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1.
FEBS J ; 288(21): 6082-6086, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34719877

RESUMO

Cancer is a leading cause of death and a major health problem worldwide, particularly in more developed countries. There is, therefore, an urgent clinical need to develop more effective therapies to treat cancer and metastatic disease. In this Editorial, the content of The FEBS Journal's Special Issue on Cancer Therapeutics is outlined. The interesting collection of recent articles in this issue covers a wide repertoire of cancer therapeutic approaches. While some of the articles discuss broad-spectrum applications such as immunotherapy and oncolytic virus therapy, others focus on a particular type of cancer or a signalling pathway that has gone awry such as aberrant Ca2+ signalling, glycosylation or pre-mRNA processing. Finally, an article featured in this issue reviews our current understanding of how cancer cells can become dormant, often for decades, and which pathways reactivate these cells to cause relapse. I am sure there is something for everyone in this issue.


Assuntos
Cálcio/metabolismo , Glicosilação , Humanos , Precursores de RNA/metabolismo
2.
J Vis Exp ; (175)2021 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-34605821

RESUMO

During gene expression, the vital step of pre-mRNA splicing involves accurate recognition of splice sites and efficient assembly of spliceosomal complexes to join exons and remove introns prior to cytoplasmic export of the mature mRNA. Splicing efficiency can be altered by the presence of mutations at splice sites, the influence of trans-acting splicing factors, or the activity of therapeutics. Here, we describe the protocol for a cellular assay that can be applied for monitoring the splicing efficiency of any given exon. The assay uses an adaptable plasmid encoded 3-exon/2-intron minigene reporter, which can be expressed in mammalian cells by transient transfection. Post-transfection, total cellular RNA is isolated, and the efficiency of exon splicing in the reporter mRNA is determined by either primer extension or semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). We describe how the impact of disease associated 5' splice-site mutations can be determined by introducing them in the reporter; and how the suppression of these mutations can be achieved by co-transfection with U1 small nuclear RNA (snRNA) construct carrying compensatory mutations in its 5' region that basepairs with the 5'-splice sites at exon-intron junctions in pre-mRNAs. Thus, the reporter can be used for the design of therapeutic U1 particles to improve recognition of mutant 5' splice-sites. Insertion of cis-acting regulatory sites, such as splicing enhancer or silencer sequences, into the reporter can also be used to examine the role of U1 snRNP in regulation mediated by a specific alternative splicing factor. Finally, reporter expressing cells can be incubated with small molecules to determine the effect of potential therapeutics on constitutive pre-mRNA splicing or on exons carrying mutant 5' splice sites. Overall, the reporter assay can be applied to monitor splicing efficiency in a variety of conditions to study fundamental splicing mechanisms and splicing-associated diseases.


Assuntos
Processamento Alternativo , Splicing de RNA , Animais , Sequência de Bases , Íntrons/genética , Precursores de RNA/genética , Precursores de RNA/metabolismo , Sítios de Splice de RNA , Splicing de RNA/genética
3.
Int J Mol Sci ; 22(19)2021 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-34638646

RESUMO

Ribonuclease P (RNase P) is an important ribonucleoprotein (RNP), responsible for the maturation of the 5' end of precursor tRNAs (pre-tRNAs). In all organisms, the cleavage activity of a single phosphodiester bond adjacent to the first nucleotide of the acceptor stem is indispensable for cell viability and lies within an essential catalytic RNA subunit. Although RNase P is a ribozyme, its kinetic efficiency in vivo, as well as its structural variability and complexity throughout evolution, requires the presence of one protein subunit in bacteria to several protein partners in archaea and eukaryotes. Moreover, the existence of protein-only RNase P (PRORP) enzymes in several organisms and organelles suggests a more complex evolutionary timeline than previously thought. Recent detailed structures of bacterial, archaeal, human and mitochondrial RNase P complexes suggest that, although apparently dissimilar enzymes, they all recognize pre-tRNAs through conserved interactions. Interestingly, individual protein subunits of the human nuclear and mitochondrial holoenzymes have additional functions and contribute to a dynamic network of elaborate interactions and cellular processes. Herein, we summarize the role of each RNase P subunit with a focus on the human nuclear RNP and its putative role in flawless gene expression in light of recent structural studies.


Assuntos
Domínio Catalítico/fisiologia , Subunidades Proteicas/metabolismo , Ribonuclease P/metabolismo , Animais , Humanos , Cinética , Precursores de RNA/metabolismo , RNA Catalítico/metabolismo
4.
Nat Commun ; 12(1): 5610, 2021 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-34584079

RESUMO

Introns of human transfer RNA precursors (pre-tRNAs) are excised by the tRNA splicing endonuclease TSEN in complex with the RNA kinase CLP1. Mutations in TSEN/CLP1 occur in patients with pontocerebellar hypoplasia (PCH), however, their role in the disease is unclear. Here, we show that intron excision is catalyzed by tetrameric TSEN assembled from inactive heterodimers independently of CLP1. Splice site recognition involves the mature domain and the anticodon-intron base pair of pre-tRNAs. The 2.1-Å resolution X-ray crystal structure of a TSEN15-34 heterodimer and differential scanning fluorimetry analyses show that PCH mutations cause thermal destabilization. While endonuclease activity in recombinant mutant TSEN is unaltered, we observe assembly defects and reduced pre-tRNA cleavage activity resulting in an imbalanced pre-tRNA pool in PCH patient-derived fibroblasts. Our work defines the molecular principles of intron excision in humans and provides evidence that modulation of TSEN stability may contribute to PCH phenotypes.


Assuntos
Doenças Cerebelares/metabolismo , Endonucleases/metabolismo , Mutação , Precursores de RNA/metabolismo , Splicing de RNA , RNA de Transferência/metabolismo , Animais , Doenças Cerebelares/genética , Cristalografia por Raios X , Endonucleases/química , Endonucleases/genética , Endorribonucleases/química , Endorribonucleases/genética , Endorribonucleases/metabolismo , Células HEK293 , Humanos , Íntrons/genética , Conformação Proteica , Multimerização Proteica , Precursores de RNA/genética , RNA de Transferência/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Células Sf9 , Spodoptera
5.
Nat Struct Mol Biol ; 28(9): 713-723, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34489609

RESUMO

Human mitochondrial transcripts contain messenger and ribosomal RNAs flanked by transfer RNAs (tRNAs), which are excised by mitochondrial RNase (mtRNase) P and Z to liberate all RNA species. In contrast to nuclear or bacterial RNase P, mtRNase P is not a ribozyme but comprises three protein subunits that carry out RNA cleavage and methylation by unknown mechanisms. Here, we present the cryo-EM structure of human mtRNase P bound to precursor tRNA, which reveals a unique mechanism of substrate recognition and processing. Subunits TRMT10C and SDR5C1 form a subcomplex that binds conserved mitochondrial tRNA elements, including the anticodon loop, and positions the tRNA for methylation. The endonuclease PRORP is recruited and activated through interactions with its PPR and nuclease domains to ensure precise pre-tRNA cleavage. The structure provides the molecular basis for the first step of RNA processing in human mitochondria.


Assuntos
3-Hidroxiacil-CoA Desidrogenases/química , Metiltransferases/química , Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA , Ribonuclease P/química , 3-Hidroxiacil-CoA Desidrogenases/metabolismo , Anticódon/química , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Proteínas Arqueais/química , Proteínas Arqueais/metabolismo , Microscopia Crioeletrônica , Humanos , Metilação , Metiltransferases/genética , Metiltransferases/metabolismo , Mitocôndrias/enzimologia , Modelos Moleculares , Mutação de Sentido Incorreto , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica , Mapeamento de Interação de Proteínas , RNA Fúngico/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Ribonuclease P/metabolismo , Especificidade da Espécie , Relação Estrutura-Atividade , Especificidade por Substrato
6.
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
7.
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
8.
Int J Mol Sci ; 22(15)2021 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-34360561

RESUMO

Pre-mRNA splicing is an essential process for gene expression in higher eukaryotes, which requires a high order of accuracy. Mutations in splicing factors or regulatory elements in pre-mRNAs often result in many human diseases. Myelodysplastic syndrome (MDS) is a heterogeneous group of chronic myeloid neoplasms characterized by many symptoms and a high risk of progression to acute myeloid leukemia. Recent findings indicate that mutations in splicing factors represent a novel class of driver mutations in human cancers and affect about 50% of Myelodysplastic syndrome (MDS) patients. Somatic mutations in MDS patients are frequently found in genes SF3B1, SRSF2, U2AF1, and ZRSR2. Interestingly, they are involved in the recognition of 3' splice sites and exons. It has been reported that mutations in these splicing regulators result in aberrant splicing of many genes. In this review article, we first describe molecular mechanism of pre-mRNA splicing as an introduction and mainly focus on those four splicing factors to describe their mutations and their associated aberrant splicing patterns.


Assuntos
Mutação , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/patologia , Precursores de RNA/genética , Fatores de Processamento de RNA/genética , Splicing de RNA , Humanos
9.
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
10.
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
11.
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
12.
Int J Cancer ; 149(10): 1787-1800, 2021 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-34346508

RESUMO

The splicing of microexons (very small exons) is frequently dysregulated in the brain of individuals with autism spectrum disorder. However, little is known of the patterns, regulatory mechanisms and roles of microexon splicing in cancer. We here examined the transcriptome-wide profile of microexon splicing in matched colorectal cancer (CRC) and normal tissue specimens. Out of 1492 microexons comprising 3 to 15 nucleotides, 21 (1%) manifested differential splicing between CRC and normal tissue. The 21 genes harboring the differentially spliced microexons were enriched in gene ontology terms related to cell adhesion and migration. RNA interference-mediated knockdown experiments identified two splicing factors, RBFOX2 and PTBP1, as regulators of microexon splicing in CRC cells. RBFOX2 and PTBP1 were found to directly bind to microexon-containing pre-mRNAs and to control their splicing in such cells. Differential microexon splicing was shown to be due, at least in part, to altered expression of RBFOX2 and PTBP1 in CRC tissue compared to matched normal tissue. Finally, we found that changes in the pattern of microexon splicing were associated with CRC metastasis. Our data thus suggest that altered expression of RBFOX2 and PTBP1 might influence CRC metastasis through the regulation of microexon splicing.


Assuntos
Processamento Alternativo , Neoplasias Colorretais/genética , Éxons/genética , Ribonucleoproteínas Nucleares Heterogêneas/genética , Proteína de Ligação a Regiões Ricas em Polipirimidinas/genética , Fatores de Processamento de RNA/genética , Proteínas Repressoras/genética , Linhagem Celular Tumoral , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica , Ontologia Genética , Células HCT116 , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Humanos , Immunoblotting , Metástase Neoplásica , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , Ligação Proteica , Precursores de RNA/genética , Precursores de RNA/metabolismo , Fatores de Processamento de RNA/metabolismo , Proteínas Repressoras/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa
13.
Nucleic Acids Res ; 49(16): 9444-9458, 2021 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-34387688

RESUMO

The ribonucleoprotein (RNP) form of archaeal RNase P comprises one catalytic RNA and five protein cofactors. To catalyze Mg2+-dependent cleavage of the 5' leader from pre-tRNAs, the catalytic (C) and specificity (S) domains of the RNase P RNA (RPR) cooperate to recognize different parts of the pre-tRNA. While ∼250-500 mM Mg2+ renders the archaeal RPR active without RNase P proteins (RPPs), addition of all RPPs lowers the Mg2+ requirement to ∼10-20 mM and improves the rate and fidelity of cleavage. To understand the Mg2+- and RPP-dependent structural changes that increase activity, we used pre-tRNA cleavage and ensemble FRET assays to characterize inter-domain interactions in Pyrococcus furiosus (Pfu) RPR, either alone or with RPPs ± pre-tRNA. Following splint ligation to doubly label the RPR (Cy3-RPRC domain and Cy5-RPRS domain), we used native mass spectrometry to verify the final product. We found that FRET correlates closely with activity, the Pfu RPR and RNase P holoenzyme (RPR + 5 RPPs) traverse different Mg2+-dependent paths to converge on similar functional states, and binding of the pre-tRNA by the holoenzyme influences Mg2+ cooperativity. Our findings highlight how Mg2+ and proteins in multi-subunit RNPs together favor RNA conformations in a dynamic ensemble for functional gains.


Assuntos
Archaea/enzimologia , Magnésio/metabolismo , RNA Arqueal/genética , Ribonuclease P/genética , Conformação de Ácido Nucleico/efeitos dos fármacos , Pyrococcus furiosus/enzimologia , Pyrococcus furiosus/genética , Precursores de RNA/genética , RNA Arqueal/ultraestrutura , RNA Catalítico , Ribonuclease P/ultraestrutura
14.
Int J Mol Sci ; 22(12)2021 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-34204574

RESUMO

Using TSG101 pre-mRNA, we previously discovered cancer-specific re-splicing of mature mRNA that generates aberrant transcripts/proteins. The fact that mRNA is aberrantly re-spliced in various cancer cells implies there must be an important mechanism to prevent deleterious re-splicing on the spliced mRNA in normal cells. We thus postulated that mRNA re-splicing is controlled by specific repressors, and we searched for repressor candidates by siRNA-based screening for mRNA re-splicing activity. We found that knock-down of EIF4A3, which is a core component of the exon junction complex (EJC), significantly promoted mRNA re-splicing. Remarkably, we could recapitulate cancer-specific mRNA re-splicing in normal cells by knock-down of any of the core EJC proteins, EIF4A3, MAGOH, or RBM8A (Y14), implicating the EJC core as the repressor of mRNA re-splicing often observed in cancer cells. We propose that the EJC core is a critical mRNA quality control factor to prevent over-splicing of mature mRNA.


Assuntos
Éxons , Regulação Neoplásica da Expressão Gênica , Neoplasias/genética , Precursores de RNA/genética , Splicing de RNA , RNA Mensageiro/genética , Linhagem Celular Tumoral , Fator de Iniciação 4A em Eucariotos/genética , Fator de Iniciação 4A em Eucariotos/metabolismo , Humanos , Modelos Biológicos , Neoplasias/metabolismo , Ligação Proteica , Transporte de RNA , Proteínas de Ligação a RNA/metabolismo
15.
Curr Protoc ; 1(7): e209, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34314573

RESUMO

The gut microbiome is recognized as a critical regulator of human diseases. Constituents of the microbiota and their individual activities can affect a broad range of disease states related to autoimmunity, cancer, infection, metabolism, mental health, and toxicant exposure. A substantial number of microbiome species are not culturable, limiting their study in vitro. Sequencing methods have allowed quantification of the composition of the microbiome, but methods to characterize the physiological status of bacterial species remain limited. Ribosomal RNA precursors (pre-rRNAs) are species-specific intermediates in bacterial ribosomal synthesis, and their levels are highly responsive to environmental changes. Immediately before and during active growth, pre-rRNA levels are high, whereas in non-dividing cells, copy numbers are orders of magnitude lower. These dynamics are conserved in all bacterial species and occur exclusively in viable cells, allowing the specific characterization of living and functional bacteria in their native states. Pre-rRNA analysis has been shown to yield valuable real-time information on the physiology of individual bacterial species within complex samples, beyond what traditional qPCR and sequencing methods can offer. Herein, we describe a PCR-based protocol to interrogate and quantify the in situ growth status of bacterial species of interest within a complex microbiome. We also describe an in vitro protocol to characterize the pre-rRNA/growth relationship for a given bacterial species to provide greater context for values obtained from natural samples. Improved understanding of microbial physiological responses to exposures could reveal novel toxicological mechanisms, biomarkers, and potential treatments. © 2021 Wiley Periodicals LLC. Basic Protocol: Targeted steady-state pre-rRNA analysis Support Protocol: Characterization of pre-rRNA/growth relationship © 2021 by John Wiley & Sons, Inc.


Assuntos
Microbiota , Precursores de RNA , Bactérias/genética , Humanos , Precursores de RNA/genética , RNA Ribossômico 16S/genética , Reação em Cadeia da Polimerase em Tempo Real
16.
Nat Commun ; 12(1): 4645, 2021 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-34330918

RESUMO

A major part of the transcriptome complexity is attributed to multiple types of DNA or RNA fusion events, which take place within a gene such as alternative splicing or between different genes such as DNA rearrangement and trans-splicing. In the present study, using the RNA deep sequencing data, we systematically survey a type of non-canonical fusions between the RNA transcripts from the two opposite DNA strands. We name the products of such fusion events cross-strand chimeric RNA (cscRNA). Hundreds to thousands of cscRNAs can be found in human normal tissues, primary cells, and cancerous cells, and in other species as well. Although cscRNAs exhibit strong tissue-specificity, our analysis identifies thousands of recurrent cscRNAs found in multiple different samples. cscRNAs are mostly originated from convergent transcriptions of the annotated genes and their anti-sense DNA. The machinery of cscRNA biogenesis is unclear, but the cross-strand junction events show some features related to RNA splicing. The present study is a comprehensive survey of the non-canonical cross-strand RNA junction events, a resource for further characterization of the originations and functions of the cscRNAs.


Assuntos
Perfilação da Expressão Gênica/métodos , Fusão Gênica , Splicing de RNA , RNA/genética , Trans-Splicing , Transcriptoma/genética , Células A549 , Linhagem Celular Tumoral , Biologia Computacional/métodos , Humanos , Hibridização in Situ Fluorescente/métodos , Modelos Genéticos , Células PC-3 , Precursores de RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos
17.
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
18.
Nucleic Acids Res ; 49(14): 7870-7883, 2021 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-34283224

RESUMO

Risdiplam is the first approved small-molecule splicing modulator for the treatment of spinal muscular atrophy (SMA). Previous studies demonstrated that risdiplam analogues have two separate binding sites in exon 7 of the SMN2 pre-mRNA: (i) the 5'-splice site and (ii) an upstream purine (GA)-rich binding site. Importantly, the sequence of this GA-rich binding site significantly enhanced the potency of risdiplam analogues. In this report, we unambiguously determined that a known risdiplam analogue, SMN-C2, binds to single-stranded GA-rich RNA in a sequence-specific manner. The minimum required binding sequence for SMN-C2 was identified as GAAGGAAGG. We performed all-atom simulations using a robust Gaussian accelerated molecular dynamics (GaMD) method, which captured spontaneous binding of a risdiplam analogue to the target nucleic acids. We uncovered, for the first time, a ligand-binding pocket formed by two sequential GAAG loop-like structures. The simulation findings were highly consistent with experimental data obtained from saturation transfer difference (STD) NMR and structure-affinity-relationship studies of the risdiplam analogues. Together, these studies illuminate us to understand the molecular basis of single-stranded purine-rich RNA recognition by small-molecule splicing modulators with an unprecedented binding mode.


Assuntos
Compostos Azo/metabolismo , Atrofia Muscular Espinal/genética , Pirimidinas/metabolismo , Precursores de RNA/genética , Splicing de RNA , Compostos Azo/química , Compostos Azo/uso terapêutico , Sequência de Bases , Sítios de Ligação/genética , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Éxons/genética , Cinética , Espectroscopia de Ressonância Magnética/métodos , Simulação de Dinâmica Molecular , Estrutura Molecular , Atrofia Muscular Espinal/tratamento farmacológico , Atrofia Muscular Espinal/metabolismo , Mutação , Fármacos Neuromusculares/química , Fármacos Neuromusculares/metabolismo , Fármacos Neuromusculares/uso terapêutico , Conformação de Ácido Nucleico , Pirimidinas/química , Pirimidinas/uso terapêutico , Precursores de RNA/química , Precursores de RNA/metabolismo , Proteína 2 de Sobrevivência do Neurônio Motor/genética
19.
Development ; 148(15)2021 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-34323273

RESUMO

Vertebrate animals usually display robust growth trajectories during juvenile stages, and reversible suspension of this growth momentum by a single genetic determinant has not been reported. Here, we report a single genetic factor that is essential for juvenile growth in zebrafish. Using a forward genetic screen, we recovered a temperature-sensitive allele, pan (after Peter Pan), that suspends whole-organism growth at juvenile stages. Remarkably, even after growth is halted for a full 8-week period, pan mutants are able to resume a robust growth trajectory after release from the restrictive temperature, eventually growing into fertile adults without apparent adverse phenotypes. Positional cloning and complementation assays revealed that pan encodes a probable ATP-dependent RNA helicase (DEAD-Box Helicase 52; ddx52) that maintains the level of 47S precursor ribosomal RNA. Furthermore, genetic silencing of ddx52 and pharmacological inhibition of bulk RNA transcription similarly suspend the growth of flies, zebrafish and mice. Our findings reveal evidence that safe, reversible pauses of juvenile growth can be mediated by targeting the activity of a single gene, and that its pausing mechanism has high evolutionary conservation.


Assuntos
RNA Helicases/genética , RNA/genética , Peixe-Zebra/genética , Alelos , Animais , Feminino , Inativação Gênica/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Precursores de RNA/genética , Ribossomos/genética , Transcrição Genética/genética
20.
EMBO Rep ; 22(9): e52320, 2021 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-34312949

RESUMO

HP1 proteins are best known as markers of heterochromatin and gene silencing. Yet, they are also RNA-binding proteins and the HP1γ/CBX3 family member is present on transcribed genes together with RNA polymerase II, where it regulates co-transcriptional processes such as alternative splicing. To gain insight in the role of the RNA-binding activity of HP1γ in transcriptionally active chromatin, we have captured and analysed RNAs associated with this protein. We find that HP1γ is specifically targeted to hexameric RNA motifs and coincidentally transposable elements of the SINE family. As these elements are abundant in introns, while essentially absent from exons, the HP1γ RNA association tethers unspliced pre-mRNA to chromatin via the intronic regions and limits the usage of intronic cryptic splice sites. Thus, our data unveil novel determinants in the relationship between chromatin and co-transcriptional splicing.


Assuntos
Precursores de RNA , Splicing de RNA , Processamento Alternativo/genética , Íntrons/genética , Precursores de RNA/genética , Precursores de RNA/metabolismo , Splicing de RNA/genética , Proteínas de Ligação a RNA
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