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1.
Nucleic Acids Res ; 49(2): 1133-1151, 2021 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-33406240

RESUMO

Alternative splicing generates multiple transcript and protein isoforms from a single gene and controls transcript intracellular localization and stability by coupling to mRNA export and nonsense-mediated mRNA decay (NMD). RNA interference (RNAi) is a potent mechanism to modulate gene expression. However, its interactions with alternative splicing are poorly understood. We used artificial microRNAs (amiRNAs, also termed shRNAmiR) to knockdown all splice variants of selected target genes in Arabidopsis thaliana. We found that splice variants, which vary by their protein-coding capacity, subcellular localization and sensitivity to NMD, are affected differentially by an amiRNA, although all of them contain the target site. Particular transcript isoforms escape amiRNA-mediated degradation due to their nuclear localization. The nuclear and NMD-sensitive isoforms mask RNAi action in alternatively spliced genes. Interestingly, Arabidopsis SPL genes, which undergo alternative splicing and are targets of miR156, are regulated in the same manner. Moreover, similar results were obtained in mammalian cells using siRNAs, indicating cross-kingdom conservation of these interactions among RNAi and splicing isoforms. Furthermore, we report that amiRNA can trigger artificial alternative splicing, thus expanding the RNAi functional repertoire. Our findings unveil novel interactions between different post-transcriptional processes in defining transcript fates and regulating gene expression.


Assuntos
Processamento Alternativo/genética , Proteínas de Arabidopsis/genética , Arabidopsis/genética , Regulação da Expressão Gênica de Plantas/genética , Técnicas de Silenciamento de Genes , Degradação do RNAm Mediada por Códon sem Sentido , Isoformas de Proteínas/genética , Interferência de RNA , Precursores de RNA/metabolismo , RNA de Plantas/metabolismo , Proteínas de Arabidopsis/biossíntese , Éxons , Genes de Plantas , Células HeLa , Humanos , MicroRNAs/genética , Plantas Geneticamente Modificadas , Isoformas de Proteínas/biossíntese , Protoplastos/metabolismo , Precursores de RNA/genética , Processamento Pós-Transcricional do RNA , RNA de Plantas/genética , Fatores de Processamento de Serina-Arginina/biossíntese , Fatores de Processamento de Serina-Arginina/genética , Transcrição Genética , Transfecção
2.
Nat Commun ; 12(1): 89, 2021 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-33397958

RESUMO

The RNA-binding protein QKI belongs to the hnRNP K-homology domain protein family, a well-known regulator of pre-mRNA alternative splicing and is associated with several neurodevelopmental disorders. Qki is found highly expressed in developing and adult hearts. By employing the human embryonic stem cell (hESC) to cardiomyocyte differentiation system and generating QKI-deficient hESCs (hESCs-QKIdel) using CRISPR/Cas9 gene editing technology, we analyze the physiological role of QKI in cardiomyocyte differentiation, maturation, and contractile function. hESCs-QKIdel largely maintain normal pluripotency and normal differentiation potential for the generation of early cardiogenic progenitors, but they fail to transition into functional cardiomyocytes. In this work, by using a series of transcriptomic, cell and biochemical analyses, and the Qki-deficient mouse model, we demonstrate that QKI is indispensable to cardiac sarcomerogenesis and cardiac function through its regulation of alternative splicing in genes involved in Z-disc formation and contractile physiology, suggesting that QKI is associated with the pathogenesis of certain forms of cardiomyopathies.


Assuntos
Processamento Alternativo/genética , Desenvolvimento Muscular/genética , Contração Miocárdica/genética , Precursores de RNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , Actinina/genética , Animais , Diferenciação Celular/genética , Embrião de Mamíferos/metabolismo , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Camundongos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Miócitos Cardíacos/ultraestrutura , Precursores de RNA/genética , Proteínas de Ligação a RNA/genética , Transcriptoma/genética
4.
Gene ; 776: 145442, 2021 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-33482283

RESUMO

Ribosome biogenesis requires the concerted activities of three nuclear RNA polymerases, (Pols) I, II, and III, to produce 25S, 18S and 5.8S ribosomal RNA (rRNA), messenger RNA (mRNA) encoding ribosomal proteins, and the 5S rRNA, respectively. The rRNA is processed and modified before being assembled with ribosomal proteins to produce a ribosome. Ribosome biogenesis requires extensive energetic investment by the cell, so it is critical that this process is tightly regulated in accord with cellular growth potential. Previous work revealed that rRNA synthesis in Saccharomyces cerevisiae is repressed prior to the cells shift from active growth (log phase) to limited/static growth (stationary phase). The mechanism(s) by which cells anticipate imminent stationary phase are unknown. In this study, we demonstrate that growing cells produce one or more compounds that accumulate in the growth medium, and that this compound induces repression of rRNA synthesis. When cells encounter this compound, rRNA synthesis is rapidly repressed. We further show that subunits of Pols I and II are degraded during the transition from log to stationary phase growth, but this degradation does not account for the observed repression of rRNA synthesis. Interestingly, repression of rRNA synthesis by spent media requires the nuclear exosome, implying that spent media stimulates rapid rRNA degradation. Together, these data suggest that yeast use quorum sensing to regulate rRNA synthesis in anticipation of high cell density in stationary phase.


Assuntos
Percepção de Quorum/genética , RNA Ribossômico/biossíntese , RNA Ribossômico/genética , Núcleo Celular/metabolismo , Precursores de RNA/genética , Estabilidade de RNA , RNA Ribossômico 5S/genética , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
5.
Nucleic Acids Res ; 49(1): 479-490, 2021 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-33330934

RESUMO

The mammalian Ate1 gene encodes an arginyl transferase enzyme with tumor suppressor function that depends on the inclusion of one of the two mutually exclusive exons (MXE), exons 7a and 7b. We report that the molecular mechanism underlying MXE splicing in Ate1 involves five conserved regulatory intronic elements R1-R5, of which R1 and R4 compete for base pairing with R3, while R2 and R5 form an ultra-long-range RNA structure spanning 30 Kb. In minigenes, single and double mutations that disrupt base pairings in R1R3 and R3R4 lead to the loss of MXE splicing, while compensatory triple mutations that restore RNA structure revert splicing to that of the wild type. In the endogenous Ate1 pre-mRNA, blocking the competing base pairings by LNA/DNA mixmers complementary to R3 leads to the loss of MXE splicing, while the disruption of R2R5 interaction changes the ratio of MXE. That is, Ate1 splicing is controlled by two independent, dynamically interacting, and functionally distinct RNA structure modules. Exon 7a becomes more included in response to RNA Pol II slowdown, however it fails to do so when the ultra-long-range R2R5 interaction is disrupted, indicating that exon 7a/7b ratio depends on co-transcriptional RNA folding. In sum, these results demonstrate that splicing is coordinated both in time and in space over very long distances, and that the interaction of these components is mediated by RNA structure.


Assuntos
Processamento Alternativo/genética , Aminoaciltransferases/genética , Conformação de Ácido Nucleico , Oligonucleotídeos Antissenso/farmacologia , Oligonucleotídeos/farmacologia , Dobramento de RNA , Precursores de RNA/genética , RNA Mensageiro/genética , Células A549 , Sequência de Bases , Linhagem Celular Tumoral , Sequência Conservada , Éxons/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Íntrons/genética , Mutagênese Sítio-Dirigida , Proteínas de Neoplasias/genética , Oligonucleotídeos/genética , Oligonucleotídeos Antissenso/genética , Especificidade de Órgãos , RNA Mensageiro/metabolismo , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico , Elongação da Transcrição Genética
6.
Gene ; 766: 145146, 2021 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-32941952

RESUMO

The removal of introns from mRNA precursors (pre-mRNAs) is an essential step in eukaryotic gene expression. The splicing machinery heavily contributes to biological complexity and especially to the ability of cells to adapt to altered cellular conditions. Hypoxia also plays a key role in the pathophysiology of many diseases, including Alzheimer's disease (AD). In the presented study, we have examined the influence of cellular hypoxia on mRNA splice variant formation from Alzheimer's disease-related Tau and APP genes in brain cells. We have shown that the hypoxic microenvironment influenced the formation of Tau mRNA splice variants, but had no effect on APP mRNA splice variant formation. Additionally, our presented results indicate that splicing factor SRSF1 but not SRSF5 alters the formation of Tau cellular mRNA splice variants in hypoxic cells. Obtained results have also shown that hypoxic brain cells possess enhanced CLK1-4 kinase mRNA levels. This study underlines that cellular hypoxia can influence disease development through changing pre-mRNA splicing.


Assuntos
Doença de Alzheimer/genética , Precursor de Proteína beta-Amiloide/genética , Hipóxia Celular/genética , RNA Mensageiro/genética , Proteínas tau/genética , Processamento Alternativo/genética , Encéfalo/metabolismo , Linhagem Celular Tumoral , Humanos , Íntrons/genética , Precursores de RNA/genética , Transcrição Genética/genética
7.
Subcell Biochem ; 96: 409-432, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33252738

RESUMO

In this chapter, the essential role of the SF3b multi-protein complex will be discussed in the context of the overall spliceosome. SF3b is critical during spliceosome assembly for recognition of the branch point (BP) adenosine and, by de facto, selection of the 3' splice site. This complex is highly dynamic, undergoing significant conformational changes upon loading of the branch duplex RNA and in its relative positioning during spliceosomal remodeling from the A, pre-B, B, Bact and B* complexes. Ultimately, during the spliceosome activation phase, SF3b must be displaced to unmask the branch point adenosine for the first splicing reaction to occur. In certain cancers, such as the hematological malignancies CML, CLL and MDS, the SF3b subunit SF3B1 is frequently mutated. Recent studies suggest these mutations lead to inappropriate branch point selection and mis-splicing events that appear to be drivers of disease. Finally, the SF3b complex is the target for at least three different classes of natural product-based inhibitors. These inhibitors bind in the BP adenosine-binding pocket and demonstrate a pre-mRNA competitive mechanism of action resulting in either intron retention or exon skipping. These compounds are extremely useful as chemical probes to isolate and characterize early stages of spliceosome assembly. They are also being explored preclinically and clinically as possible agents for hematological cancers.


Assuntos
Produtos Biológicos/farmacologia , Complexos Multiproteicos/antagonistas & inibidores , Spliceossomos/química , Spliceossomos/efeitos dos fármacos , Neoplasias Hematológicas/tratamento farmacológico , Humanos , Complexos Multiproteicos/genética , Precursores de RNA/genética , Precursores de RNA/metabolismo , Processamento de RNA , Spliceossomos/genética , Spliceossomos/metabolismo
8.
Nat Commun ; 11(1): 4455, 2020 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-32901005

RESUMO

Dysregulated alternative splicing (AS) driving carcinogenetic mitosis remains poorly understood. Here, we demonstrate that cancer metastasis-associated antigen 1 (MTA1), a well-known oncogenic chromatin modifier, broadly interacts and co-expresses with RBPs across cancers, contributing to cancerous mitosis-related AS. Using developed fCLIP-seq technology, we show that MTA1 binds abundant transcripts, preferentially at splicing-responsible motifs, influencing the abundance and AS pattern of target transcripts. MTA1 regulates the mRNA level and guides the AS of a series of mitosis regulators. MTA1 deletion abrogated the dynamic AS switches of variants for ATRX and MYBL2 at mitotic stage, which are relevant to mitosis-related tumorigenesis. MTA1 dysfunction causes defective mitotic arrest, leads to aberrant chromosome segregation, and results in chromosomal instability (CIN), eventually contributing to tumorigenesis. Currently, little is known about the RNA splicing during mitosis; here, we uncover that MTA1 binds transcripts and orchestrates dynamic splicing of mitosis regulators in tumorigenesis.


Assuntos
Carcinogênese/genética , Carcinogênese/metabolismo , Montagem e Desmontagem da Cromatina/fisiologia , Mitose/fisiologia , RNA Mensageiro/metabolismo , Proteínas Repressoras/metabolismo , Transativadores/metabolismo , Processamento Alternativo , Animais , Sítios de Ligação/genética , Montagem e Desmontagem da Cromatina/genética , Instabilidade Cromossômica , Feminino , Células HCT116 , Xenoenxertos , Humanos , Camundongos , Camundongos Nus , Mitose/genética , Neoplasias/genética , Neoplasias/metabolismo , Precursores de RNA/genética , Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/genética , Transativadores/antagonistas & inibidores , Transativadores/genética
9.
Mol Cell ; 79(6): 978-990.e5, 2020 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-32857953

RESUMO

Processing bodies (PBs) and stress granules (SGs) are prominent examples of subcellular, membraneless compartments that are observed under physiological and stress conditions, respectively. We observe that the trimeric PB protein DCP1A rapidly (within ∼10 s) phase-separates in mammalian cells during hyperosmotic stress and dissolves upon isosmotic rescue (over ∼100 s) with minimal effect on cell viability even after multiple cycles of osmotic perturbation. Strikingly, this rapid intracellular hyperosmotic phase separation (HOPS) correlates with the degree of cell volume compression, distinct from SG assembly, and is exhibited broadly by homo-multimeric (valency ≥ 2) proteins across several cell types. Notably, HOPS sequesters pre-mRNA cleavage factor components from actively transcribing genomic loci, providing a mechanism for hyperosmolarity-induced global impairment of transcription termination. Our data suggest that the multimeric proteome rapidly responds to changes in hydration and molecular crowding, revealing an unexpected mode of globally programmed phase separation and sequestration.


Assuntos
Endorribonucleases/genética , Precursores de RNA/genética , Estresse Fisiológico/genética , Transativadores/genética , Terminação da Transcrição Genética , Animais , Tamanho Celular , Sobrevivência Celular/genética , Humanos , Pressão Osmótica/fisiologia , Proteoma/genética
10.
Nucleic Acids Res ; 48(16): 9250-9261, 2020 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-32813009

RESUMO

N 6-methylation of 2'-O-methyladenosine (Am) in RNA occurs in eukaryotic cells to generate N6,2'-O-dimethyladenosine (m6Am). Identification of the methyltransferase responsible for m6Am catalysis has accelerated studies on the function of m6Am in RNA processing. While m6Am is generally found in the first transcribed nucleotide of mRNAs, the modification is also found internally within U2 snRNA. However, the writer required for catalyzing internal m6Am formation had remained elusive. By sequencing transcriptome-wide RNA methylation at single-base-resolution, we identified human METTL4 as the writer that directly methylates Am at U2 snRNA position 30 into m6Am. We found that METTL4 localizes to the nucleus and its conserved methyltransferase catalytic site is required for U2 snRNA methylation. By sequencing human cells with overexpressed Mettl4, we determined METTL4's in vivo target RNA motif specificity. In the absence of Mettl4 in human cells, U2 snRNA lacks m6Am thereby affecting a subset of splicing events that exhibit specific features such as 3' splice-site weakness and an increase in exon inclusion. These findings suggest that METTL4 methylation of U2 snRNA regulates splicing of specific pre-mRNA transcripts.


Assuntos
Adenosina/análogos & derivados , Metiltransferases/genética , Processamento de RNA/genética , RNA Nuclear Pequeno/genética , Adenosina/genética , Catálise , Éxons/genética , Humanos , Metilação , Precursores de RNA/genética , Sítios de Splice de RNA/genética , RNA Mensageiro/genética , Spliceossomos/genética
11.
Signal Transduct Target Ther ; 5(1): 125, 2020 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-32661235

RESUMO

Stress proteins (SPs) including heat-shock proteins (HSPs), RNA chaperones, and ER associated stress proteins are molecular chaperones essential for cellular homeostasis. The major functions of HSPs include chaperoning misfolded or unfolded polypeptides, protecting cells from toxic stress, and presenting immune and inflammatory cytokines. Regarded as a double-edged sword, HSPs also cooperate with numerous viruses and cancer cells to promote their survival. RNA chaperones are a group of heterogeneous nuclear ribonucleoproteins (hnRNPs), which are essential factors for manipulating both the functions and metabolisms of pre-mRNAs/hnRNAs transcribed by RNA polymerase II. hnRNPs involve in a large number of cellular processes, including chromatin remodelling, transcription regulation, RNP assembly and stabilization, RNA export, virus replication, histone-like nucleoid structuring, and even intracellular immunity. Dysregulation of stress proteins is associated with many human diseases including human cancer, cardiovascular diseases, neurodegenerative diseases (e.g., Parkinson's diseases, Alzheimer disease), stroke and infectious diseases. In this review, we summarized the biologic function of stress proteins, and current progress on their mechanisms related to virus reproduction and diseases caused by virus infections. As SPs also attract a great interest as potential antiviral targets (e.g., COVID-19), we also discuss the present progress and challenges in this area of HSP-based drug development, as well as with compounds already under clinical evaluation.


Assuntos
Antivirais/farmacologia , Betacoronavirus/efeitos dos fármacos , Infecções por Coronavirus/tratamento farmacológico , Proteínas de Choque Térmico/genética , Ribonucleoproteínas Nucleares Heterogêneas/genética , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Pneumonia Viral/tratamento farmacológico , Antivirais/síntese química , Betacoronavirus/genética , Betacoronavirus/patogenicidade , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Infecções por Coronavirus/genética , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Regulação da Expressão Gênica , Proteínas de Choque Térmico/agonistas , Proteínas de Choque Térmico/antagonistas & inibidores , Proteínas de Choque Térmico/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas/agonistas , Ribonucleoproteínas Nucleares Heterogêneas/antagonistas & inibidores , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Interações Hospedeiro-Patógeno/genética , Humanos , Terapia de Alvo Molecular/métodos , Pandemias , Pneumonia Viral/genética , Pneumonia Viral/patologia , Pneumonia Viral/virologia , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Precursores de RNA/genética , Precursores de RNA/metabolismo , Índice de Gravidade de Doença , Transdução de Sinais , Transcrição Genética/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos
12.
Nucleic Acids Res ; 48(15): 8645-8662, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32614436

RESUMO

In Trypanosoma brucei, mitochondrial pre-mRNAs undergo 3'-5' exonucleolytic processing, 3' adenylation and uridylation, 5' pyrophosphate removal, and, often, U-insertion/deletion editing. The 3' modifications are modulated by pentatricopeptide repeat (PPR) Kinetoplast Polyadenylation Factors (KPAFs). We have shown that KPAF3 binding to the 3' region stabilizes properly trimmed transcripts and stimulates their A-tailing by KPAP1 poly(A) polymerase. Conversely, poly(A) binding KPAF4 shields the nascent A-tail from uridylation and decay thereby protecting pre-mRNA upon KPAF3 displacement by editing. While editing concludes in the 5' region, KPAF1/2 dimer induces A/U-tailing to activate translation. Remarkably, 5' end recognition and pyrophosphate hydrolysis by the PPsome complex also contribute to mRNA stabilization. Here, we demonstrate that KPAF4 functions as a heterodimer with KPAF5, a protein lacking discernable motifs. We show that KPAF5 stabilizes KPAF4 to enable poly(A) tail recognition, which likely leads to mRNA stabilization during the editing process and impedes spontaneous translational activation of partially-edited transcripts. Thus, KPAF4/5 represents a poly(A) binding element of the mitochondrial polyadenylation complex. We present evidence that RNA editing substrate binding complex bridges the 5' end-bound PPsome and 3' end-bound polyadenylation complexes. This interaction may enable mRNA circularization, an apparently critical element of mitochondrial mRNA stability and quality control.


Assuntos
Polinucleotídeo Adenililtransferase/genética , Proteínas de Protozoários/genética , RNA de Protozoário/genética , Trypanosoma brucei brucei/genética , Mitocôndrias/genética , Poliadenilação/genética , Proteínas de Protozoários/química , Edição de RNA/genética , Precursores de RNA/genética , Estabilidade de RNA , RNA Mensageiro/química , RNA Mensageiro/genética , RNA de Protozoário/química , Fatores de Poliadenilação e Clivagem de mRNA/genética
13.
Nucleic Acids Res ; 48(15): 8349-8359, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32621610

RESUMO

Alternative splicing (AS) and alternative polyadenylation (APA) generate diverse transcripts in mammalian genomes during development and differentiation. Epigenetic marks such as trimethylation of histone H3 lysine 36 (H3K36me3) and DNA methylation play a role in generating transcriptome diversity. Intragenic CpG islands (iCGIs) and their corresponding host genes exhibit dynamic epigenetic and gene expression patterns during development and between different tissues. We hypothesise that iCGI-associated H3K36me3, DNA methylation and transcription can influence host gene AS and/or APA. We investigate H3K36me3 and find that this histone mark is not a major regulator of AS or APA in our model system. Genomewide, we identify over 4000 host genes that harbour an iCGI in the mammalian genome, including both previously annotated and novel iCGI/host gene pairs. The transcriptional activity of these iCGIs is tissue- and developmental stage-specific and, for the first time, we demonstrate that the premature termination of host gene transcripts upstream of iCGIs is closely correlated with the level of iCGI transcription in a DNA-methylation independent manner. These studies suggest that iCGI transcription, rather than H3K36me3 or DNA methylation, interfere with host gene transcription and pre-mRNA processing genomewide and contributes to the spatiotemporal diversification of both the transcriptome and proteome.


Assuntos
Epigênese Genética , Processamento de Proteína Pós-Traducional/genética , Precursores de RNA/genética , Transcrição Genética , Animais , Diferenciação Celular/genética , Cromatina/genética , Ilhas de CpG/genética , Metilação de DNA/genética , Genoma/genética , Código das Histonas/genética , Humanos , Regiões Promotoras Genéticas , Pseudogenes/genética , Precursores de RNA/metabolismo
14.
RNA ; 26(10): 1389-1399, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32522889

RESUMO

Alternative splicing is responsible for much of the transcriptomic and proteomic diversity observed in eukaryotes and involves combinatorial regulation by many cis-acting elements and trans-acting factors. SR and hnRNP splicing regulatory proteins often have opposing effects on splicing efficiency depending on where they bind the pre-mRNA relative to the splice site. Position-dependent splicing repression occurs at spliceosomal E-complex, suggesting that U1 snRNP binds but cannot facilitate higher order spliceosomal assembly. To test the hypothesis that the structure of U1 snRNA changes during activation or repression, we developed a method to structure-probe native U1 snRNP in enriched conformations that mimic activated or repressed spliceosomal E-complexes. While the core of U1 snRNA is highly structured, the 5' end of U1 snRNA shows different SHAPE reactivities and psoralen crosslinking efficiencies depending on where splicing regulatory elements are located relative to the 5' splice site. A motif within the 5' splice site binding region of U1 snRNA is more reactive toward SHAPE electrophiles when repressors are bound, suggesting U1 snRNA is bound, but less base-paired. These observations demonstrate that splicing regulators modulate splice site selection allosterically.


Assuntos
Regulação Alostérica/genética , Processamento Alternativo/genética , RNA Nuclear Pequeno/genética , Ribonucleoproteína Nuclear Pequena U1/genética , Spliceossomos/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas/genética , Proteômica/métodos , Precursores de RNA/genética , Sítios de Splice de RNA/genética , RNA Mensageiro/genética
15.
RNA ; 26(10): 1345-1359, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32554553

RESUMO

Metazoan replication-dependent histone pre-mRNAs are cleaved at the 3' end by U7 snRNP, an RNA-guided endonuclease that contains U7 snRNA, seven proteins of the Sm ring, FLASH, and four polyadenylation factors: symplekin, CPSF73, CPSF100, and CstF64. A fully recombinant U7 snRNP was recently reconstituted from all 13 components for functional and structural studies and shown to accurately cleave histone pre-mRNAs. Here, we analyzed the activity of recombinant U7 snRNP in more detail. We demonstrate that in addition to cleaving histone pre-mRNAs endonucleolytically, reconstituted U7 snRNP acts as a 5'-3' exonuclease that degrades the downstream product generated from histone pre-mRNAs as a result of the endonucleolytic cleavage. Surprisingly, recombinant U7 snRNP also acts as an endonuclease on single-stranded DNA substrates. All these activities depend on the ability of U7 snRNA to base-pair with the substrate and on the presence of the amino-terminal domain (NTD) of symplekin in either cis or trans, and are abolished by mutations within the catalytic center of CPSF73, or by binding of the NTD to the SSU72 phosphatase of RNA polymerase II. Altogether, our results demonstrate that recombinant U7 snRNP functionally mimics its endogenous counterpart and provide evidence that CPSF73 is both an endonuclease and a 5'-3' exonuclease, consistent with the activity of other members of the ß-CASP family. Our results also raise the intriguing possibility that CPSF73 may be involved in some aspects of DNA metabolism in vivo.


Assuntos
Fator de Especificidade de Clivagem e Poliadenilação/genética , Endonucleases/genética , Exonucleases/genética , RNA Nuclear Pequeno/genética , Ribonucleoproteína Nuclear Pequena U7/genética , Animais , Histonas/genética , Camundongos , Precursores de RNA/genética , Processamento Pós-Transcricional do RNA/genética
16.
Nat Commun ; 11(1): 2837, 2020 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-32503992

RESUMO

Group II introns are ubiquitous self-splicing ribozymes and retrotransposable elements evolutionarily and chemically related to the eukaryotic spliceosome, with potential applications as gene-editing tools. Recent biochemical and structural data have captured the intron in multiple conformations at different stages of catalysis. Here, we employ enzymatic assays, X-ray crystallography, and molecular simulations to resolve the spatiotemporal location and function of conformational changes occurring between the first and the second step of splicing. We show that the first residue of the highly-conserved catalytic triad is protonated upon 5'-splice-site scission, promoting a reversible structural rearrangement of the active site (toggling). Protonation and active site dynamics induced by the first step of splicing facilitate the progression to the second step. Our insights into the mechanism of group II intron splicing parallels functional data on the spliceosome, thus reinforcing the notion that these evolutionarily-related molecular machines share the same enzymatic strategy.


Assuntos
Íntrons/genética , Precursores de RNA/metabolismo , Processamento de RNA , RNA Bacteriano/metabolismo , Spliceossomos/metabolismo , Bacillaceae/genética , Domínio Catalítico/genética , Cristalografia por Raios X , Simulação de Dinâmica Molecular , Mutagênese , Conformação de Ácido Nucleico , Precursores de RNA/genética , RNA Bacteriano/genética , Análise Espaço-Temporal
17.
Nucleic Acids Res ; 48(11): 6294-6309, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32402057

RESUMO

Recognition of highly degenerate mammalian splice sites by the core spliceosomal machinery is regulated by several protein factors that predominantly bind exonic splicing motifs. These are postulated to be single-stranded in order to be functional, yet knowledge of secondary structural features that regulate the exposure of exonic splicing motifs across the transcriptome is not currently available. Using transcriptome-wide RNA structural information we show that retained introns in mouse are commonly flanked by a short (≲70 nucleotide), highly base-paired segment upstream and a predominantly single-stranded exonic segment downstream. Splicing assays with select pre-mRNA substrates demonstrate that loops immediately upstream of the introns contain pre-mRNA-specific splicing enhancers, the substitution or hybridization of which impedes splicing. Additionally, the exonic segments flanking the retained introns appeared to be more enriched in a previously identified set of hexameric exonic splicing enhancer (ESE) sequences compared to their spliced counterparts, suggesting that base-pairing in the exonic segments upstream of retained introns could be a means for occlusion of ESEs. The upstream exonic loops of the test substrate promoted recruitment of splicing factors and consequent pre-mRNA structural remodeling, leading up to assembly of the early spliceosome. These results suggest that disruption of exonic stem-loop structures immediately upstream (but not downstream) of the introns regulate alternative splicing events, likely through modulating accessibility of splicing factors.


Assuntos
Pareamento de Bases , Éxons , Íntrons , Processamento de RNA , Adenoviridae/genética , Animais , Sequência de Bases , Elementos Facilitadores Genéticos , Éxons/genética , Inativação Gênica , Íntrons/genética , Camundongos , Células-Tronco Embrionárias Murinas , Mutação , Precursores de RNA/genética , Precursores de RNA/metabolismo , Processamento de RNA/genética , Spliceossomos/metabolismo , Transcriptoma/genética , Globinas beta/genética
18.
PLoS One ; 15(5): e0233672, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32469945

RESUMO

Agents that modulate pre-mRNA splicing are of interest in multiple therapeutic areas, including cancer. We report our recent screening results with the application of a cell-based Triple Exon Skipping Luciferase Reporter (TESLR) using a library that is composed of FDA approved drugs, clinical compounds, and mechanistically characterized tool compounds. Confirmatory assays showed that three clinical antitumor therapeutic candidates (milciclib, PF-3758309 and PF-562271) are potent splicing modulators and that these drugs are, in fact, nanomolar inhibitors of multiple kinases involved in the regulation the spliceosome. We also report the identification of new SF3B1 antagonists (sudemycinol C and E) and show that these antagonists can be used to develop a displacement assay for SF3B1 small molecule ligands. These results further support the broad potential for the development of agents that target the spliceosome for the treatment of cancer and other diseases, as well as new avenues for the discovery of new chemotherapeutic agents for a range of diseases.


Assuntos
Antineoplásicos/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Éxons/efeitos dos fármacos , Precursores de RNA/genética , Processamento de RNA/efeitos dos fármacos , Linhagem Celular Tumoral , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Inibidores de Proteínas Quinases/farmacologia
19.
Nucleic Acids Res ; 48(12): 6839-6854, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32449937

RESUMO

SERRATE/ARS2 is a conserved RNA effector protein involved in transcription, processing and export of different types of RNAs. In Arabidopsis, the best-studied function of SERRATE (SE) is to promote miRNA processing. Here, we report that SE interacts with the nuclear exosome targeting (NEXT) complex, comprising the RNA helicase HEN2, the RNA binding protein RBM7 and one of the two zinc-knuckle proteins ZCCHC8A/ZCCHC8B. The identification of common targets of SE and HEN2 by RNA-seq supports the idea that SE cooperates with NEXT for RNA surveillance by the nuclear exosome. Among the RNA targets accumulating in absence of SE or NEXT are miRNA precursors. Loss of NEXT components results in the accumulation of pri-miRNAs without affecting levels of miRNAs, indicating that NEXT is, unlike SE, not required for miRNA processing. As compared to se-2, se-2 hen2-2 double mutants showed increased accumulation of pri-miRNAs, but partially restored levels of mature miRNAs and attenuated developmental defects. We propose that the slow degradation of pri-miRNAs caused by loss of HEN2 compensates for the poor miRNA processing efficiency in se-2 mutants, and that SE regulates miRNA biogenesis through its double contribution in promoting miRNA processing but also pri-miRNA degradation through the recruitment of the NEXT complex.


Assuntos
Proteínas de Arabidopsis/genética , MicroRNAs/genética , RNA Helicases/genética , Processamento Pós-Transcricional do RNA/genética , Proteínas de Ligação a RNA/genética , Arabidopsis/genética , Proteínas de Ligação ao Cálcio/genética , Núcleo Celular/genética , Exossomos/genética , Regulação da Expressão Gênica de Plantas/genética , Mutação/genética , Precursores de RNA/genética , Estabilidade de RNA/genética , Ribonuclease III/genética
20.
RNA ; 26(9): 1081-1085, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32439718

RESUMO

We reported previously that, in budding yeast, transcription rate affects both the efficiency and fidelity of pre-mRNA splicing, especially of ribosomal protein transcripts. Here, we report that the majority of ribosomal protein transcripts with non-consensus 5' splice sites are spliced less efficiently when transcription is faster, and more efficiently with slower transcription. These results support the "window of opportunity" model, and we suggest a possible mechanism to explain these findings.


Assuntos
Processamento de RNA/genética , Saccharomycetales/genética , Precursores de RNA/genética , Ribossomos/genética , Transcrição Genética/genética
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