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1.
Int J Mol Sci ; 21(13)2020 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-32645951

RESUMO

The SARS-CoV-2 virus is a recently-emerged zoonotic pathogen already well adapted to transmission and replication in humans. Although the mutation rate is limited, recently introduced mutations in SARS-CoV-2 have the potential to alter viral fitness. In addition to amino acid changes, mutations could affect RNA secondary structure critical to viral life cycle, or interfere with sequences targeted by host miRNAs. We have analysed subsets of genomes from SARS-CoV-2 isolates from around the globe and show that several mutations introduce changes in Watson-Crick pairing, with resultant changes in predicted secondary structure. Filtering to targets matching miRNAs expressed in SARS-CoV-2-permissive host cells, we identified ten separate target sequences in the SARS-CoV-2 genome; three of these targets have been lost through conserved mutations. A genomic site targeted by the highly abundant miR-197-5p, overexpressed in patients with cardiovascular disease, is lost by a conserved mutation. Our results are compatible with a model that SARS-CoV-2 replication within the human host is constrained by host miRNA defences. The impact of these and further mutations on secondary structures, miRNA targets or potential splice sites offers a new context in which to view future SARS-CoV-2 evolution, and a potential platform for engineering conditional attenuation to vaccine development, as well as providing a better understanding of viral tropism and pathogenesis.


Assuntos
Betacoronavirus/genética , Genoma Viral , MicroRNAs/metabolismo , RNA Viral/química , Regiões 3' não Traduzidas , Sequência de Bases , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Bases de Dados Genéticas , Humanos , MicroRNAs/química , MicroRNAs/genética , Mutação , Conformação de Ácido Nucleico , Pandemias , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Sítios de Splice de RNA , Processamento de RNA , Alinhamento de Sequência , Proteínas não Estruturais Virais/genética , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/metabolismo
2.
Rinsho Ketsueki ; 61(6): 634-642, 2020.
Artigo em Japonês | MEDLINE | ID: mdl-32624537

RESUMO

Dysregulation of pre-mRNA splicing and transcription is a key step in gene expression control in patients with leukemia. Herein, we discuss the occurrence of frequent overlap of mutations affecting epigenetic regulation and pre-mRNA splicing in patients with leukemia, which together promote leukemogenesis through coordinated effects on the epigenome and pre-mRNA splicing. In particular, we have determined an important pathogenic role of cross-talk between altered epigenetic state and pre-mRNA splicing, provided functional evidence that mutations in pre-mRNA splicing factors drive leukemia development, and uncovered spliceosomal changes as a novel mediator of IDH2 mutant leukemogenesis. By isolating specific pre-mRNA splicing events that functionally contribute to IDH2/SRSF2 double-mutant leukemogenesis, we found that loss of the Integrator complex plays an important role in leukemia development. Our studies provided new evidence that defects in the Integrator complex remarkably affect several gene expression programs associated with hematopoietic differentiation and signaling pathways via transcriptional pause-release dysregulation, blockade of myeloid differentiation, and promotion of leukemogenesis in the Idh2 mutant background in vivo. Moreover, our results revealed important translational implications, given the substantial efforts to pharmacologically inhibit mutant IDH1/2 and splicing factors.


Assuntos
Neoplasias Hematológicas , Processamento de RNA , Epigênese Genética , Humanos , Mutação , Fatores de Processamento de Serina-Arginina
3.
Rinsho Ketsueki ; 61(6): 643-650, 2020.
Artigo em Japonês | MEDLINE | ID: mdl-32624538

RESUMO

Splicing factor 3b subunit 1 (SF3B1) is the most commonly mutated RNA splicing factor identified in myelodysplastic syndrome (MDS), chronic lymphocytic leukemia, and uveal melanoma. The mechanisms by which SF3B1 mutations promote malignancy are poorly understood. Here, we integrated pan-cancer RNA sequencing to identify mutant SF3B1-dependent aberrant splicing events with a positive CRISPR screen to prioritize alterations that functionally promote oncogenesis. Our results indicated that diverse, recurrent SF3B1 mutations converge on the repression of bromodomain containing 9 (BRD9), a core component of the recently described non-canonical barrier-to-autointegration factor complex (ncBAF). Mutant SF3B1 recognizes intronic sequences within BRD9 as exons, thereby permitting inclusion of aberrant sequence (i.e., poison exon) that will result in the degradation of BRD9 mRNA. BRD9 depletion results in significant loss of ncBAF at CCCTC-binding factor (CTCF)-binding loci but has no impact on the localization of canonical BAF. These actions resulted in disturbed myeloid/erythroid differentiation and promoted the development of MDS and melanoma. Of note, correcting BRD9 mis-splicing in SF3B1-mutant cells with antisense oligonucleotides (ASOs), by targeting the poison exon with CRISPR-directed mutagenesis, or via the use of spliceosomal inhibitors are all potential therapeutic options. Our results implicate disruption of ncBAF as a critical factor promoting the development of the diverse array of cancers that carry SF3B1 mutations and suggest a mechanism-based therapeutic approach for treating these malignancies.


Assuntos
Processamento de RNA , Carcinogênese , Humanos , Mutação , Fosfoproteínas , Fatores de Processamento de RNA , RNA Mensageiro , Fatores de Transcrição
4.
Gene ; 753: 144793, 2020 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-32446918

RESUMO

BACKGROUND: Fragile X syndrome (FXS) is a monogenic disorder and a common cause of intellectual disability (ID). Up to now, very few pathological variants other than the typical CGG-repeat expansion have been reported in the FMR1 gene. METHODS: A panel of 56 intellectual disability (ID) genes including the FMR1 gene was sequenced in a cohort of 300 patients with unexplained ID. To determine the effect of a new FMR1 variant, total RNA from peripheral blood cells was reverse transcribed, amplified by polymerase chain reaction and sequenced. RESULTS: We report a novel G to A point variant (c.801G > A) located at the last nucleotide of exon 8 in the FMR1 gene in one patient with ID. Direct sequencing of the RT-PCR products revealed that the transcript from the allele with G to A variant skips exon 8 entirely, resulting in a joining of exons 7 and 9. Skipping of exon 8 may result in an abnormal FMR1 protein (FMRP), which removes the highly conserved region that encoding the KH1 domain of FMRP. CONCLUSIONS: This report describes for the first time that a synonymous variant in the FMR1 gene is associated with an error in mRNA processing, leading preferentially to the production of an aberrant transcript without exon 8. This splice variant was associated with an unspecific clinical presentation, suggesting the need for more detailed investigation of silent variants in ID patients with a large spectrum of phenotypes.


Assuntos
Proteína do X Frágil de Retardo Mental/genética , Síndrome do Cromossomo X Frágil/genética , Deficiência Intelectual/genética , Adolescente , Adulto , Alelos , Estudos de Coortes , Éxons , Humanos , Masculino , Mutação , Fenótipo , Polimorfismo de Nucleotídeo Único , Domínios Proteicos , Processamento de RNA , Análise de Sequência de RNA/métodos , Mutação Silenciosa , Expansão das Repetições de Trinucleotídeos
5.
Nature ; 583(7816): 469-472, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32408336

RESUMO

A new coronavirus was recently discovered and named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Infection with SARS-CoV-2 in humans causes coronavirus disease 2019 (COVID-19) and has been rapidly spreading around the globe1,2. SARS-CoV-2 shows some similarities to other coronaviruses; however, treatment options and an understanding of how SARS-CoV-2 infects cells are lacking. Here we identify the host cell pathways that are modulated by SARS-CoV-2 and show that inhibition of these pathways prevents viral replication in human cells. We established a human cell-culture model for infection with a clinical isolate of SARS-CoV-2. Using this cell-culture system, we determined the infection profile of SARS-CoV-2 by translatome3 and proteome proteomics at different times after infection. These analyses revealed that SARS-CoV-2 reshapes central cellular pathways such as translation, splicing, carbon metabolism, protein homeostasis (proteostasis) and nucleic acid metabolism. Small-molecule inhibitors that target these pathways prevented viral replication in cells. Our results reveal the cellular infection profile of SARS-CoV-2 and have enabled the identification of drugs that inhibit viral replication. We anticipate that our results will guide efforts to understand the molecular mechanisms that underlie the modulation of host cells after infection with SARS-CoV-2. Furthermore, our findings provide insights for the development of therapies for the treatment of COVID-19.


Assuntos
Betacoronavirus/efeitos dos fármacos , Betacoronavirus/metabolismo , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/metabolismo , Terapia de Alvo Molecular , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/metabolismo , Proteômica , Betacoronavirus/genética , Betacoronavirus/crescimento & desenvolvimento , Células CACO-2 , Carbono/metabolismo , Infecções por Coronavirus/genética , Infecções por Coronavirus/virologia , Avaliação Pré-Clínica de Medicamentos , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Interações Hospedeiro-Patógeno/genética , Humanos , Técnicas In Vitro , Cinética , Pandemias , Pneumonia Viral/genética , Pneumonia Viral/virologia , Biossíntese de Proteínas/efeitos dos fármacos , Proteoma/metabolismo , Proteostase , Processamento de RNA , Fatores de Tempo , Replicação Viral/efeitos dos fármacos
6.
PLoS One ; 15(4): e0229315, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32320410

RESUMO

Mutations in the splicing machinery have been implicated in a number of human diseases. Most notably, the U2 small nuclear ribonucleoprotein (snRNP) component SF3b1 has been found to be frequently mutated in blood cancers such as myelodysplastic syndromes (MDS). SF3b1 is a highly conserved HEAT repeat (HR)-containing protein and most of these blood cancer mutations cluster in a hot spot located in HR4-8. Recently, a second mutational hotspot has been identified in SF3b1 located in HR9-12 and is associated with acute myeloid leukemias, bladder urothelial carcinomas, and uterine corpus endometrial carcinomas. The consequences of these mutations on SF3b1 functions during splicing have not yet been tested. We incorporated the corresponding mutations into the yeast homolog of SF3b1 and tested their impact on splicing. We find that all of these HR9-12 mutations can support splicing in yeast, and this suggests that none of them are loss of function alleles in humans. The Hsh155V502F mutation alters splicing of several pre-mRNA reporters containing weak branch sites as well as a genetic interaction with Prp2 and physical interactions with Prp5 and Prp3. The ability of a single allele of Hsh155 to perturb interactions with multiple factors functioning at different stages of the splicing reaction suggests that some SF3b1-mutant disease phenotypes may have a complex origin on the spliceosome.


Assuntos
Mutação/genética , Fosfoproteínas/genética , Precursores de RNA/genética , Fatores de Processamento de RNA/genética , Processamento de RNA/genética , Sequências Repetitivas de Aminoácidos , Ribonucleoproteína Nuclear Pequena U2/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Sequência Consenso/genética , Epistasia Genética , Humanos , Fosfoproteínas/química , Ligação Proteica , Fatores de Processamento de RNA/química , Ribonucleoproteína Nuclear Pequena U2/química , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/química
7.
Nat Commun ; 11(1): 1768, 2020 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-32286305

RESUMO

The differential production of transcript isoforms from gene loci is a key cellular mechanism. Yet, its impact in protein production remains an open question. Here, we describe ORQAS (ORF quantification pipeline for alternative splicing), a pipeline for the translation quantification of individual transcript isoforms using ribosome-protected mRNA fragments (ribosome profiling). We find evidence of translation for 40-50% of the expressed isoforms in human and mouse, with 53% of the expressed genes having more than one translated isoform in human, and 33% in mouse. Differential splicing analysis revealed that about 40% of the splicing changes at RNA level are concordant with changes in translation. Furthermore, orthologous cassette exons between human and mouse preserve the directionality of the change, and are enriched in microexons in a comparison between glia and glioma. ORQAS leverages ribosome profiling to uncover a widespread and evolutionarily conserved impact of differential splicing on translation, particularly of microexon-containing isoforms.


Assuntos
Proteoma/metabolismo , Processamento de RNA/fisiologia , Processamento Alternativo/genética , Animais , Biologia Computacional/métodos , Humanos , Camundongos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteoma/genética , Processamento de RNA/genética , Ribossomos/genética , Ribossomos/metabolismo , Transcriptoma/genética
8.
Proc Natl Acad Sci U S A ; 117(19): 10305-10312, 2020 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-32332164

RESUMO

The gene encoding the core spliceosomal protein SF3B1 is the most frequently mutated gene encoding a splicing factor in a variety of hematologic malignancies and solid tumors. SF3B1 mutations induce use of cryptic 3' splice sites (3'ss), and these splicing errors contribute to tumorigenesis. However, it is unclear how widespread this type of cryptic 3'ss usage is in cancers and what is the full spectrum of genetic mutations that cause such missplicing. To address this issue, we performed an unbiased pan-cancer analysis to identify genetic alterations that lead to the same aberrant splicing as observed with SF3B1 mutations. This analysis identified multiple mutations in another spliceosomal gene, SUGP1, that correlated with significant usage of cryptic 3'ss known to be utilized in mutant SF3B1 expressing cells. Remarkably, this is consistent with recent biochemical studies that identified a defective interaction between mutant SF3B1 and SUGP1 as the molecular defect responsible for cryptic 3'ss usage. Experimental validation revealed that five different SUGP1 mutations completely or partially recapitulated the 3'ss defects. Our analysis suggests that SUGP1 mutations in cancers can induce missplicing identical or similar to that observed in mutant SF3B1 cancers.


Assuntos
Biologia Computacional/métodos , Mutação , Neoplasias/genética , Fosfoproteínas/genética , Sítios de Splice de RNA , Fatores de Processamento de RNA/genética , Processamento de RNA , Análise Mutacional de DNA , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias/patologia , Spliceossomos
9.
Nat Commun ; 11(1): 1931, 2020 04 22.
Artigo em Inglês | MEDLINE | ID: mdl-32321912

RESUMO

Enhancing the efficacy of proteasome inhibitors (PI) is a central goal in myeloma therapy. We proposed that signaling-level responses after PI may reveal new mechanisms of action that can be therapeutically exploited. Unbiased phosphoproteomics after treatment with the PI carfilzomib surprisingly demonstrates the most prominent phosphorylation changes on splicing related proteins. Spliceosome modulation is invisible to RNA or protein abundance alone. Transcriptome analysis after PI demonstrates broad-scale intron retention, suggestive of spliceosome interference, as well as specific alternative splicing of protein homeostasis machinery components. These findings lead us to evaluate direct spliceosome inhibition in myeloma, which synergizes with carfilzomib and shows potent anti-tumor activity. Functional genomics and exome sequencing further support the spliceosome as a specific vulnerability in myeloma. Our results propose splicing interference as an unrecognized modality of PI mechanism, reveal additional modes of spliceosome modulation, and suggest spliceosome targeting as a promising therapeutic strategy in myeloma.


Assuntos
Mieloma Múltiplo/tratamento farmacológico , Inibidores de Proteassoma/administração & dosagem , Spliceossomos/efeitos dos fármacos , Animais , Antineoplásicos/administração & dosagem , Feminino , Humanos , Camundongos , Mieloma Múltiplo/genética , Mieloma Múltiplo/metabolismo , Oligopeptídeos/administração & dosagem , Processamento de RNA/efeitos dos fármacos , Spliceossomos/genética , Spliceossomos/metabolismo , Spliceossomos/microbiologia
10.
PLoS One ; 15(4): e0231450, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32302342

RESUMO

RNA-binding proteins (RBPs) control many types of post-transcriptional regulation, including mRNA splicing, mRNA stability, and translational efficiency, by directly binding to their target RNAs and their mutation and dysfunction are often associated with several human neurological diseases and tumorigenesis. Crosslinking immunoprecipitation (CLIP), coupled with high-throughput sequencing (HITS-CLIP), is a powerful technique for investigating the molecular mechanisms underlying disease pathogenesis by comprehensive identification of RBP target sequences at the transcriptome level. However, HITS-CLIP protocol is still required for some optimization due to experimental complication, low efficiency and time-consuming, whose library has to be generated from very small amounts of RNAs. Here we improved a more efficient, rapid, and reproducible CLIP method by optimizing BrdU-CLIP. Our protocol produced a 10-fold greater yield of pre-amplified CLIP library, which resulted in a low duplicate rate of CLIP-tag reads because the number of PCR cycles required for library amplification was reduced. Variance of the yields was also reduced, and the experimental period was shortened by 2 days. Using this, we validated IL-6 expression by a nuclear RBP, HNRNPU, which directly binds the 3'-UTR of IL-6 mRNA in HeLa cells. Importantly, this interaction was only observed in the cytoplasmic fraction, suggesting a role of cytoplasmic HNRNPU in mRNA stability control. This optimized method enables us to accurately identify target genes and provides a snapshot of the protein-RNA interactions of nucleocytoplasmic shuttling RBPs.


Assuntos
Núcleo Celular/metabolismo , Citoplasma/metabolismo , Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , Regiões 3' não Traduzidas/fisiologia , Transporte Ativo do Núcleo Celular/fisiologia , Linhagem Celular Tumoral , Sequenciamento de Cromatina por Imunoprecipitação/métodos , Perfilação da Expressão Gênica/métodos , Biblioteca Gênica , Células HeLa , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Imunoprecipitação/métodos , Interleucina-6/metabolismo , Processamento de RNA/fisiologia , RNA Mensageiro/metabolismo , Análise de Sequência de RNA/métodos , Transcriptoma/fisiologia
11.
PLoS Genet ; 16(4): e1008643, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32294086

RESUMO

Hereditary hearing loss is challenging to diagnose because of the heterogeneity of the causative genes. Further, some genes involved in hereditary hearing loss have yet to be identified. Using whole-exome analysis of three families with congenital, severe-to-profound hearing loss, we identified a missense variant of SLC12A2 in five affected members of one family showing a dominant inheritance mode, along with de novo splice-site and missense variants of SLC12A2 in two sporadic cases, as promising candidates associated with hearing loss. Furthermore, we detected another de novo missense variant of SLC12A2 in a sporadic case. SLC12A2 encodes Na+, K+, 2Cl- cotransporter (NKCC) 1 and plays critical roles in the homeostasis of K+-enriched endolymph. Slc12a2-deficient mice have congenital, profound deafness; however, no human variant of SLC12A2 has been reported as associated with hearing loss. All identified SLC12A2 variants mapped to exon 21 or its 3'-splice site. In vitro analysis indicated that the splice-site variant generates an exon 21-skipped SLC12A2 mRNA transcript expressed at much lower levels than the exon 21-included transcript in the cochlea, suggesting a tissue-specific role for the exon 21-encoded region in the carboy-terminal domain. In vitro functional analysis demonstrated that Cl- influx was significantly decreased in all SLC12A2 variants studied. Immunohistochemistry revealed that SLC12A2 is located on the plasma membrane of several types of cells in the cochlea, including the strial marginal cells, which are critical for endolymph homeostasis. Overall, this study suggests that variants affecting exon 21 of the SLC12A2 transcript are responsible for hereditary hearing loss in humans.


Assuntos
Perda Auditiva Neurossensorial/congênito , Perda Auditiva Neurossensorial/genética , Mutação , Domínios Proteicos/genética , Membro 2 da Família 12 de Carreador de Soluto/química , Membro 2 da Família 12 de Carreador de Soluto/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Cloretos/metabolismo , Cóclea/metabolismo , Cóclea/patologia , Surdez/congênito , Surdez/genética , Éxons/genética , Feminino , Expressão Gênica , Células HEK293 , Humanos , Lactente , Macaca fascicularis , Masculino , Linhagem , Processamento de RNA , RNA Mensageiro/análise , RNA Mensageiro/genética , Membro 2 da Família 12 de Carreador de Soluto/metabolismo
12.
Mol Cell ; 78(1): 5-8, 2020 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-32243831

RESUMO

Elegant studies by Hasler et al. (2020) and Wang et al. (2020) uncover a novel role of LARP7 in facilitating the 2'-O-methylation of the spliceosomal U6 snRNA, which is functionally required for fidelity of pre-mRNA splicing and development of male germ cells.


Assuntos
Processamento de RNA , RNA Nuclear Pequeno , Animais , Sequência de Bases , Masculino , Camundongos , Conformação de Ácido Nucleico , Espermatogênese
13.
Int J Mol Sci ; 21(7)2020 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-32276369

RESUMO

Sex determination and differentiation are nearly universal to all eukaryotic organisms, encompassing diverse systems and mechanisms. Here, we identified a spliceosomal protein gene BmSPX involved in sex determination of the lepidopeteran insect, Bombyx mori. In a transgenic silkworm line that overexpressed the BmSPX gene, transgenic silkworm males exhibited differences in their external genitalia compared to wild-type males, but normal internal genitalia. Additionally, transgenic silkworm females exhibited a developmental disorder of the reproductive organs. Upregulation of BmSPX significantly increased the expression levels of sex-determining genes (BmMasc and BmIMP) and reduced the female-type splice isoform of Bmdsx, which is a key switch gene downstream of the sex-determination pathway. Additionally, co-immunoprecipitation assays confirmed an interaction between the BmSPX protein and BmPSI, an upstream regulatory factor of Bmdsx. Quantitative real-time PCR showed that BmSPX over-expression upregulated the expression of the Hox gene abdominal-B (Adb-B), which is required for specification of the posterior abdomen, external genitalia, and gonads of insects, as well as the genes in the Receptor Tyrosine Kinase (RTK) signaling pathway. In conclusion, our study suggested the involvement of BmSPX, identified as a novel regulatory factor, in the sex-determination pathway and regulation of reproductive organ development in silkworms.


Assuntos
Bombyx/fisiologia , Genitália/metabolismo , Proteínas de Insetos/metabolismo , Processos de Determinação Sexual , Animais , Animais Geneticamente Modificados , Bombyx/genética , Regulação da Expressão Gênica , Gônadas/metabolismo , Proteínas de Insetos/fisiologia , Masculino , Processamento de RNA , Spliceossomos
14.
Nucleic Acids Res ; 48(8): 4572-4584, 2020 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-32196113

RESUMO

The single G protein of the spliceosome, Snu114, has been proposed to facilitate splicing as a molecular motor or as a regulatory G protein. However, available structures of spliceosomal complexes show Snu114 in the same GTP-bound state, and presently no Snu114 GTPase-regulatory protein is known. We determined a crystal structure of Snu114 with a Snu114-binding region of the Prp8 protein, in which Snu114 again adopts the same GTP-bound conformation seen in spliceosomes. Snu114 and the Snu114-Prp8 complex co-purified with endogenous GTP. Snu114 exhibited weak, intrinsic GTPase activity that was abolished by the Prp8 Snu114-binding region. Exchange of GTP-contacting residues in Snu114, or of Prp8 residues lining the Snu114 GTP-binding pocket, led to temperature-sensitive yeast growth and affected the same set of splicing events in vivo. Consistent with dynamic Snu114-mediated protein interactions during splicing, our results suggest that the Snu114-GTP-Prp8 module serves as a relay station during spliceosome activation and disassembly, but that GTPase activity may be dispensable for splicing.


Assuntos
Guanosina Trifosfato/química , Processamento de RNA , Ribonucleoproteína Nuclear Pequena U4-U6/química , Ribonucleoproteína Nuclear Pequena U5/química , Proteínas de Saccharomyces cerevisiae/química , GTP Fosfo-Hidrolases/química , GTP Fosfo-Hidrolases/metabolismo , Modelos Moleculares , Conformação Proteica , Ribonucleoproteína Nuclear Pequena U4-U6/metabolismo , Ribonucleoproteína Nuclear Pequena U5/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
15.
RNA ; 26(5): 664-673, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32127385

RESUMO

Group II introns are mobile genetic elements that perform both self-splicing and intron mobility reactions. These ribozymes are comprised of a catalytic RNA core that binds to an intron-encoded protein (IEP) to form a ribonucleoprotein (RNP) complex. Splicing proceeds through two competing reactions: hydrolysis or branching. Group IIC intron ribozymes have a minimal RNA architecture, and splice almost exclusively through hydrolysis in ribozyme reactions. Addition of the IEP allows the splicing reaction to form branched lariat RNPs capable of intron mobility. Here we examine ribozyme splicing, IEP-dependent splicing, and mobility reactions of a group IIC intron from the thermophilic bacterium Thermoanerobacter italicus (Ta.it.I1). We show that Ta.it.I1 is highly active for ribozyme activity, forming linear hydrolytic intron products. Addition of purified IEP switches activity to the canonical lariat forming splicing reaction. We demonstrate that the Ta.it.I1 group IIC intron coordinates the progression of the forward splicing reaction through a π-π' interaction between intron domains II and VI. We further show that branched splicing is supported in the absence of the IEP when the π-π' interaction is mutated. We also investigated the regulation of the two steps of reverse splicing during intron mobility into DNA substrates. Using a fluorescent mobility assay that simultaneously visualizes all steps of intron integration into DNA, we show that completion of reverse splicing is tightly coupled to cDNA synthesis regardless of mutation of the π-π' interaction.


Assuntos
Processamento de RNA/genética , RNA Catalítico/genética , RNA/genética , Ribonucleoproteínas/genética , Éxons/genética , Sequências Repetitivas Dispersas/genética , Íntrons/genética , Mutação/genética , Conformação de Ácido Nucleico , Ribonucleoproteínas/química
16.
RNA ; 26(6): 708-714, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32205324

RESUMO

Human immunodeficiency virus type 1 (HIV-1) transcripts have three fates: to serve as genomic RNAs, unspliced mRNAs, or spliced subgenomic mRNAs. Recent structural studies have shown that sequences near the 5' end of HIV-1 RNA can adopt at least two alternate three-dimensional conformations, and that these structures dictate genome versus unspliced mRNA fates. HIV-1's use of alternate transcription start sites (TSS) can influence which RNA conformer is generated, and this choice, in turn, dictates the fate of the unspliced RNA. The structural context of HIV-1's major 5' splice site differs in these two RNA conformers, suggesting that the conformers may differ in their ability to support HIV-1 splicing events. Here, we tested the hypothesis that TSS that shift the RNA monomer/dimer structural equilibrium away from the splice site sequestering dimer-competent fold would favor splicing. Consistent with this hypothesis, the results showed that the 5' ends of spliced HIV-1 RNAs were enriched in 3GCap structures and depleted of 1GCap RNAs relative to the total intracellular RNA population. These findings expand the functional significance of HIV-1 RNA structural dynamics by demonstrating roles for RNA structure in defining all three classes of HIV-1 RNAs, and suggest that HIV-1 TSS choice initiates a cascade of molecular events that dictate the fates of nascent HIV-1 RNAs.


Assuntos
HIV-1/genética , Processamento de RNA , RNA Viral/química , Sítio de Iniciação de Transcrição , Regiões 5' não Traduzidas , Células HEK293 , HIV-1/metabolismo , Humanos , Conformação de Ácido Nucleico , RNA Viral/metabolismo
17.
Am J Pathol ; 190(6): 1236-1255, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32201263

RESUMO

Hyaluronidase (HYAL)-2 is a weak, acid-active, hyaluronan-degrading enzyme broadly expressed in somatic tissues. Aberrant HYAL2 expression is implicated in diverse pathology. However, a significant proportion of HYAL2 is enzymatically inactive; thus the mechanisms through which HYAL2 dysregulation influences pathobiology are unclear. Recently, nonenzymatic HYAL2 functions have been described, and nuclear HYAL2 has been shown to influence mRNA splicing to prevent myofibroblast differentiation. Myofibroblasts drive fibrosis, thereby promoting progressive tissue damage and leading to multimorbidity. This study identifies a novel HYAL2 cytoplasmic function in myofibroblasts that is unrelated to its enzymatic activity. In fibroblasts and myofibroblasts, HYAL2 interacts with the GTPase-signaling small molecule ras homolog family member A (RhoA). Transforming growth factor beta 1-driven fibroblast-to-myofibroblast differentiation promotes HYAL2 cytoplasmic relocalization to bind to the actin cytoskeleton. Cytoskeletal-bound HYAL2 functions as a key regulator of downstream RhoA signaling and influences profibrotic myofibroblast functions, including myosin light-chain kinase-mediated myofibroblast contractility, myofibroblast migration, myofibroblast collagen/fibronectin deposition, as well as connective tissue growth factor and matrix metalloproteinase-2 expression. These data demonstrate that, in certain biological contexts, the nonenzymatic effects of HYAL2 are crucial in orchestrating RhoA signaling and downstream pathways that are important for full profibrotic myofibroblast functionality. In conjunction with previous data demonstrating the influence of HYAL2 on RNA splicing, these findings begin to explain the broad biological effects of HYAL2.


Assuntos
Fibroblastos/metabolismo , Hialuronoglucosaminidase/metabolismo , Miofibroblastos/metabolismo , Transdução de Sinais/fisiologia , Proteína rhoA de Ligação ao GTP/metabolismo , Animais , Fibrose/metabolismo , Humanos , Masculino , Processamento de RNA , Ratos
18.
Nat Commun ; 11(1): 1334, 2020 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-32170079

RESUMO

Prolonged expression of the CRISPR-Cas9 nuclease and gRNA from viral vectors may cause off-target mutagenesis and immunogenicity. Thus, a transient delivery system is needed for therapeutic genome editing applications. Here, we develop an extracellular nanovesicle-based ribonucleoprotein delivery system named NanoMEDIC by utilizing two distinct homing mechanisms. Chemical induced dimerization recruits Cas9 protein into extracellular nanovesicles, and then a viral RNA packaging signal and two self-cleaving riboswitches tether and release sgRNA into nanovesicles. We demonstrate efficient genome editing in various hard-to-transfect cell types, including human induced pluripotent stem (iPS) cells, neurons, and myoblasts. NanoMEDIC also achieves over 90% exon skipping efficiencies in skeletal muscle cells derived from Duchenne muscular dystrophy (DMD) patient iPS cells. Finally, single intramuscular injection of NanoMEDIC induces permanent genomic exon skipping in a luciferase reporter mouse and in mdx mice, indicating its utility for in vivo genome editing therapy of DMD and beyond.


Assuntos
Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas , Éxons/genética , Vesículas Extracelulares/metabolismo , Nanopartículas/química , RNA Guia/metabolismo , Sequência de Bases , Sobrevivência Celular , Dimerização , Edição de Genes , Vetores Genéticos/metabolismo , Células HEK293 , Protease de HIV/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Ligantes , Luciferases/metabolismo , Processamento de RNA/genética , RNA Catalítico/metabolismo , Ribonucleoproteínas/metabolismo , Doadores de Tecidos , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo
19.
PLoS Comput Biol ; 16(3): e1007717, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32210422

RESUMO

Spatial organization is a characteristic of all cells, achieved in eukaryotic cells by utilizing both membrane-bound and membrane-less organelles. One of the key processes in eukaryotes is RNA splicing, which readies mRNA for translation. This complex and highly dynamical chemical process involves assembly and disassembly of many molecules in multiple cellular compartments and their transport among compartments. Our goal is to model the effect of spatial organization of membrane-less organelles (specifically nuclear speckles) and of organelle heterogeneity on splicing particle biogenesis in mammalian cells. Based on multiple sources of complementary experimental data, we constructed a spatial model of a HeLa cell to capture intracellular crowding effects. We then developed chemical reaction networks to describe the formation of RNA splicing machinery complexes and splicing processes within nuclear speckles (specific type of non-membrane-bound organelles). We incorporated these networks into our spatially-resolved human cell model and performed stochastic simulations for up to 15 minutes of biological time, the longest thus far for a eukaryotic cell. We find that an increase (decrease) in the number of nuclear pore complexes increases (decreases) the number of assembled splicing particles; and that compartmentalization is critical for the yield of correctly-assembled particles. We also show that a slight increase of splicing particle localization into nuclear speckles leads to a disproportionate enhancement of mRNA splicing and a reduction in the noise of generated mRNA. Our model also predicts that the distance between genes and speckles has a considerable effect on the mRNA production rate, with genes located closer to speckles producing mRNA at higher levels, emphasizing the importance of genome organization around speckles. The HeLa cell model, including organelles and sub-compartments, provides a flexible foundation to study other cellular processes that are strongly modulated by spatiotemporal heterogeneity.


Assuntos
Modelos Biológicos , Processamento de RNA/fisiologia , RNA Mensageiro/metabolismo , Spliceossomos , Biologia Computacional , Simulação por Computador , Células HeLa , Humanos , Espaço Intracelular/química , Espaço Intracelular/metabolismo , Espaço Intracelular/fisiologia , Cinética , RNA Mensageiro/química , Spliceossomos/química , Spliceossomos/metabolismo , Spliceossomos/fisiologia
20.
Am J Hum Genet ; 106(4): 438-452, 2020 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-32197073

RESUMO

The neuro-oncological ventral antigen 2 (NOVA2) protein is a major factor regulating neuron-specific alternative splicing (AS), previously associated with an acquired neurologic condition, the paraneoplastic opsoclonus-myoclonus ataxia (POMA). We report here six individuals with de novo frameshift variants in NOVA2 affected with a severe neurodevelopmental disorder characterized by intellectual disability (ID), motor and speech delay, autistic features, hypotonia, feeding difficulties, spasticity or ataxic gait, and abnormal brain MRI. The six variants lead to the same reading frame, adding a common proline rich C-terminal part instead of the last KH RNA binding domain. We detected 41 genes differentially spliced after NOVA2 downregulation in human neural cells. The NOVA2 variant protein shows decreased ability to bind target RNA sequences and to regulate target AS events. It also fails to complement the effect on neurite outgrowth induced by NOVA2 downregulation in vitro and to rescue alterations of retinotectal axonal pathfinding induced by loss of NOVA2 ortholog in zebrafish. Our results suggest a partial loss-of-function mechanism rather than a full heterozygous loss-of-function, although a specific contribution of the novel C-terminal extension cannot be excluded.


Assuntos
Mutação da Fase de Leitura/genética , Proteínas do Tecido Nervoso/genética , Transtornos do Neurodesenvolvimento/genética , Neurônios/fisiologia , Processamento de RNA/genética , Proteínas de Ligação a RNA/genética , Processamento Alternativo/genética , Animais , Orientação de Axônios/genética , Sequência de Bases/genética , Células Cultivadas , Pré-Escolar , Regulação para Baixo/genética , Feminino , Heterozigoto , Humanos , Deficiência Intelectual/genética , Transtornos do Desenvolvimento da Linguagem/genética , Masculino , Camundongos , Hipotonia Muscular/genética , Peixe-Zebra/genética
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