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1.
Nat Commun ; 10(1): 4424, 2019 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-31562313

RESUMO

Plant microRNAs (miRNAs) associate with ARGONAUTE1 (AGO1) to direct post-transcriptional gene silencing and regulate numerous biological processes. Although AGO1 predominantly binds miRNAs in vivo, it also associates with endogenous small interfering RNAs (siRNAs). It is unclear whether the miRNA/siRNA balance affects miRNA activities. Here we report that FIERY1 (FRY1), which is involved in 5'-3' RNA degradation, regulates miRNA abundance and function by suppressing the biogenesis of ribosomal RNA-derived siRNAs (risiRNAs). In mutants of FRY1 and the nuclear 5'-3' exonuclease genes XRN2 and XRN3, we find that a large number of 21-nt risiRNAs are generated through an endogenous siRNA biogenesis pathway. The production of risiRNAs correlates with pre-rRNA processing defects in these mutants. We also show that these risiRNAs are loaded into AGO1, causing reduced loading of miRNAs. This study reveals a previously unknown link between rRNA processing and miRNA accumulation.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas Argonauta/metabolismo , MicroRNAs/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , RNA Ribossômico/metabolismo , RNA Interferente Pequeno/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Exorribonucleases/genética , Genes de Plantas , Mutagênese , Proteínas Nucleares/genética , Monoéster Fosfórico Hidrolases/genética , Interferência de RNA , Estabilidade de RNA
2.
Nucleic Acids Res ; 47(17): 9216-9230, 2019 09 26.
Artigo em Inglês | MEDLINE | ID: mdl-31428786

RESUMO

XRN4, the plant cytoplasmic homolog of yeast and metazoan XRN1, catalyzes exoribonucleolytic degradation of uncapped mRNAs from the 5' end. Most studies of cytoplasmic XRN substrates have focused on polyadenylated transcripts, although many substrates are likely first deadenylated. Here, we report the global investigation of XRN4 substrates in both polyadenylated and nonpolyadenylated RNA to better understand the impact of the enzyme in Arabidopsis. RNA degradome analysis demonstrated that xrn4 mutants overaccumulate many more decapped deadenylated intermediates than those that are polyadenylated. Among these XRN4 substrates that have 5' ends precisely at cap sites, those associated with photosynthesis, nitrogen responses and auxin responses were enriched. Moreover, xrn4 was found to be defective in the dark stress response and lateral root growth during N resupply, demonstrating that XRN4 is required during both processes. XRN4 also contributes to nonsense-mediated decay (NMD) and xrn4 accumulates 3' fragments of select NMD targets, despite the lack of the metazoan endoribonuclease SMG6 in plants. Beyond demonstrating that XRN4 is a major player in multiple decay pathways, this study identified intriguing molecular impacts of the enzyme, including those that led to new insights about mRNA decay and discovery of functional contributions at the whole-plant level.


Assuntos
Arabidopsis/genética , Exorribonucleases/genética , Degradação do RNAm Mediada por Códon sem Sentido/genética , Proteínas de Plantas/genética , Estabilidade de RNA/genética , Arabidopsis/crescimento & desenvolvimento , Regulação da Expressão Gênica de Plantas/genética , Nitrogênio/metabolismo , RNA Mensageiro/genética
3.
Nucleic Acids Res ; 47(18): 9524-9541, 2019 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-31392315

RESUMO

Co-transcriptional imprinting of mRNA by Rpb4 and Rpb7 subunits of RNA polymerase II (RNAPII) and by the Ccr4-Not complex conditions its post-transcriptional fate. In turn, mRNA degradation factors like Xrn1 are able to influence RNAPII-dependent transcription, making a feedback loop that contributes to mRNA homeostasis. In this work, we have used repressible yeast GAL genes to perform accurate measurements of transcription and mRNA degradation in a set of mutants. This genetic analysis uncovered a link from mRNA decay to transcription elongation. We combined this experimental approach with computational multi-agent modelling and tested different possibilities of Xrn1 and Ccr4 action in gene transcription. This double strategy brought us to conclude that both Xrn1-decaysome and Ccr4-Not regulate RNAPII elongation, and that they do it in parallel. We validated this conclusion measuring TFIIS genome-wide recruitment to elongating RNAPII. We found that xrn1Δ and ccr4Δ exhibited very different patterns of TFIIS versus RNAPII occupancy, which confirmed their distinct role in controlling transcription elongation. We also found that the relative influence of Xrn1 and Ccr4 is different in the genes encoding ribosomal proteins as compared to the rest of the genome.


Assuntos
Exorribonucleases/genética , RNA Polimerase II/genética , Estabilidade de RNA/genética , Ribonucleases/genética , Proteínas de Saccharomyces cerevisiae/genética , Regulação Fúngica da Expressão Gênica , Genoma Fúngico/genética , Impressão Genômica , Proteínas Ribossômicas/genética , Saccharomyces cerevisiae/genética , Fatores de Elongação da Transcrição/genética
4.
Nucleic Acids Res ; 47(17): 9329-9342, 2019 09 26.
Artigo em Inglês | MEDLINE | ID: mdl-31392982

RESUMO

Plus-strand RNA viruses can accumulate viral RNA degradation products during infections. Some of these decay intermediates are generated by the cytosolic 5'-to-3' exoribonuclease Xrn1 (mammals and yeast) or Xrn4 (plants) and are formed when the enzyme stalls on substrate RNAs upon encountering inhibitory RNA structures. Many Xrn-generated RNAs correspond to 3'-terminal segments within the 3'-UTR of viral genomes and perform important functions during infections. Here we have investigated a 3'-terminal small viral RNA (svRNA) generated by Xrn during infections with Tobacco necrosis virus-D (family Tombusviridae). Our results indicate that (i) unlike known stalling RNA structures that are compact and modular, the TNV-D structure encompasses the entire 212 nt of the svRNA and is not functionally transposable, (ii) at least two tertiary interactions within the RNA structure are required for effective Xrn blocking and (iii) most of the svRNA generated in infections is derived from viral polymerase-generated subgenomic mRNA1. In vitro and in vivo analyses allowed for inferences on roles for the svRNA. Our findings provide a new and distinct addition to the growing list of Xrn-resistant viral RNAs and stalling structures found associated with different plant and animal RNA viruses.


Assuntos
Exorribonucleases/genética , Doenças das Plantas/genética , RNA Viral/genética , Tombusviridae/genética , Regiões 3' não Traduzidas , Genoma Viral/genética , Conformação de Ácido Nucleico , Doenças das Plantas/virologia , Biossíntese de Proteínas/genética , Estabilidade de RNA/genética , Tabaco/genética , Tabaco/virologia , Tombusviridae/patogenicidade
5.
Nucleic Acids Res ; 47(17): 9282-9295, 2019 09 26.
Artigo em Inglês | MEDLINE | ID: mdl-31340047

RESUMO

XRN1 is the major cytoplasmic exoribonuclease in eukaryotes, which degrades deadenylated and decapped mRNAs in the last step of the 5'-3' mRNA decay pathway. Metazoan XRN1 interacts with decapping factors coupling the final stages of decay. Here, we reveal a direct interaction between XRN1 and the CCR4-NOT deadenylase complex mediated by a low-complexity region in XRN1, which we term the 'C-terminal interacting region' or CIR. The CIR represses reporter mRNA deadenylation in human cells when overexpressed and inhibits CCR4-NOT and isolated CAF1 deadenylase activity in vitro. Through complementation studies in an XRN1-null cell line, we dissect the specific contributions of XRN1 domains and regions toward decay of an mRNA reporter. We observe that XRN1 binding to the decapping activator EDC4 counteracts the dominant negative effect of CIR overexpression on decay. Another decapping activator PatL1 directly interacts with CIR and alleviates the CIR-mediated inhibition of CCR4-NOT activity in vitro. Ribosome profiling revealed that XRN1 loss impacts not only on mRNA levels but also on the translational efficiency of many cellular transcripts likely as a consequence of incomplete decay. Our findings reveal an additional layer of direct interactions in a tightly integrated network of factors mediating deadenylation, decapping and 5'-3' exonucleolytic decay.


Assuntos
Proteínas de Ligação a DNA/genética , Exorribonucleases/genética , Proteínas Associadas aos Microtúbulos/genética , Capuzes de RNA/genética , Estabilidade de RNA/genética , Endorribonucleases/genética , Humanos , Complexos Multiproteicos/genética , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , Proteínas/genética , RNA Mensageiro/química , RNA Mensageiro/genética , Receptores CCR4/genética , Proteínas Repressoras/genética , Transativadores/genética , Fatores de Transcrição/genética
6.
Nucleic Acids Res ; 47(16): 8746-8754, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31287870

RESUMO

RNase P RNA (RPR), the catalytic subunit of the essential RNase P ribonucleoprotein, removes the 5' leader from precursor tRNAs. The ancestral eukaryotic RPR is a Pol III transcript generated with mature termini. In the branch of the arthropod lineage that led to the insects and crustaceans, however, a new allele arose in which RPR is embedded in an intron of a Pol II transcript and requires processing from intron sequences for maturation. We demonstrate here that the Drosophila intronic-RPR precursor is trimmed to the mature form by the ubiquitous nuclease Rat1/Xrn2 (5') and the RNA exosome (3'). Processing is regulated by a subset of RNase P proteins (Rpps) that protects the nascent RPR from degradation, the typical fate of excised introns. Our results indicate that the biogenesis of RPR in vivo entails interaction of Rpps with the nascent RNA to form the RNase P holoenzyme and suggests that a new pathway arose in arthropods by coopting ancient mechanisms common to processing of other noncoding RNAs.


Assuntos
Processamento Alternativo , Proteínas de Drosophila/genética , Subunidades Proteicas/genética , RNA Mensageiro/genética , RNA de Transferência/genética , Ribonuclease P/genética , Animais , Evolução Biológica , Linhagem Celular , Biologia Computacional/métodos , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimologia , Drosophila melanogaster/genética , Embrião não Mamífero , Éxons , Exorribonucleases/genética , Exorribonucleases/metabolismo , Íntrons , Macrófagos/citologia , Macrófagos/enzimologia , Masculino , Conformação de Ácido Nucleico , Subunidades Proteicas/metabolismo , Proteólise , RNA Mensageiro/metabolismo , RNA de Transferência/metabolismo , Ribonuclease P/metabolismo
7.
Nucleic Acids Res ; 47(15): 8224-8238, 2019 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-31180491

RESUMO

The CCR4-NOT complex plays an important role in the translational repression and deadenylation of mRNAs. However, little is known about the specific roles of interacting factors. We demonstrate that the DEAD-box helicases eIF4A2 and DDX6 interact directly with the MA3 and MIF domains of CNOT1 and compete for binding. Furthermore, we now show that incorporation of eIF4A2 into the CCR4-NOT complex inhibits CNOT7 deadenylation activity in contrast to DDX6 which enhances CNOT7 activity. Polyadenylation tests (PAT) on endogenous mRNAs determined that eIF4A2 bound mRNAs have longer poly(A) tails than DDX6 bound mRNAs. Immunoprecipitation experiments show that eIF4A2 does not inhibit CNOT7 association with the CCR4-NOT complex but instead inhibits CNOT7 activity. We identified a CCR4-NOT interacting factor, TAB182, that modulates helicase recruitment into the CCR4-NOT complex, potentially affecting the outcome for the targeted mRNA. Together, these data show that the fate of an mRNA is dependent on the specific recruitment of either eIF4A2 or DDX6 to the CCR4-NOT complex which results in different pathways for translational repression and mRNA deadenylation.


Assuntos
RNA Helicases DEAD-box/metabolismo , Exorribonucleases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , RNA Mensageiro/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Sítios de Ligação/genética , Ligação Competitiva , RNA Helicases DEAD-box/química , RNA Helicases DEAD-box/genética , Exorribonucleases/genética , Células HEK293 , Células HeLa , Humanos , Modelos Genéticos , Ligação Proteica , Domínios Proteicos , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/genética , RNA Mensageiro/genética , Proteínas Repressoras/genética , Proteína 1 de Ligação a Repetições Teloméricas/genética , Proteína 1 de Ligação a Repetições Teloméricas/metabolismo , Fatores de Transcrição/genética
8.
Gene ; 706: 172-180, 2019 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-31082499

RESUMO

Molecular mechanisms of aging and longevity are still mostly unknown. Mitochondria play central roles in cellular metabolism and aging. In this study, we identified three deletion mutants of mitochondrial metabolism genes (ppa2∆, dss1∆, and afg3∆) that live longer than wild-type cells. These long-lived cells harbored significantly decreased amount of mitochondrial DNA (mtDNA) and reactive oxygen species (ROS). Compared to the serpentine nature of wild-type mitochondria, a different dynamics and distribution pattern of mitochondria were observed in the mutants. Both young and old long-lived cells produced relatively low but adequate levels of ATP for cellular activities. The status of the retrograde signaling was checked by expression of CIT2 gene and found activated in long-lived mutants. The mutant cells were also profiled for their gene expression patterns, and genes that were differentially regulated were determined. All long-lived cells comprised similar pleiotropic phenotype regarding mitochondrial dynamics and functions. Thus, this study suggests that DSS1, PPA2, and AFG3 genes modulate the lifespan by altering the mitochondrial morphology and functions.


Assuntos
Longevidade/genética , Mitocôndrias/genética , Mitocôndrias/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Envelhecimento , DNA Mitocondrial/metabolismo , Exorribonucleases/genética , Exorribonucleases/metabolismo , Genes Mitocondriais/genética , Genótipo , Proteínas Mitocondriais/genética , Estresse Oxidativo , Fenótipo , Bombas de Próton/genética , Bombas de Próton/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Deleção de Sequência , Transdução de Sinais
9.
Nat Commun ; 10(1): 2135, 2019 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-31086179

RESUMO

The exosome is a ribonucleolytic complex that plays important roles in RNA metabolism. Here we show that the exosome is necessary for the repair of DNA double-strand breaks (DSBs) in human cells and that RNA clearance is an essential step in homologous recombination. Transcription of DSB-flanking sequences results in the production of damage-induced long non-coding RNAs (dilncRNAs) that engage in DNA-RNA hybrid formation. Depletion of EXOSC10, an exosome catalytic subunit, leads to increased dilncRNA and DNA-RNA hybrid levels. Moreover, the targeting of the ssDNA-binding protein RPA to sites of DNA damage is impaired whereas DNA end resection is hyper-stimulated in EXOSC10-depleted cells. The DNA end resection deregulation is abolished by transcription inhibitors, and RNase H1 overexpression restores the RPA recruitment defect caused by EXOSC10 depletion, which suggests that RNA clearance of newly synthesized dilncRNAs is required for RPA recruitment, controlled DNA end resection and assembly of the homologous recombination machinery.


Assuntos
Quebras de DNA de Cadeia Dupla , Exorribonucleases/metabolismo , Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , Recombinação Homóloga , Proteína de Replicação A/metabolismo , DNA/genética , Exorribonucleases/genética , Complexo Multienzimático de Ribonucleases do Exossomo/genética , Exossomos/metabolismo , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , RNA Longo não Codificante/genética , RNA Interferente Pequeno/metabolismo , Rad51 Recombinase/metabolismo , Ribonuclease H/metabolismo
10.
Cancer Sci ; 110(8): 2431-2441, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31144406

RESUMO

The landscape of genetic alterations in disease models such as transgenic mice or mice with carcinogen-induced tumors has provided a huge amount of information that has shed light on the process of tumorigenesis in human non-small-cell lung cancer (NSCLC). We have previously identified stratifin (SFN) as a potent oncogene, and generated SFN-transgenic (Tg-SPC-SFN+/- ) mice, which express human SFN (hSFN) only in the lung. Here, we have found that carcinogen nicotine-derived nitrosaminoketone (NNK)-induced tumors developing in Tg-SPC-SFN+/- mice show a similar histology to human lung adenocarcinoma and exhibit high hSFN expression. In order to compare the genetic characteristics of Tg-SPC-SFN+/- tumors and human lung adenocarcinoma, the former were subjected to whole-exome sequencing. Interestingly, Tg-SPC-SFN+/- tumors showed the distinct distribution of exonic mutations and high number of mutated genes and transversion. Moreover, Tg-SPC-SFN+/- tumors showed 73 genes that were commonly detected in more than 2 tumors, mutations of which were also found in human lung adenocarcinoma. The expression levels of some of these genes were significantly associated with the clinical outcome of lung adenocarcinoma patients. Additionally, mutated genes in Tg-SPC-SFN+/- tumors were closely associated with key canonical pathways such as PI3K/AKT signaling and apoptosis signaling. These results suggest that SFN overexpression is a universal abnormality in human lung adenocarcinogenesis and Tg-SPC-SFN+/- tumors recapitulate key features of major human lung adenocarcinoma. Therefore, Tg-SPC-SFN+/- mice provide a useful model for clarifying the molecular mechanism underlying lung adenocarcinogenesis.


Assuntos
Proteínas 14-3-3/genética , Adenocarcinoma de Pulmão/induzido quimicamente , Adenocarcinoma de Pulmão/genética , Biomarcadores Tumorais/genética , Carcinógenos/farmacologia , Exorribonucleases/genética , Neoplasias Pulmonares/induzido quimicamente , Neoplasias Pulmonares/genética , Mutação/genética , Células A549 , Animais , Apoptose/genética , Carcinoma Pulmonar de Células não Pequenas/induzido quimicamente , Carcinoma Pulmonar de Células não Pequenas/genética , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos ICR , Camundongos Transgênicos , Fosfatidilinositol 3-Quinases/genética , Proteínas Proto-Oncogênicas c-akt/genética , Transdução de Sinais/genética , Sequenciamento Completo do Exoma/métodos
11.
Nucleic Acids Res ; 47(9): 4751-4764, 2019 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-30949699

RESUMO

The DXO family of proteins participates in eukaryotic mRNA 5'-end quality control, removal of non-canonical NAD+ cap and maturation of fungal rRNA precursors. In this work, we characterize the Arabidopsis thaliana DXO homolog, DXO1. We demonstrate that the plant-specific modification within the active site negatively affects 5'-end capping surveillance properties of DXO1, but has only a minor impact on its strong deNADding activity. Unexpectedly, catalytic activity does not contribute to striking morphological and molecular aberrations observed upon DXO1 knockout in plants, which include growth and pigmentation deficiency, global transcriptomic changes and accumulation of RNA quality control siRNAs. Conversely, these phenotypes depend on the plant-specific N-terminal extension of DXO1. Pale-green coloration of DXO1-deficient plants and our RNA-seq data reveal that DXO1 affects chloroplast-localized processes. We propose that DXO1 mediates the connection between RNA turnover and retrograde chloroplast-to-nucleus signaling independently of its deNADding properties.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Proteínas de Cloroplastos/genética , Exorribonucleases/genética , Precursores de RNA/genética , RNA/genética , Arabidopsis/enzimologia , Proteínas de Arabidopsis/química , Proteínas de Cloroplastos/química , Cloroplastos/genética , Exorribonucleases/química , Técnicas de Inativação de Genes , Mutação , NAD/genética , RNA/química , Precursores de RNA/química , Processamento Pós-Transcricional do RNA , Estabilidade de RNA/genética , RNA Mensageiro/genética , RNA Interferente Pequeno/genética
12.
Mol Cell ; 74(4): 688-700.e3, 2019 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-30930056

RESUMO

Mutations in RNA-processing enzymes are increasingly linked to human disease. Telomerase RNA and related noncoding RNAs require 3' end-processing steps, including oligoadenylation. Germline mutations in poly(A)ribonuclease (PARN) cause accumulation of extended human telomerase RNA (hTR) species and precipitate dyskeratosis congenita and pulmonary fibrosis. Here, we develop nascent RNAend-seq to measure processing rates of RNA precursors. We find that mature hTR derives from extended precursors but that in PARN-mutant cells hTR maturation kinetically stalls and unprocessed precursors are degraded. Loss of poly(A)polymerase PAPD5 in PARN-mutant cells accelerates hTR maturation and restores hTR processing, indicating that oligoadenylation and deadenylation set rates of hTR maturation. The H/ACA domain mediates hTR maturation by precisely defining the 3' end, recruiting poly(A)polymerase activity, and conferring sensitivity to PARN regulation. These data reveal a feedforward circuit in which post-transcriptional oligoadenylation controls RNA maturation kinetics. Similar alterations in RNA processing rates may contribute to mechanisms of RNA-based human disease.


Assuntos
Disceratose Congênita/genética , Exorribonucleases/genética , RNA Nucleotidiltransferases/genética , RNA/genética , Telomerase/genética , Disceratose Congênita/patologia , Mutação em Linhagem Germinativa/genética , Células HeLa , Humanos , Cinética , Processamento Pós-Transcricional do RNA/genética
13.
Nat Struct Mol Biol ; 26(4): 275-280, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30911188

RESUMO

Messenger RNA (mRNA) homeostasis represents an essential part of gene expression, in which the generation of mRNA by RNA polymerase is counter-balanced by its degradation by nucleases. The conserved 5'-to-3' exoribonuclease Xrn1 has a crucial role in eukaryotic mRNA homeostasis by degrading decapped or cleaved mRNAs post-translationally and, more surprisingly, also co-translationally. Here we report that active Xrn1 can directly and specifically interact with the translation machinery. A cryo-electron microscopy structure of a programmed Saccharomyces cerevisiae 80S ribosome-Xrn1 nuclease complex reveals how the conserved core of Xrn1 enables binding at the mRNA exit site of the ribosome. This interface provides a conduit for channelling of the mRNA from the ribosomal decoding site directly into the active center of the nuclease, thus separating mRNA decoding from degradation by only 17 ± 1 nucleotides. These findings explain how rapid 5'-to-3' mRNA degradation is coupled efficiently to its final round of mRNA translation.


Assuntos
Exorribonucleases/metabolismo , Ribossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Microscopia Crioeletrônica , Exorribonucleases/genética , Exorribonucleases/ultraestrutura , RNA Mensageiro/metabolismo , Ribossomos/genética , Ribossomos/ultraestrutura , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/ultraestrutura
14.
Nat Commun ; 10(1): 1298, 2019 03 21.
Artigo em Inglês | MEDLINE | ID: mdl-30899024

RESUMO

The highly conserved 5'-3' exonuclease Xrn1 regulates gene expression in eukaryotes by coupling nuclear DNA transcription to cytosolic mRNA decay. By integrating transcriptome-wide analyses of translation with biochemical and functional studies, we demonstrate an unanticipated regulatory role of Xrn1 in protein synthesis. Xrn1 promotes translation of a specific group of transcripts encoding membrane proteins. Xrn1-dependence for translation is linked to poor structural RNA contexts for translation initiation, is mediated by interactions with components of the translation initiation machinery and correlates with an Xrn1-dependence for mRNA localization at the endoplasmic reticulum, the translation compartment of membrane proteins. Importantly, for this group of mRNAs, Xrn1 stimulates transcription, mRNA translation and decay. Our results uncover a crosstalk between the three major stages of gene expression coordinated by Xrn1 to maintain appropriate levels of membrane proteins.


Assuntos
Exorribonucleases/genética , Regulação Fúngica da Expressão Gênica , Proteínas de Membrana/genética , Biossíntese de Proteínas , RNA Mensageiro/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Transcrição Genética , Clonagem Molecular , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Exorribonucleases/metabolismo , Expressão Gênica , Perfilação da Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Proteínas de Membrana/metabolismo , Estabilidade de RNA , RNA Mensageiro/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais
15.
RNA ; 25(6): 737-746, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30926754

RESUMO

Human RNA exoribonuclease 2 (Rexo2) is an evolutionarily conserved 3'-to-5' DEDDh-family exonuclease located primarily in mitochondria. Rexo2 degrades small RNA oligonucleotides of <5 nucleotides (nanoRNA) in a way similar to Escherichia coli Oligoribonuclease (ORN), suggesting that it plays a role in RNA turnover in mitochondria. However, how Rexo2 preferentially binds and degrades nanoRNA remains elusive. Here, we show that Rexo2 binds small RNA and DNA oligonucleotides with the highest affinity, and it is most robust in degrading small nanoRNA into mononucleotides in the presence of magnesium ions. We further determined three crystal structures of Rexo2 in complex with single-stranded RNA or DNA at resolutions of 1.8-2.2 Å. Rexo2 forms a homodimer and interacts mainly with the last two 3'-end nucleobases of substrates by hydrophobic and π-π stacking interactions via Leu53, Trp96, and Tyr164, signifying its preference in binding and degrading short oligonucleotides without sequence specificity. Crystal structure of Rexo2 is highly similar to that of the RNA-degrading enzyme ORN, revealing a two-magnesium-ion-dependent hydrolysis mechanism. This study thus provides the molecular basis for human Rexo2, showing how it binds and degrades nanoRNA into nucleoside monophosphates and plays a crucial role in RNA salvage pathways in mammalian mitochondria.


Assuntos
Proteínas 14-3-3/química , Biomarcadores Tumorais/química , DNA de Cadeia Simples/química , Exorribonucleases/química , Magnésio/química , Proteínas Mitocondriais/química , Oligorribonucleotídeos/química , RNA/química , Proteínas 14-3-3/genética , Proteínas 14-3-3/metabolismo , Sítios de Ligação , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Cátions Bivalentes , Clonagem Molecular , Cristalografia por Raios X , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Exorribonucleases/genética , Exorribonucleases/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Hidrólise , Interações Hidrofóbicas e Hidrofílicas , Magnésio/metabolismo , Mitocôndrias/química , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Modelos Moleculares , Oligorribonucleotídeos/genética , Oligorribonucleotídeos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , RNA/genética , RNA/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
16.
Nucleic Acids Res ; 47(7): 3631-3639, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-30828718

RESUMO

Correct synthesis and maintenance of functional tRNA 3'-CCA-ends is a crucial prerequisite for aminoacylation and must be achieved by the phylogenetically diverse group of tRNA nucleotidyltransferases. While numerous reports on the in vitro characterization exist, robust analysis under in vivo conditions is lacking. Here, we utilize Escherichia coli RNase T, a tRNA-processing enzyme responsible for the tRNA-CCA-end turnover, to generate an in vivo system for the evaluation of A-adding activity. Expression of RNase T results in a prominent growth phenotype that renders the presence of a CCA- or A-adding enzyme essential for cell survival in an E. coli Δcca background. The distinct growth fitness allows for both complementation and selection of enzyme variants in a natural environment. We demonstrate the potential of our system via detection of altered catalytic efficiency and temperature sensitivity. Furthermore, we select functional enzyme variants out of a sequence pool carrying a randomized codon for a highly conserved position essential for catalysis. The presented E. coli-based approach opens up a wide field of future studies including the investigation of tRNA nucleotidyltransferases from all domains of life and the biological relevance of in vitro data concerning their functionality and mode of operation.


Assuntos
Escherichia coli/genética , Exorribonucleases/genética , Nucleotidiltransferases/genética , RNA Nucleotidiltransferases/genética , Aminoacilação/genética , Escherichia coli/crescimento & desenvolvimento , Exorribonucleases/química , Cinética , Conformação de Ácido Nucleico , Nucleotidiltransferases/química , RNA Nucleotidiltransferases/química , Processamento Pós-Transcricional do RNA/genética
17.
Nucleic Acids Res ; 47(5): 2229-2243, 2019 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-30859196

RESUMO

DNA replication is a stochastic process with replication forks emanating from multiple replication origins. The origins must be licenced in G1, and the replisome activated at licenced origins in order to generate bi-directional replication forks in S-phase. Differential firing times lead to origin interference, where a replication fork from an origin can replicate through and inactivate neighbouring origins (origin obscuring). We developed a Bayesian algorithm to characterize origin firing statistics from Okazaki fragment (OF) sequencing data. Our algorithm infers the distributions of firing times and the licencing probabilities for three consecutive origins. We demonstrate that our algorithm can distinguish partial origin licencing and origin obscuring in OF sequencing data from Saccharomyces cerevisiae and human cell types. We used our method to analyse the decreased origin efficiency under loss of Rat1 activity in S. cerevisiae, demonstrating that both reduced licencing and increased obscuring contribute. Moreover, we show that robust analysis is possible using only local data (across three neighbouring origins), and analysis of the whole chromosome is not required. Our algorithm utilizes an approximate likelihood and a reversible jump sampling technique, a methodology that can be extended to analysis of other mechanistic processes measurable through Next Generation Sequencing data.


Assuntos
Algoritmos , Replicação do DNA/genética , Sequenciamento de Nucleotídeos em Larga Escala , Origem de Replicação/genética , Teorema de Bayes , Cromossomos/genética , DNA/biossíntese , DNA/genética , Exorribonucleases/genética , Humanos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Processos Estocásticos
18.
Nucleic Acids Res ; 47(6): 3045-3057, 2019 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-30715470

RESUMO

Uridylation-dependent RNA decay is a widespread eukaryotic pathway modulating RNA homeostasis. Terminal uridylyltransferases (Tutases) add untemplated uridyl residues to RNA 3'-ends, marking them for degradation by the U-specific exonuclease Dis3L2. In Schizosaccharomyces pombe, Cid1 uridylates a variety of RNAs. In this study, we investigate the prevalence and impact of uridylation-dependent RNA decay in S. pombe by transcriptionally profiling cid1 and dis3L2 deletion strains. We found that the exonuclease Dis3L2 represents a bottleneck in uridylation-dependent mRNA decay, whereas Cid1 plays a redundant role that can be complemented by other Tutases. Deletion of dis3L2 elicits a cellular stress response, upregulating transcription of genes involved in protein folding and degradation. Misfolded proteins accumulate in both deletion strains, yet only trigger a strong stress response in dis3L2 deficient cells. While a deletion of cid1 increases sensitivity to protein misfolding stress, a dis3L2 deletion showed no increased sensitivity or was even protective. We furthermore show that uridylyl- and adenylyltransferases cooperate to generate a 5'-NxAUUAAAA-3' RNA motif on dak2 mRNA. Our studies elucidate the role of uridylation-dependent RNA decay as part of a global mRNA surveillance, and we found that perturbation of this pathway leads to the accumulation of misfolded proteins and elicits cellular stress responses.


Assuntos
RNA Nucleotidiltransferases/genética , Estabilidade de RNA/genética , Proteínas de Schizosaccharomyces pombe/genética , Schizosaccharomyces/genética , Exorribonucleases/genética , Complexo Multienzimático de Ribonucleases do Exossomo/genética , Nucleotidiltransferases/genética , RNA Fúngico/genética , RNA Mensageiro/genética , Uridina/genética
19.
Mol Cell ; 74(1): 196-211.e11, 2019 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-30799147

RESUMO

The compendium of RNA-binding proteins (RBPs) has been greatly expanded by the development of RNA-interactome capture (RIC). However, it remained unknown if the complement of RBPs changes in response to environmental perturbations and whether these rearrangements are important. To answer these questions, we developed "comparative RIC" and applied it to cells challenged with an RNA virus called sindbis (SINV). Over 200 RBPs display differential interaction with RNA upon SINV infection. These alterations are mainly driven by the loss of cellular mRNAs and the emergence of viral RNA. RBPs stimulated by the infection redistribute to viral replication factories and regulate the capacity of the virus to infect. For example, ablation of XRN1 causes cells to be refractory to SINV, while GEMIN5 moonlights as a regulator of SINV gene expression. In summary, RNA availability controls RBP localization and function in SINV-infected cells.


Assuntos
Células Epiteliais/virologia , Perfilação da Expressão Gênica/métodos , RNA Viral/genética , Proteínas de Ligação a RNA/genética , Vírus Sindbis/genética , Transcriptoma , Neoplasias do Colo do Útero/virologia , Regiões 5' não Traduzidas , Sítios de Ligação , Células Epiteliais/metabolismo , Exorribonucleases/genética , Exorribonucleases/metabolismo , Feminino , Regulação Viral da Expressão Gênica , Células HEK293 , Células HeLa , Interações Hospedeiro-Patógeno , Humanos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Ligação Proteica , RNA Viral/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas Nucleares Pequenas/genética , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Vírus Sindbis/crescimento & desenvolvimento , Vírus Sindbis/metabolismo , Vírus Sindbis/patogenicidade , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/metabolismo , Replicação Viral
20.
Funct Integr Genomics ; 19(3): 437-452, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30671704

RESUMO

Significance of microRNAs in regulating gene expression in higher eukaryotes as well as in pathogens like fungi to suppress host defense is a well-established phenomenon. The present study focuses on leaf rust fungi Puccinia triticina (Pathotype 77-5) mediated RNAi to make wheat (Triticum aestivum L.) more susceptible. To reach such conclusions, we first confirmed the presence of argonaute (AGO) and dicer-like protein (DCL) family sequences in Puccinia. Bioinformatic tools were applied to retrieve the sequences from Puccinia genome followed by cloning and sequencing from P. triticina pathotype 77-5 cDNA to obtain the specific sequences. Their homologs were searched in other 14 Puccinia races to relate them with pathogenesis. Further, precursor sequences for three miRNA-like RNA molecules (milRs) were cloned from P. triticina cDNA. Their target genes like MAP kinase were successfully predicted and validated through degradome mapping and qRT-PCR. Gradual increase in milR2 (milR and milR*) expression over progressive time point of infection and positive expression for all the milRs within 77-5 urediniospores confirmed a complete host- independent RNAi activity by P. triticina.


Assuntos
Basidiomycota/genética , Inativação Gênica , Interações Hospedeiro-Patógeno/genética , MicroRNAs/genética , Imunidade Vegetal/genética , Triticum/genética , Proteínas Argonauta/genética , Proteínas Argonauta/metabolismo , Basidiomycota/patogenicidade , Exorribonucleases/genética , Exorribonucleases/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Triticum/microbiologia
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