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1.
Genes Dev ; 34(15-16): 1075-1088, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32616520

RESUMO

Nonsense-mediated decay (NMD) is a translation-dependent RNA quality control mechanism that occurs in the cytoplasm. However, it is unknown how NMD regulates the stability of RNAs translated at the endoplasmic reticulum (ER). Here, we identify a localized NMD pathway dedicated to ER-translated mRNAs. We previously identified NBAS, a component of the Syntaxin 18 complex involved in Golgi-to-ER trafficking, as a novel NMD factor. Furthermore, we show that NBAS fulfills an independent function in NMD. This ER-NMD pathway requires the interaction of NBAS with the core NMD factor UPF1, which is partially localized at the ER in the proximity of the translocon. NBAS and UPF1 coregulate the stability of ER-associated transcripts, in particular those associated with the cellular stress response. We propose a model where NBAS recruits UPF1 to the membrane of the ER and activates an ER-dedicated NMD pathway, thus providing an ER-protective function by ensuring quality control of ER-translated mRNAs.


Assuntos
Retículo Endoplasmático/metabolismo , Degradação do RNAm Mediada por Códon sem Sentido , Retículo Endoplasmático/enzimologia , Complexo de Golgi/metabolismo , Células HeLa , Humanos , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/fisiologia , Biossíntese de Proteínas , RNA Helicases/metabolismo
2.
EMBO J ; 42(19): e114378, 2023 Oct 04.
Artigo em Inglês | MEDLINE | ID: mdl-37605642

RESUMO

mRNA surveillance pathways are essential for accurate gene expression and to maintain translation homeostasis, ensuring the production of fully functional proteins. Future insights into mRNA quality control pathways will enable us to understand how cellular mRNA levels are controlled, how defective or unwanted mRNAs can be eliminated, and how dysregulation of these can contribute to human disease. Here we review translation-coupled mRNA quality control mechanisms, including the non-stop and no-go mRNA decay pathways, describing their mechanisms, shared trans-acting factors, and differences. We also describe advances in our understanding of the nonsense-mediated mRNA decay (NMD) pathway, highlighting recent mechanistic findings, the discovery of novel factors, as well as the role of NMD in cellular physiology and its impact on human disease.

3.
RNA ; 28(9): 1224-1238, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-35768279

RESUMO

The DExD/H-box RNA helicase DHX34 is a nonsense-mediated decay (NMD) factor that together with core NMD factors coregulates NMD targets in nematodes and in vertebrates. Here, we show that DHX34 is also associated with the human spliceosomal catalytic C complex. Mapping of DHX34 endogenous binding sites using cross-linking immunoprecipitation (CLIP) revealed that DHX34 is preferentially associated with pre-mRNAs and locates at exon-intron boundaries. Accordingly, we observed that DHX34 regulates a large number of alternative splicing (AS) events in mammalian cells in culture, establishing a dual role for DHX34 in both NMD and pre-mRNA splicing. We previously showed that germline DHX34 mutations associated to familial myelodysplasia (MDS)/acute myeloid leukemia (AML) predisposition abrogate its activity in NMD. Interestingly, we observe now that DHX34 regulates the splicing of pre-mRNAs that have been linked to AML/MDS predisposition. This is consistent with silencing experiments in hematopoietic stem/progenitor cells (HSPCs) showing that loss of DHX34 results in differentiation blockade of both erythroid and myeloid lineages, which is a hallmark of AML development. Altogether, these data unveil new cellular functions of DHX34 and suggest that alterations in the levels and/or activity of DHX34 could contribute to human disease.


Assuntos
Leucemia Mieloide Aguda , Síndromes Mielodisplásicas , Processamento Alternativo , Animais , Humanos , Leucemia Mieloide Aguda/genética , Mamíferos/genética , Síndromes Mielodisplásicas/genética , Degradação do RNAm Mediada por Códon sem Sentido , RNA Helicases/genética , RNA Helicases/metabolismo , Precursores de RNA/genética , Precursores de RNA/metabolismo , Splicing de RNA , RNA Mensageiro/genética
4.
PLoS Biol ; 18(12): e3001030, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33320856

RESUMO

With the ongoing COVID-19 (Coronavirus Disease 2019) pandemic, caused by the novel coronavirus SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2), there is a need for sensitive, specific, and affordable diagnostic tests to identify infected individuals, not all of whom are symptomatic. The most sensitive test involves the detection of viral RNA using RT-qPCR (quantitative reverse transcription PCR), with many commercial kits now available for this purpose. However, these are expensive, and supply of such kits in sufficient numbers cannot always be guaranteed. We therefore developed a multiplex assay using well-established SARS-CoV-2 targets alongside a human cellular control (RPP30) and a viral spike-in control (Phocine Herpes Virus 1 [PhHV-1]), which monitor sample quality and nucleic acid extraction efficiency, respectively. Here, we establish that this test performs as well as widely used commercial assays, but at substantially reduced cost. Furthermore, we demonstrate >1,000-fold variability in material routinely collected by combined nose and throat swabbing and establish a statistically significant correlation between the detected level of human and SARS-CoV-2 nucleic acids. The inclusion of the human control probe in our assay therefore provides a quantitative measure of sample quality that could help reduce false-negative rates. We demonstrate the feasibility of establishing a robust RT-qPCR assay at approximately 10% of the cost of equivalent commercial assays, which could benefit low-resource environments and make high-volume testing affordable.


Assuntos
Teste para COVID-19/métodos , COVID-19/diagnóstico , RNA Viral/análise , SARS-CoV-2/isolamento & purificação , Teste para COVID-19/economia , Humanos , Reação em Cadeia da Polimerase Multiplex/economia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/economia , SARS-CoV-2/genética
5.
Nucleic Acids Res ; 44(4): 1483-95, 2016 Feb 29.
Artigo em Inglês | MEDLINE | ID: mdl-26773057

RESUMO

The Nonsense-mediated mRNA decay (NMD) pathway selectively degrades mRNAs harboring premature termination codons (PTCs) but also regulates the abundance of a large number of cellular RNAs. The central role of NMD in the control of gene expression requires the existence of buffering mechanisms that tightly regulate the magnitude of this pathway. Here, we will focus on the mechanism of NMD with an emphasis on the role of RNA helicases in the transition from NMD complexes that recognize a PTC to those that promote mRNA decay. We will also review recent strategies aimed at uncovering novel trans-acting factors and their functional role in the NMD pathway. Finally, we will describe recent progress in the study of the physiological role of the NMD response.


Assuntos
Códon sem Sentido/genética , Regulação da Expressão Gênica/genética , Degradação do RNAm Mediada por Códon sem Sentido/genética , Estabilidade de RNA/genética , Humanos , Redes e Vias Metabólicas/genética , RNA Helicases/genética , RNA Mensageiro/genética
6.
EMBO Rep ; 16(1): 71-8, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25452588

RESUMO

Nonsense-mediated mRNA decay (NMD) is a surveillance mechanism that degrades mRNAs harboring premature termination codons (PTCs). We have conducted a genome-wide RNAi screen in Caenorhabditis elegans that resulted in the identification of five novel NMD genes that are conserved throughout evolution. Two of their human homologs, GNL2 (ngp-1) and SEC13 (npp-20), are also required for NMD in human cells. We also show that the C. elegans gene noah-2, which is present in Drosophila melanogaster but absent in humans, is an NMD factor in fruit flies. Altogether, these data identify novel NMD factors that are conserved throughout evolution, highlighting the complexity of the NMD pathway and suggesting that yet uncovered novel factors may act to regulate this process.


Assuntos
Caenorhabditis elegans/genética , Proteínas de Transporte/metabolismo , Drosophila melanogaster/genética , Proteínas de Ligação ao GTP/metabolismo , Degradação do RNAm Mediada por Códon sem Sentido/fisiologia , Proteínas Nucleares/metabolismo , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/crescimento & desenvolvimento , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Transporte/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriologia , Proteínas do Ovo/genética , Proteínas do Ovo/metabolismo , Embrião não Mamífero , Evolução Molecular , Proteínas de Ligação ao GTP/genética , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Proteínas Nucleares/genética , Interferência de RNA , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
7.
Nucleic Acids Res ; 41(17): 8319-31, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23828042

RESUMO

The nonsense-mediated mRNA decay (NMD) pathway selectively degrades mRNAs harboring premature termination codons but also regulates the abundance of cellular RNAs. We sought to identify transcripts that are regulated by two novel NMD factors, DHX34 and neuroblastoma amplified sequence (NBAS), which were identified in a genome-wide RNA interference screen in Caenorhabditis elegans and later shown to mediate NMD in vertebrates. We performed microarray expression profile analysis in human cells, zebrafish embryos and C. elegans that were individually depleted of these factors. Our analysis revealed that a significant proportion of genes are co-regulated by DHX34, NBAS and core NMD factors in these three organisms. Further analysis indicates that NMD modulates cellular stress response pathways and membrane trafficking across species. Interestingly, transcripts encoding different NMD factors were sensitive to DHX34 and NBAS depletion, suggesting that these factors participate in a conserved NMD negative feedback regulatory loop, as was recently described for core NMD factors. In summary, we find that DHX34 and NBAS act in concert with core NMD factors to co-regulate a large number of endogenous RNA targets. Furthermore, the conservation of a mechanism to tightly control NMD homeostasis across different species highlights the importance of the NMD response in the control of gene expression.


Assuntos
Proteínas de Caenorhabditis elegans/fisiologia , Degradação do RNAm Mediada por Códon sem Sentido , RNA Helicases/fisiologia , Proteínas de Peixe-Zebra/fisiologia , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/antagonistas & inibidores , Evolução Molecular , Perfilação da Expressão Gênica , Células HeLa , Homeostase , Humanos , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/fisiologia , RNA Helicases/antagonistas & inibidores , RNA Mensageiro/metabolismo , Transativadores/fisiologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/antagonistas & inibidores
8.
Nucleic Acids Res ; 39(9): 3686-94, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21227923

RESUMO

The nonsense-mediated mRNA decay (NMD) pathway is a highly conserved surveillance mechanism that is present in all eukaryotes. It prevents the synthesis of truncated proteins by selectively degrading mRNAs harbouring premature termination codons (PTCs). The core NMD effectors were originally identified in genetic screens in Saccharomyces cerevisae and in the nematode Caenorhabditis elegans, and subsequently by homology searches in other metazoans. A genome-wide RNAi screen in C. elegans resulted in the identification of two novel NMD genes that are essential for proper embryonic development. Their human orthologues, DHX34 and NAG/NBAS, are required for NMD in human cells. Here, we find that the zebrafish genome encodes orthologues of DHX34 and NAG/NBAS. We show that the morpholino-induced depletion of zebrafish Dhx34 and Nbas proteins results in severe developmental defects and reduced embryonic viability. We also found that Dhx34 and Nbas are required for degradation of PTC-containing mRNAs in zebrafish embryos. The phenotypes observed in both Dhx34 and Nbas morphants are similar to defects in Upf1, Smg-5- or Smg-6- depleted embryos, suggesting that these factors affect the same pathway and confirming that zebrafish embryogenesis requires an active NMD pathway.


Assuntos
Códon sem Sentido , Desenvolvimento Embrionário/genética , RNA Helicases/fisiologia , Estabilidade de RNA , RNA Mensageiro/metabolismo , Proteínas de Peixe-Zebra/fisiologia , Animais , Embrião não Mamífero/embriologia , Embrião não Mamífero/metabolismo , Humanos , Proteínas de Neoplasias/metabolismo , RNA Helicases/genética , RNA Helicases/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
9.
FEBS Lett ; 581(16): 3087-97, 2007 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-17560998

RESUMO

Precursor (pre)-mRNA splicing can impact the efficiency of coupled steps in gene expression. SRm160 (SR-related nuclear matrix protein of 160 kDa), is a splicing coactivator that also functions as a 3'-end cleavage-stimulatory factor. Here, we have identified an evolutionary-conserved SRm160-interacting protein, referred to as hRED120 (for human Arg/Glu/Asp-rich protein of 120 kDa). hRED120 contains a conventional RNA recognition motif and, like SRm160, a PWI nucleic acid binding domain, suggesting that it has the potential to bridge different RNP complexes. Also, similar to SRm160, hRED120 associates with snRNP components, and remains associated with mRNA after splicing. Simultaneous suppression in Caenorhabditis elegans of the ortholog of hRED120 with the orthologs of splicing and 3'-end processing factors results in aberrant growth or developmental defects. These results suggest that RED120 may function to couple splicing with mRNA 3'-end formation.


Assuntos
Processamento de Terminações 3' de RNA/genética , Precursores de RNA/metabolismo , Splicing de RNA/genética , Proteínas de Ligação a RNA/genética , Sequência de Aminoácidos , Animais , Antígenos Nucleares/metabolismo , Caenorhabditis elegans/genética , Núcleo Celular/metabolismo , Clonagem Molecular , Sequência Conservada , Células HeLa , Humanos , Dados de Sequência Molecular , Proteínas Associadas à Matriz Nuclear/metabolismo , Proteínas Nucleares , Filogenia , Ligação Proteica , Estrutura Terciária de Proteína/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Homologia de Sequência de Aminoácidos
10.
Mol Cell Biol ; 22(14): 5141-56, 2002 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-12077342

RESUMO

A growing body of evidence supports the coordination of pre-mRNA processing and transcriptional regulation. We demonstrate here that mammalian PRP4 kinase (PRP4K) is associated with complexes involved in both of these processes. PRP4K is implicated in pre-mRNA splicing as the homologue of the Schizosaccharomyces pombe pre-mRNA splicing kinase Prp4p, and it is enriched in SC35-containing nuclear splicing speckles. RNA interference of Caenorhabditis elegans PRP4K indicates that it is essential in metazoans. In support of a role for PRP4K in pre-mRNA splicing, we identified PRP6, SWAP, and pinin as interacting proteins and demonstrated that PRP4K is a U5 snRNP-associated kinase. In addition, BRG1 and N-CoR, components of nuclear hormone coactivator and corepressor complexes, also interact with PRP4K. PRP4K coimmunoprecipitates with N-CoR, BRG1, pinin, and PRP6, and we present data suggesting that PRP6 and BRG1 are substrates of this kinase. Lastly, PRP4K, BRG1, and PRP6 can be purified as components of the N-CoR-2 complex, and affinity-purified PRP4K/N-CoR complexes exhibit deacetylase activity. We suggest that PRP4K is an essential kinase that, in association with the both U5 snRNP and N-CoR deacetylase complexes, demonstrates a possible coordination of pre-mRNA splicing with chromatin remodeling events involved in transcriptional regulation.


Assuntos
Proteínas de Drosophila , Proteínas Nucleares/isolamento & purificação , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/isolamento & purificação , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Repressoras/isolamento & purificação , Proteínas Repressoras/metabolismo , Ribonucleoproteína Nuclear Pequena U4-U6/isolamento & purificação , Ribonucleoproteína Nuclear Pequena U4-U6/metabolismo , Ribonucleoproteína Nuclear Pequena U5/isolamento & purificação , Ribonucleoproteína Nuclear Pequena U5/metabolismo , Proteínas de Schizosaccharomyces pombe , Sequência de Aminoácidos , Animais , Caenorhabditis elegans/enzimologia , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Transporte/metabolismo , Moléculas de Adesão Celular/metabolismo , Clonagem Molecular , DNA Helicases , Proteínas de Ligação a DNA , Genes de Helmintos , Histona Desacetilases/genética , Histona Desacetilases/isolamento & purificação , Histona Desacetilases/metabolismo , Humanos , Técnicas In Vitro , Camundongos , Dados de Sequência Molecular , Correpressor 1 de Receptor Nuclear , Proteínas Serina-Treonina Quinases/genética , Proteínas/metabolismo , Processamento Pós-Transcricional do RNA , Splicing de RNA , Fatores de Processamento de RNA , Proteínas de Ligação a RNA , Ribonucleoproteína Nuclear Pequena U4-U6/genética , Schizosaccharomyces/enzimologia , Fatores de Transcrição/metabolismo , Transcrição Gênica , Técnicas do Sistema de Duplo-Híbrido
11.
Methods Enzymol ; 449: 149-64, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-19215757

RESUMO

The nonsense-mediated mRNA decay (NMD) pathway is a surveillance mechanism that targets the degradation of mRNAs harboring premature termination codons (PTCs). Two key aspects of NMD are the definition of a PTC codon and the identification of the molecular machinery dedicated to this mechanism. This chapter describes the development of transgenic reporters as well as the use of genome-wide RNAi and genetic screens to identify novel components of the NMD pathway in the nematode Caenorhabditis elegans.


Assuntos
Caenorhabditis elegans/genética , Estabilidade de RNA/genética , Animais , Códon sem Sentido/genética , Interferência de RNA/fisiologia
12.
Genes Dev ; 21(9): 1075-85, 2007 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-17437990

RESUMO

The nonsense-mediated mRNA decay (NMD) pathway selectively degrades mRNAs harboring premature termination codons (PTCs). Seven genes (smg-1-7, for suppressor with morphological effect on genitalia) that are essential for NMD were originally identified in the nematode Caenorhabditis elegans, and orthologs of these genes have been found in several species. Whereas in humans NMD is linked to splicing, PTC definition occurs independently of exon boundaries in Drosophila. Here, we have conducted an analysis of the cis-acting sequences and trans-acting factors that are required for NMD in C. elegans. We show that a PTC codon is defined independently of introns in C. elegans and, consequently, components of the exon junction complex (EJC) are dispensable for NMD. We also show a distance-dependent effect, whereby PTCs that are closer to the 3' end of the mRNA are less sensitive to NMD. We also provide evidence for the existence of previously unidentified components of the NMD pathway that, unlike known smg genes, are essential for viability in C. elegans. A genome-wide RNA interference (RNAi) screen resulted in the identification of two such novel NMD genes, which are essential for proper embryonic development, and as such represent a new class of essential NMD genes in C. elegans that we have termed smgl (for smg lethal). We show that the encoded proteins are conserved throughout evolution and are required for NMD in C. elegans and also in human cells.


Assuntos
Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Códon sem Sentido , RNA de Helmintos/genética , RNA de Helmintos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Animais , Animais Geneticamente Modificados , Sequência de Bases , Caenorhabditis elegans/embriologia , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Sequência Conservada , Éxons , Genes de Helmintos , Genes Reporter , Células HeLa , Humanos , Íntrons , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases , Interferência de RNA , Splicing de RNA
13.
J Biol Chem ; 280(51): 42227-36, 2005 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-16159877

RESUMO

In this study, we describe a rapid immunoaffinity purification procedure for gel-free tandem mass spectrometry-based analysis of endogenous protein complexes and apply it to the characterization of complexes containing the SRm160 (serine/arginine repeat-related nuclear matrix protein of 160 kDa) splicing coactivator. In addition to promoting splicing, SRm160 stimulates 3'-end processing via its N-terminal PWI nucleic acid-binding domain and is found in a post-splicing exon junction complex that has been implicated in coupling splicing with mRNA turnover, export, and translation. Consistent with these known functional associations, we found that the majority of proteins identified in SRm160-containing complexes are associated with pre-mRNA processing. Interestingly, SRm160 is also associated with factors involved in chromatin regulation and sister chromatid cohesion, specifically the cohesin subunits SMC1alpha, SMC3, RAD21, and SA2. Gradient fractionation suggested that there are two predominant SRm160-containing complexes, one enriched in splicing components and the other enriched in cohesin subunits. Co-immunoprecipitation and co-localization experiments, as well as combinatorial RNA interference in Caenorhabditis elegans, support the existence of conserved and functional interactions between SRm160 and cohesin.


Assuntos
Antígenos Nucleares/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas Associadas à Matriz Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Proteoma , Proteínas de Ligação a RNA/metabolismo , Animais , Antígenos Nucleares/química , Antígenos Nucleares/genética , Caenorhabditis elegans/genética , Proteínas Cromossômicas não Histona , Células HeLa , Humanos , Imunoprecipitação , Espectrometria de Massas , Proteínas Associadas à Matriz Nuclear/química , Proteínas Associadas à Matriz Nuclear/genética , Ligação Proteica , Splicing de RNA , RNA Mensageiro/genética , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Coesinas
14.
RNA ; 9(7): 881-91, 2003 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-12810921

RESUMO

The mRNA export pathway is highly conserved throughout evolution. We have used RNA interference (RNAi) to functionally characterize bona fide RNA export factors and components of the exon-exon junction complex (EJC) in Caenorhabditis elegans. RNAi of CeNXT1/p15, the binding partner of CeNXF1/TAP, caused early embryonic lethality, demonstrating an essential function of this gene during C. elegans development. Moreover, depletion of this protein resulted in nuclear accumulation of poly(A)(+) RNAs, supporting a direct role of NXT1/p15 in mRNA export in C. elegans. Previously, we have shown that RNAi of CeSRm160, a protein of the EJC complex, resulted in wild-type phenotype; in the present study, we demonstrate that RNAi of CeY14, another component of this complex, results in embryonic lethality. In contrast, depletion of the EJC component CeRNPS1 results in no discernible phenotype. Proteins of the REF/Aly family act as adaptor proteins mediating the recruitment of the mRNA export factor, NXF1/TAP, to mRNAs. The C. elegans genome encodes three members of the REF/Aly family. RNAi of individual Ref genes, or codepletion of two Ref genes in different combinations, resulted in wild-type phenotype. Simultaneous suppression of all three Ref genes did not compromise viability or progression through developmental stages in the affected progeny, and only caused a minor defect in larval mobility. Furthermore, no defects in mRNA export were observed upon simultaneous depletion of all three REF proteins. These results suggest the existence of multiple adaptor proteins that mediate mRNA export in C. elegans.


Assuntos
Caenorhabditis elegans/genética , RNA Mensageiro/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Animais , Caenorhabditis elegans/embriologia , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Dimerização , Embrião não Mamífero/citologia , Modelos Genéticos , Dados de Sequência Molecular , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/química , Fatores de Transcrição/genética
15.
J Biol Chem ; 278(45): 44153-60, 2003 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-12944400

RESUMO

SRm160 (the SR-related nuclear matrix protein of 160 kDa) functions as a splicing coactivator and 3'-end cleavage-stimulatory factor. It is also a component of the splicing-dependent exon-junction complex (EJC), which has been implicated in coupling of pre-mRNA splicing with mRNA turnover and mRNA export. We have investigated whether the association of SRm160 with the EJC is important for efficient 3'-end cleavage. The EJC components RNPS1, REF, UAP56, and Y14 interact with SRm160. However, when these factors were tethered to transcripts, only SRm160 and RNPS1 stimulated 3'-end cleavage. Whereas SRm160 stimulated cleavage to a similar extent in the presence or absence of an active intron, stimulation of 3'-end cleavage by tethered RNPS1 is dependent on an active intron. Assembly of an EJC adjacent to the cleavage and polyadenylation signal in vitro did not significantly affect cleavage efficiency. These results suggest that SRm160 stimulates cleavage independently of its association with EJC components and that the cleavage-stimulatory activity of RNPS1 may be an indirect consequence of its ability to stimulate splicing. Using RNA interference (RNAi) in Caenorhabditis elegans, we determined whether interactions between SRm160 and the cleavage machinery are important in a whole organism context. Simultaneous RNAi of SRm160 and the cleavage factor CstF-50 (Cleavage stimulation factor 50-kDa subunit) resulted in late embryonic developmental arrest. In contrast, RNAi of CstF-50 in combination with RNPS1 or REFs did not result in an apparent phenotype. Our combined results provide evidence for an evolutionarily conserved interaction between SRm160 and the 3'-end cleavage machinery that functions independently of EJC formation.


Assuntos
Antígenos Nucleares/metabolismo , Éxons/fisiologia , Proteínas Associadas à Matriz Nuclear/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Antígenos Nucleares/química , Antígenos Nucleares/genética , Caenorhabditis elegans/embriologia , Caenorhabditis elegans/genética , Caenorhabditis elegans/crescimento & desenvolvimento , Linhagem Celular , Fator Estimulador de Clivagem/genética , Fator Estimulador de Clivagem/fisiologia , Sequência Conservada , Evolução Molecular , Humanos , Técnicas de Imunoadsorção , Íntrons/fisiologia , Proteínas Associadas à Matriz Nuclear/química , Proteínas Associadas à Matriz Nuclear/genética , Reação em Cadeia da Polimerase , Subunidades Proteicas/genética , Subunidades Proteicas/fisiologia , Interferência de RNA/fisiologia , Precursores de RNA/metabolismo , Splicing de RNA , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas Recombinantes de Fusão , Transfecção
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