de RNA/química , Precursores de RNA/metabolismo , RNA Ribossômico/química , RNA Ribossômico/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Subunidades Ribossômicas Maiores de Eucariotos/química , Ribossomos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética
4.
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
5.
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
6.
Nat Commun ; 12(1): 4545, 2021 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-34315864

RESUMO

In the earliest step of spliceosome assembly, the two splice sites flanking an intron are brought into proximity by U1 snRNP and U2AF along with other proteins. The mechanism that facilitates this intron looping is poorly understood. Using a CRISPR interference-based approach to halt RNA polymerase II transcription in the middle of introns in human cells, we discovered that the nascent 5' splice site base pairs with a U1 snRNA that is tethered to RNA polymerase II during intron synthesis. This association functionally corresponds with splicing outcome, involves bona fide 5' splice sites and cryptic intronic sites, and occurs transcriptome-wide. Overall, our findings reveal that the upstream 5' splice sites remain attached to the transcriptional machinery during intron synthesis and are thus brought into proximity of the 3' splice sites; potentially mediating the rapid splicing of long introns.


Assuntos
Íntrons/genética , Sítios de Splice de RNA/genética , Transcrição Genética , Pareamento de Bases/genética , Sequência de Bases , Éxons/genética , Células HEK293 , Células HeLa , Humanos , Proteínas dos Microfilamentos/genética , RNA Polimerase II/metabolismo , Precursores de RNA/genética , Precursores de RNA/metabolismo , RNA Nuclear Pequeno/genética , RNA Nuclear Pequeno/metabolismo , Proteínas de Ligação a RNA/genética , Ribonucleoproteína Nuclear Pequena U1/metabolismo , Transcriptoma/genética
7.
RNA ; 27(10): 1148-1154, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34230059

RESUMO

CPSF73 is the endonuclease that catalyzes the cleavage reaction for 3'-end processing of mRNA precursors (pre-mRNAs) in two distinct machineries, a canonical machinery for the majority of pre-mRNAs and a U7 snRNP (U7 machinery) for replication-dependent histone pre-mRNAs in animal cells. CPSF73 also possesses 5'-3' exonuclease activity in the U7 machinery, degrading the downstream cleavage product after the endonucleolytic cleavage. Recent studies show that CPSF73 is a potential target for developing anticancer, antimalarial, and antiprotozoal drugs, spurring interest in identifying new small-molecule inhibitors against this enzyme. CPSF73 nuclease activity has so far been demonstrated using a gel-based end-point assay, using radiolabeled or fluorescently labeled RNA substrates. By taking advantage of unique properties of the U7 machinery, we have developed a novel, real-time fluorescence assay for the nuclease activity of CPSF73. This assay is facile and high-throughput, and should also be helpful for the discovery of new CPSF73 inhibitors.


Assuntos
Bioensaio , Fator de Especificidade de Clivagem e Poliadenilação/metabolismo , Histonas/metabolismo , Processamento de Terminações 3' de RNA , Precursores de RNA/metabolismo , Ribonucleoproteína Nuclear Pequena U7/metabolismo , Sistema Livre de Células , Fator de Especificidade de Clivagem e Poliadenilação/química , Fator de Especificidade de Clivagem e Poliadenilação/genética , Ensaios Enzimáticos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Fluorescência , Corantes Fluorescentes/química , Histonas/química , Histonas/genética , Humanos , Modelos Moleculares , Fenilalanina/análogos & derivados , Fenilalanina/química , Fenilalanina/farmacologia , Piperazinas/química , Piperazinas/farmacologia , Proteólise , Precursores de RNA/química , Precursores de RNA/genética , Rodaminas/química , Ribonucleoproteína Nuclear Pequena U7/química , Ribonucleoproteína Nuclear Pequena U7/genética , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia
8.
Nucleic Acids Res ; 49(12): 7103-7121, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34161584

RESUMO

The specific recognition of splice signals at or near exon-intron junctions is not explained by their weak conservation and instead is postulated to require a multitude of features embedded in the pre-mRNA strand. We explored the possibility of 3D structural scaffold of AdML-a model pre-mRNA substrate-guiding early spliceosomal components to the splice signal sequences. We find that mutations in the non-cognate splice signal sequences impede recruitment of early spliceosomal components due to disruption of the global structure of the pre-mRNA. We further find that the pre-mRNA segments potentially interacting with the early spliceosomal component U1 snRNP are distributed across the intron, that there is a spatial proximity of 5' and 3' splice sites within the pre-mRNA scaffold, and that an interplay exists between the structural scaffold and splicing regulatory elements in recruiting early spliceosomal components. These results suggest that early spliceosomal components can recognize a 3D structural scaffold beyond the short splice signal sequences, and that in our model pre-mRNA, this scaffold is formed across the intron involving the major splice signals. This provides a conceptual basis to analyze the contribution of recognizable 3D structural scaffolds to the splicing code across the mammalian transcriptome.


Assuntos
Precursores de RNA/química , Splicing de RNA , RNA Mensageiro/química , Células HeLa , Humanos , Íntrons , Mutação , Conformação de Ácido Nucleico , Domínios Proteicos , Precursores de RNA/metabolismo , Sítios de Splice de RNA , RNA Mensageiro/metabolismo , Ribonucleoproteína Nuclear Pequena U1/metabolismo , Fatores de Processamento de Serina-Arginina/química , Fatores de Processamento de Serina-Arginina/metabolismo , Fator de Processamento U2AF/metabolismo
9.
Nat Commun ; 12(1): 3308, 2021 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-34083519

RESUMO

The spatial partitioning of the transcriptome in the cell is an important form of gene-expression regulation. Here, we address how intron retention influences the spatio-temporal dynamics of transcripts from two clinically relevant genes: TERT (Telomerase Reverse Transcriptase) pre-mRNA and TUG1 (Taurine-Upregulated Gene 1) lncRNA. Single molecule RNA FISH reveals that nuclear TERT transcripts uniformly and robustly retain specific introns. Our data suggest that the splicing of TERT retained introns occurs during mitosis. In contrast, TUG1 has a bimodal distribution of fully spliced cytoplasmic and intron-retained nuclear transcripts. We further test the functionality of intron-retention events using RNA-targeting thiomorpholino antisense oligonucleotides to block intron excision. We show that intron retention is the driving force for the nuclear compartmentalization of these RNAs. For both RNAs, altering this splicing-driven subcellular distribution has significant effects on cell viability. Together, these findings show that stable retention of specific introns can orchestrate spatial compartmentalization of these RNAs within the cell. This process reveals that modulating RNA localization via targeted intron retention can be utilized for RNA-based therapies.


Assuntos
Núcleo Celular/genética , Núcleo Celular/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Telomerase/genética , Animais , Compartimento Celular , Linhagem Celular , Linhagem Celular Tumoral , Células HCT116 , Células HEK293 , Células HeLa , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Hibridização in Situ Fluorescente , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Íntrons , Camundongos , Mitose , Precursores de RNA/genética , Precursores de RNA/metabolismo , Splicing de RNA , Estabilidade de RNA , Especificidade da Espécie
10.
Int J Mol Sci ; 22(11)2021 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-34071057

RESUMO

Cytosolic ribosomes (cytoribosomes) are macromolecular ribonucleoprotein complexes that are assembled from ribosomal RNA and ribosomal proteins, which are essential for protein biosynthesis. Mitochondrial ribosomes (mitoribosomes) perform translation of the proteins essential for the oxidative phosphorylation system. The biogenesis of cytoribosomes and mitoribosomes includes ribosomal RNA processing, modification and binding to ribosomal proteins and is assisted by numerous biogenesis factors. This is a major energy-consuming process in the cell and, therefore, is highly coordinated and sensitive to several cellular stressors. In mitochondria, the regulation of mitoribosome biogenesis is essential for cellular respiration, a process linked to cell growth and proliferation. This review briefly overviews the key stages of cytosolic and mitochondrial ribosome biogenesis; summarizes the main steps of ribosome biogenesis alterations occurring during tumorigenesis, highlighting the changes in the expression level of cytosolic ribosomal proteins (CRPs) and mitochondrial ribosomal proteins (MRPs) in different types of tumors; focuses on the currently available information regarding the extra-ribosomal functions of CRPs and MRPs correlated to cancer; and discusses the role of CRPs and MRPs as biomarkers and/or molecular targets in cancer treatment.


Assuntos
Transformação Celular Neoplásica , Neoplasias/metabolismo , Biogênese de Organelas , Ribossomos , Animais , Apoptose , Autofagia , Ciclo Celular , Movimento Celular , Nucléolo Celular/metabolismo , Citosol/metabolismo , Reparo do DNA , Estresse do Retículo Endoplasmático , Células Eucarióticas/metabolismo , Células Eucarióticas/ultraestrutura , Regulação Neoplásica da Expressão Gênica , Terapia Genética/métodos , Humanos , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias/diagnóstico , Neoplasias/genética , Neoplasias/terapia , Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA , RNA Mitocondrial/metabolismo , RNA Ribossômico/metabolismo , Proteínas Ribossômicas/biossíntese , Proteínas Ribossômicas/fisiologia , Ribossomos/fisiologia
11.
Methods Enzymol ; 655: 291-324, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34183127

RESUMO

In animal cells, replication-dependent histone pre-mRNAs are processed at the 3'-end by an endonucleolytic cleavage carried out by the U7 snRNP, a machinery that contains the U7 snRNA and many protein subunits. Studies on the composition of this machinery and understanding of its role in 3'-end processing were greatly facilitated by the development of an in vitro system utilizing nuclear extracts from mammalian cells 35 years ago and later from Drosophila cells. Most recently, recombinant expression and purification of the components of the machinery have enabled the full reconstitution of an active machinery and its complex with a model pre-mRNA substrate, using 13 proteins and 2 RNAs, and the determination of the structure of this active machinery. This chapter presents protocols for preparing nuclear extracts containing endogenous processing machinery, for assembling semi-recombinant and fully reconstituted machineries, and for histone pre-mRNA 3'-end processing assays with these samples.


Assuntos
Histonas , Precursores de RNA , Animais , Drosophila/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Precursores de RNA/genética , Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA , Ribonucleoproteína Nuclear Pequena U7/genética , Ribonucleoproteína Nuclear Pequena U7/metabolismo
12.
Nucleic Acids Res ; 49(11): 6267-6280, 2021 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-34096575

RESUMO

Prefoldin is a heterohexameric complex conserved from archaea to humans that plays a cochaperone role during the co-translational folding of actin and tubulin monomers. Additional functions of prefoldin have been described, including a positive contribution to transcription elongation and chromatin dynamics in yeast. Here we show that prefoldin perturbations provoked transcriptional alterations across the human genome. Severe pre-mRNA splicing defects were also detected, particularly after serum stimulation. We found impairment of co-transcriptional splicing during transcription elongation, which explains why the induction of long genes with a high number of introns was affected the most. We detected genome-wide prefoldin binding to transcribed genes and found that it correlated with the negative impact of prefoldin depletion on gene expression. Lack of prefoldin caused global decrease in Ser2 and Ser5 phosphorylation of the RNA polymerase II carboxy-terminal domain. It also reduced the recruitment of the CTD kinase CDK9 to transcribed genes, and the association of splicing factors PRP19 and U2AF65 to chromatin, which is known to depend on CTD phosphorylation. Altogether the reported results indicate that human prefoldin is able to act locally on the genome to modulate gene expression by influencing phosphorylation of elongating RNA polymerase II, and thereby regulating co-transcriptional splicing.


Assuntos
Chaperonas Moleculares/fisiologia , Splicing de RNA , RNA Mensageiro/metabolismo , Transcrição Genética , Linhagem Celular , Humanos , Íntrons , RNA Polimerase II/metabolismo , Precursores de RNA/metabolismo , Fatores de Processamento de RNA/metabolismo , Proteínas Repressoras/fisiologia , Transcriptoma
13.
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
14.
J Med Chem ; 64(11): 7110-7155, 2021 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-34060847

RESUMO

RNAs are involved in an enormous range of cellular processes, including gene regulation, protein synthesis, and cell differentiation, and dysfunctional RNAs are associated with disorders such as cancers, neurodegenerative diseases, and viral infections. Thus, the identification of compounds with the ability to bind RNAs and modulate their functions is an exciting approach for developing next-generation therapies. Numerous RNA-binding agents have been reported over the past decade, but the design of synthetic molecules with selectivity for specific RNA sequences is still in its infancy. In this perspective, we highlight recent advances in targeting RNAs with synthetic molecules, and we discuss the potential value of this approach for the development of innovative therapeutic agents.


Assuntos
Descoberta de Drogas , RNA/metabolismo , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Humanos , MicroRNAs/antagonistas & inibidores , MicroRNAs/metabolismo , RNA/antagonistas & inibidores , Precursores de RNA/metabolismo , Splicing de RNA/efeitos dos fármacos , RNA Longo não Codificante/antagonistas & inibidores , RNA Longo não Codificante/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia
15.
Int J Mol Sci ; 22(9)2021 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-34064331

RESUMO

Metastasis is the process whereby cancer cells migrate from the primary tumour site to colonise the surrounding or distant tissue or organ. Metastasis is the primary cause of cancer-related mortality and approximately half of all cancer patients present at diagnosis with some form of metastasis. Consequently, there is a clear need to better understand metastasis in order to develop new tools to combat this process. MicroRNAs (miRNAs) regulate gene expression and play an important role in cancer development and progression including in the metastatic process. Particularly important are the roles that miRNAs play in the interaction between tumour cells and non-tumoral cells of the tumour microenvironment (TME), a process mediated largely by circulating miRNAs contained primarily in extracellular vesicles (EVs). In this review, we outline the accumulating evidence for the importance of miRNAs in the communication between tumour cells and the cells of the TME in the context of the pre-metastatic and metastatic niche.


Assuntos
MicroRNAs/genética , Proteínas de Neoplasias/genética , Neoplasias/genética , RNA Neoplásico/genética , Microambiente Tumoral/genética , Animais , Comunicação Celular , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Vesículas Extracelulares/genética , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/patologia , Fibroblastos/metabolismo , Fibroblastos/patologia , Regulação Neoplásica da Expressão Gênica , Humanos , Metástase Linfática , MicroRNAs/classificação , MicroRNAs/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Células Neoplásicas Circulantes/metabolismo , Células Neoplásicas Circulantes/patologia , Precursores de RNA/genética , Precursores de RNA/metabolismo , RNA Neoplásico/metabolismo , Macrófagos Associados a Tumor/metabolismo , Macrófagos Associados a Tumor/patologia
16.
Nat Commun ; 12(1): 2899, 2021 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-34006838

RESUMO

There is urgent need for new drug regimens that more rapidly cure tuberculosis (TB). Existing TB drugs and regimens vary in treatment-shortening activity, but the molecular basis of these differences is unclear, and no existing assay directly quantifies the ability of a drug or regimen to shorten treatment. Here, we show that drugs historically classified as sterilizing and non-sterilizing have distinct impacts on a fundamental aspect of Mycobacterium tuberculosis physiology: ribosomal RNA (rRNA) synthesis. In culture, in mice, and in human studies, measurement of precursor rRNA reveals that sterilizing drugs and highly effective drug regimens profoundly suppress M. tuberculosis rRNA synthesis, whereas non-sterilizing drugs and weaker regimens do not. The rRNA synthesis ratio provides a readout of drug effect that is orthogonal to traditional measures of bacterial burden. We propose that this metric of drug activity may accelerate the development of shorter TB regimens.


Assuntos
Antituberculosos/administração & dosagem , Mycobacterium tuberculosis/efeitos dos fármacos , Precursores de RNA/metabolismo , RNA Ribossômico/metabolismo , Tuberculose/tratamento farmacológico , Animais , Modelos Animais de Doenças , Feminino , Humanos , Camundongos Endogâmicos BALB C , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/fisiologia , Precursores de RNA/genética , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Ribossômico/genética , Resultado do Tratamento , Tuberculose/diagnóstico , Tuberculose/microbiologia
17.
Nat Commun ; 12(1): 3244, 2021 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-34050143

RESUMO

N6-methyladenosine (m6A) is a modification that plays pivotal roles in RNA metabolism and function, although its functions in spliceosomal U6 snRNA remain unknown. To elucidate its role, we conduct a large-scale transcriptome analysis of a Schizosaccharomyces pombe strain lacking this modification and found a global change of pre-mRNA splicing. The most significantly impacted introns are enriched for adenosine at the fourth position pairing the m6A in U6 snRNA, and exon sequences weakly recognized by U5 snRNA. This suggests cooperative recognition of 5' splice site by U6 and U5 snRNPs, and also a role of m6A facilitating efficient recognition of the splice sites weakly interacting with U5 snRNA, indicating that U6 snRNA m6A relaxes the 5' exon constraint and allows protein sequence diversity along with explosively increasing number of introns over the course of eukaryotic evolution.


Assuntos
Regulação Fúngica da Expressão Gênica , Splicing de RNA , RNA Fúngico/metabolismo , RNA Nuclear Pequeno/metabolismo , Schizosaccharomyces/genética , Regiões 3' não Traduzidas/genética , Regiões 5' não Traduzidas/genética , Adenosina/análogos & derivados , Adenosina/metabolismo , Éxons/genética , Espectrometria de Massas , Metiltransferases/genética , Metiltransferases/isolamento & purificação , Metiltransferases/metabolismo , Precursores de RNA/genética , Precursores de RNA/metabolismo , Sítios de Splice de RNA/genética , RNA Fúngico/genética , RNA Nuclear Pequeno/genética , RNA-Seq , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Ribonucleoproteína Nuclear Pequena U4-U6 , Ribonucleoproteína Nuclear Pequena U5 , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/isolamento & purificação , Proteínas de Schizosaccharomyces pombe/metabolismo
18.
Nat Commun ; 12(1): 2661, 2021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-33976182

RESUMO

Precursor messenger RNA (pre-mRNA) splicing is an essential and tightly regulated process in eukaryotic cells; however, the regulatory mechanisms for the splicing are not well understood. Here, we characterize a RNA binding protein named FgRbp1 in Fusarium graminearum, a fungal pathogen of cereal crops worldwide. Deletion of FgRbp1 leads to reduced splicing efficiency in 47% of the F. graminearum intron-containing gene transcripts that are involved in various cellular processes including vegetative growth, development, and virulence. The human ortholog RBM42 is able to fully rescue the growth defects of ΔFgRbp1. FgRbp1 binds to the motif CAAGR in its target mRNAs, and interacts with the splicing factor FgU2AF23, a highly conserved protein involved in 3' splice site recognition, leading to enhanced recruitment of FgU2AF23 to the target mRNAs. This study demonstrates that FgRbp1 is a splicing regulator and regulates the pre-mRNA splicing in a sequence-dependent manner in F. graminearum.


Assuntos
Proteínas Fúngicas/metabolismo , Fusarium/metabolismo , Precursores de RNA/metabolismo , Splicing de RNA , Proteínas de Ligação a RNA/metabolismo , Fator de Processamento U2AF/metabolismo , Sequência de Bases , Sítios de Ligação/genética , Grão Comestível/microbiologia , Proteínas Fúngicas/genética , Fusarium/genética , Fusarium/patogenicidade , Humanos , Íntrons/genética , Ligação Proteica , Precursores de RNA/genética , Sítios de Splice de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Virulência
19.
Int J Mol Sci ; 22(9)2021 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-33946990

RESUMO

Ubiquitination and deubiquitination are protein post-translational modification processes that have been recognized as crucial mediators of many complex cellular networks, including maintaining ubiquitin homeostasis, controlling protein stability, and regulating several signaling pathways. Therefore, some of the enzymes involved in ubiquitination and deubiquitination, particularly E3 ligases and deubiquitinases, have attracted attention for drug discovery. Here, we review recent findings on USP15, one of the deubiquitinases, which regulates diverse signaling pathways by deubiquitinating vital target proteins. Even though several basic previous studies have uncovered the versatile roles of USP15 in different signaling networks, those have not yet been systematically and specifically reviewed, which can provide important information about possible disease markers and clinical applications. This review will provide a comprehensive overview of our current understanding of the regulatory mechanisms of USP15 on different signaling pathways for which dynamic reverse ubiquitination is a key regulator.


Assuntos
Processamento de Proteína Pós-Traducional/fisiologia , Transdução de Sinais/fisiologia , Proteases Específicas de Ubiquitina/fisiologia , Animais , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/metabolismo , Proteínas Morfogenéticas Ósseas/fisiologia , Complexo do Signalossomo COP9/fisiologia , Humanos , Imunidade Inata , Masculino , Camundongos , NF-kappa B/metabolismo , Neoplasias da Próstata/metabolismo , Domínios Proteicos , Isoformas de Proteínas , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Precursores de RNA/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo I/metabolismo , Proteínas Smad/metabolismo , Fator de Crescimento Transformador beta/fisiologia , Proteína Supressora de Tumor p53/metabolismo , Proteases Específicas de Ubiquitina/química , Proteases Específicas de Ubiquitina/genética , Ubiquitinação , Via de Sinalização Wnt/fisiologia , Proteínas de Xenopus/metabolismo
20.
Mol Cell ; 81(9): 1859-1860, 2021 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-33961773

RESUMO

Daniels et al. (2021) and Jourdain et al. (2021) identify LUC7L2 as a component of the U1 snRNP capable of reprogramming cellular metabolism through changes in alternative pre-mRNA splicing.


Assuntos
Processamento Alternativo , Neoplasias , Humanos , Precursores de RNA/genética , Precursores de RNA/metabolismo , Splicing de RNA , Ribonucleoproteína Nuclear Pequena U1/metabolismo
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