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1.
Mol Cell ; 82(9): 1678-1690.e12, 2022 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-35305312

RESUMO

The functional consequence of N6-methyladenosine (m6A) RNA modification is mediated by "reader" proteins of the YTH family. YTH domain-containing 2 (YTHDC2) is essential for mammalian fertility, but its molecular function is poorly understood. Here, we identify U-rich motifs as binding sites of YTHDC2 on 3' UTRs of mouse testicular RNA targets. Although its YTH domain is an m6A-binder in vitro, the YTH point mutant mice are fertile. Significantly, the loss of its 3'→5' RNA helicase activity causes mouse infertility, with the catalytic-dead mutation being dominant negative. Biochemical studies reveal that the weak helicase activity of YTHDC2 is enhanced by its interaction with the 5'→3' exoribonuclease XRN1. Single-cell transcriptomics indicate that Ythdc2 mutant mitotic germ cells transition into meiosis but accumulate a transcriptome with mixed mitotic/meiotic identity that fail to progress further into meiosis. Finally, our demonstration that ythdc2 mutant zebrafish are infertile highlights its conserved role in animal germ cell development.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Exorribonucleases/metabolismo , RNA Helicases , Peixe-Zebra , Animais , Fertilidade/genética , Mamíferos/metabolismo , Meiose , Camundongos , RNA/genética , RNA Helicases/genética , RNA Helicases/metabolismo , Peixe-Zebra/genética
2.
EMBO J ; 2024 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-39394354

RESUMO

Complete cytoplasmic polyadenosine tail (polyA-tail) deadenylation is thought to be essential for initiating mRNA decapping and subsequent degradation. To investigate this prevalent model, we conducted direct RNA sequencing of S. cerevisiae mRNAs derived from chase experiments under steady-state and stress condition. Subsequently, we developed a numerical model based on a modified gamma distribution function, which estimated the transcriptomic deadenylation rate at 10 A/min. A simplified independent method, based on the delineation of quantile polyA-tail values, showed a correlation between the decay and deadenylation rates of individual mRNAs, which appeared consistent within functional transcript groups and associated with codon optimality. Notably, these rates varied during the stress response. Detailed analysis of ribosomal protein-coding mRNAs (RPG mRNAs), constituting 40% of the transcriptome, singled out this transcript group. While deadenylation and decay of RPG mRNAs accelerated under heat stress, their degradation could proceed even when deadenylation was blocked, depending entirely on ongoing nuclear export. Our findings support the general primary function of deadenylation in dictating the onset of decapping, while also demonstrating complex relations between these processes.

3.
Mol Cell ; 75(2): 324-339.e11, 2019 07 25.
Artigo em Inglês | MEDLINE | ID: mdl-31155380

RESUMO

Nonsense-mediated decay (NMD) is a surveillance system that degrades mRNAs containing a premature termination codon (PTC) and plays important roles in protein homeostasis and disease. The efficiency of NMD is variable, impacting the clinical outcome of genetic mutations. However, limited resolution of bulk analyses has hampered the study of NMD efficiency. Here, we develop an assay to visualize NMD of individual mRNA molecules in real time. We find that NMD occurs with equal probability during each round of translation of an mRNA molecule. However, this probability is variable and depends on the exon sequence downstream of the PTC, the PTC-to-intron distance, and the number of introns both upstream and downstream of the PTC. Additionally, a subpopulation of mRNAs can escape NMD, further contributing to variation in NMD efficiency. Our study uncovers real-time dynamics of NMD, reveals key mechanisms that influence NMD efficiency, and provides a powerful method to study NMD.


Assuntos
Códon sem Sentido/genética , Degradação do RNAm Mediada por Códon sem Sentido/genética , RNA Mensageiro/genética , Códon sem Sentido/química , Éxons/genética , Humanos , Íntrons/genética , Mutação/genética , Estabilidade de RNA/genética , RNA Mensageiro/química , Imagem Individual de Molécula
4.
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 , Sindbis virus/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 , Proteínas do Complexo SMN , Sindbis virus/crescimento & desenvolvimento , Sindbis virus/metabolismo , Sindbis virus/patogenicidade , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/metabolismo , Replicação Viral
5.
J Cell Sci ; 137(11)2024 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-38841902

RESUMO

The model of RNA stability has undergone a transformative shift with the revelation of a cytoplasmic capping activity that means a subset of transcripts are recapped autonomously of their nuclear counterparts. The present study demonstrates nucleo-cytoplasmic shuttling of the mRNA-capping enzyme (CE, also known as RNA guanylyltransferase and 5'-phosphatase; RNGTT), traditionally acknowledged for its nuclear localization and functions, elucidating its contribution to cytoplasmic capping activities. A unique nuclear export sequence in CE mediates XPO1-dependent nuclear export of CE. Notably, during sodium arsenite-induced oxidative stress, cytoplasmic CE (cCE) congregates within stress granules (SGs). Through an integrated approach involving molecular docking and subsequent co-immunoprecipitation, we identify eIF3b, a constituent of SGs, as an interactive associate of CE, implying that it has a potential role in guiding cCE to SGs. We measured the cap status of specific mRNA transcripts from U2OS cells that were non-stressed, stressed and recovered from stress, which indicated that cCE-target transcripts lost their caps during stress but remarkably regained cap stability during the recovery phase. This comprehensive study thus uncovers a novel facet of cytoplasmic CE, which facilitates cellular recovery from stress by maintaining cap homeostasis of target mRNAs.


Assuntos
Citoplasma , Homeostase , RNA Mensageiro , Grânulos de Estresse , Humanos , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Grânulos de Estresse/metabolismo , Citoplasma/metabolismo , Capuzes de RNA/metabolismo , Arsenitos/farmacologia , Estresse Oxidativo , Transporte Ativo do Núcleo Celular , RNA Nucleotidiltransferases/metabolismo , RNA Nucleotidiltransferases/genética , Compostos de Sódio/farmacologia , Proteína Exportina 1 , Carioferinas/metabolismo , Carioferinas/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Grânulos Citoplasmáticos/metabolismo , Estabilidade de RNA , Núcleo Celular/metabolismo , Linhagem Celular Tumoral , Nucleotidiltransferases
6.
RNA ; 30(6): 662-679, 2024 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-38443115

RESUMO

Despite being predicted to lack coding potential, cytoplasmic long noncoding (lnc)RNAs can associate with ribosomes. However, the landscape and biological relevance of lncRNA translation remain poorly studied. In yeast, cytoplasmic Xrn1-sensitive unstable transcripts (XUTs) are targeted by nonsense-mediated mRNA decay (NMD), suggesting a translation-dependent degradation process. Here, we report that XUTs are pervasively translated, which impacts their decay. We show that XUTs globally accumulate upon translation elongation inhibition, but not when initial ribosome loading is impaired. Ribo-seq confirmed ribosomes binding to XUTs and identified ribosome-associated 5'-proximal small ORFs. Mechanistically, the NMD-sensitivity of XUTs mainly depends on the 3'-untranslated region length. Finally, we show that the peptide resulting from the translation of an NMD-sensitive XUT reporter exists in NMD-competent cells. Our work highlights the role of translation in the posttranscriptional metabolism of XUTs. We propose that XUT-derived peptides could be exposed to natural selection, while NMD restricts XUT levels.


Assuntos
Exorribonucleases , Degradação do RNAm Mediada por Códon sem Sentido , Biossíntese de Proteínas , RNA Longo não Codificante , Ribossomos , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Exorribonucleases/metabolismo , Exorribonucleases/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Ribossomos/metabolismo , Ribossomos/genética , Regiões 3' não Traduzidas , Fases de Leitura Aberta , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Estabilidade de RNA
7.
Mol Cell ; 72(1): 10-17, 2018 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-30290147

RESUMO

Transcript buffering involves reciprocal adjustments between overall rates in mRNA synthesis and degradation to maintain similar cellular concentrations of mRNAs. This phenomenon was first discovered in yeast and encompasses coordination between the nuclear and cytoplasmic compartments. Transcript buffering was revealed by novel methods for pulse labeling of RNA to determine in vivo synthesis and degradation rates. In this Perspective, we discuss the current knowledge of transcript buffering. Emphasis is placed on the future challenges to determine the nature and directionality of the buffering signals, the generality of transcript buffering beyond yeast, and the molecular mechanisms responsible for this balancing.


Assuntos
Estabilidade de RNA/genética , RNA Mensageiro/biossíntese , Transcrição Gênica , Núcleo Celular/genética , Citoplasma/genética , Capuzes de RNA/genética , RNA Mensageiro/genética , Saccharomyces cerevisiae/genética
8.
Mol Cell ; 68(3): 615-625.e9, 2017 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-29056324

RESUMO

RNA degradation plays a fundamental role in regulating gene expression. In order to characterize the spatiotemporal dynamics of RNA turnover in single cells, we developed a fluorescent biosensor based on dual-color, single-molecule RNA imaging that allows intact transcripts to be distinguished from stabilized degradation intermediates. Using this method, we measured mRNA decay in single cells and found that individual degradation events occur independently within the cytosol and are not enriched within processing bodies. We show that slicing of an mRNA targeted for endonucleolytic cleavage by the RNA-induced silencing complex can be observed in real time in living cells. This methodology provides a framework for investigating the entire life history of individual mRNAs from birth to death in single cells.


Assuntos
Microscopia de Fluorescência , Estabilidade de RNA , RNA Mensageiro/metabolismo , Imagem Individual de Molécula/métodos , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Regulação da Expressão Gênica , Genes Reporter , Células HeLa , Humanos , Cinética , Microscopia de Vídeo , Modelos Genéticos , RNA Mensageiro/genética , Transfecção
9.
Mol Cell ; 68(2): 374-387.e12, 2017 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-29033321

RESUMO

N6-methyladenosine (m6A) is an essential internal RNA modification that is critical for gene expression control in most organisms. Proteins with a YTH domain recognize m6A marks and are mediators of molecular functions like RNA splicing, mRNA decay, and translation control. Here we demonstrate that YTH domain-containing 2 (YTHDC2) is an m6A reader that is essential for male and female fertility in mice. High-throughput mapping of the m6A transcriptome and expression analysis in the Yhtdc2 mutant testes reveal an upregulation of m6A-enriched transcripts. Our biochemical studies indicate that YTHDC2 is an RNA-induced ATPase with a 3'→5' RNA helicase activity. Furthermore, YTHDC2 recruits the 5'→3' exoribonuclease XRN1 via Ankyrin repeats that are inserted in between the RecA modules of the RNA helicase domain. Our studies reveal a role for YTHDC2 in modulating the levels of m6A-modified germline transcripts to maintain a gene expression program that is conducive for progression through meiosis.


Assuntos
Adenosina/análogos & derivados , Regulação da Expressão Gênica/fisiologia , Meiose/fisiologia , RNA Helicases/metabolismo , RNA Mensageiro/metabolismo , Adenosina/genética , Adenosina/metabolismo , Animais , Repetição de Anquirina , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Exorribonucleases/genética , Exorribonucleases/metabolismo , Masculino , Camundongos , Camundongos Mutantes , Domínios Proteicos , RNA Helicases/genética , RNA Mensageiro/genética
10.
Biochem J ; 481(7): 481-498, 2024 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-38440860

RESUMO

The protein kinase Gcn2 and its effector protein Gcn1 are part of the general amino acid control signalling (GAAC) pathway best known in yeast for its function in maintaining amino acid homeostasis. Under amino acid limitation, Gcn2 becomes activated, subsequently increasing the levels of phosphorylated eIF2α (eIF2α-P). This leads to the increased translation of transcriptional regulators, such as Gcn4 in yeast and ATF4 in mammals, and subsequent re-programming of the cell's gene transcription profile, thereby allowing cells to cope with starvation. Xrn1 is involved in RNA decay, quality control and processing. We found that Xrn1 co-precipitates Gcn1 and Gcn2, suggesting that these three proteins are in the same complex. Growth under starvation conditions was dependent on Xrn1 but not on Xrn1-ribosome association, and this correlated with reduced eIF2α-P levels. Constitutively active Gcn2 leads to a growth defect due to eIF2α-hyperphosphorylation, and we found that this phenotype was independent of Xrn1, suggesting that xrn1 deletion does not enhance eIF2α de-phosphorylation. Our study provides evidence that Xrn1 is required for efficient Gcn2 activation, directly or indirectly. Thus, we have uncovered a potential new link between RNA metabolism and the GAAC.


Assuntos
Fator de Iniciação 2 em Eucariotos , Exorribonucleases , Fatores de Alongamento de Peptídeos , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Aminoácidos/metabolismo , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Mamíferos/metabolismo , Fatores de Alongamento de Peptídeos/genética , Fatores de Alongamento de Peptídeos/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Exorribonucleases/genética , Exorribonucleases/metabolismo
11.
Mol Cell ; 61(3): 379-392, 2016 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-26805575

RESUMO

Antisense long non-coding (aslnc)RNAs represent a substantial part of eukaryotic transcriptomes that are, in yeast, controlled by the Xrn1 exonuclease. Nonsense-Mediated Decay (NMD) destabilizes the Xrn1-sensitive aslncRNAs (XUT), but what determines their sensitivity remains unclear. We report that 3' single-stranded (3'-ss) extension mediates XUTs degradation by NMD, assisted by the Mtr4 and Dbp2 helicases. Single-gene investigation, genome-wide RNA analyses, and double-stranded (ds)RNA mapping revealed that 3'-ss extensions discriminate the NMD-targeted XUTs from stable lncRNAs. Ribosome profiling showed that XUT are translated, locking them for NMD activity. Interestingly, mutants of the Mtr4 and Dbp2 helicases accumulated XUTs, suggesting that dsRNA unwinding is a critical step for degradation. Indeed, expression of anticomplementary transcripts protects cryptic intergenic lncRNAs from NMD. Our results indicate that aslncRNAs form dsRNA that are only translated and targeted to NMD if dissociated by Mtr4 and Dbp2. We propose that NMD buffers genome expression by discarding pervasive regulatory transcripts.


Assuntos
Degradação do RNAm Mediada por Códon sem Sentido , RNA de Cadeia Dupla/metabolismo , RNA Fúngico/metabolismo , RNA Longo não Codificante/metabolismo , Saccharomyces cerevisiae/metabolismo , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Exorribonucleases/genética , Exorribonucleases/metabolismo , Regulação Fúngica da Expressão Gênica , Genoma Fúngico , Mutação , Conformação de Ácido Nucleico , Fases de Leitura Aberta , RNA Antissenso/genética , RNA Antissenso/metabolismo , RNA de Cadeia Dupla/química , RNA de Cadeia Dupla/genética , RNA Fúngico/química , RNA Fúngico/genética , RNA Longo não Codificante/química , RNA Longo não Codificante/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transcrição Gênica
12.
J Gen Virol ; 104(12)2023 12.
Artigo em Inglês | MEDLINE | ID: mdl-38059479

RESUMO

Flavivirids are small, enveloped, positive-sense RNA viruses from the family Flaviviridae with genomes of ~9-13 kb. Metatranscriptomic analyses of metazoan organisms have revealed a diversity of flavivirus-like or flavivirid viral sequences in fish and marine invertebrate groups. However, no flavivirus-like virus has been identified in amphibians. To remedy this, we investigated the virome of the European common frog (Rana temporaria) in the UK, utilizing high-throughput sequencing at six catch locations. De novo assembly revealed a coding-complete virus contig of a novel flavivirid ~11.2 kb in length. The virus encodes a single ORF of 3456 aa and 5' and 3' untranslated regions (UTRs) of 227 and 666 nt, respectively. We named this virus Rana tamanavirus (RaTV), as BLASTp analysis of the polyprotein showed the closest relationships to Tamana bat virus (TABV) and Cyclopterus lumpus virus from Pteronotus parnellii and Cyclopterus lumpus, respectively. Phylogenetic analysis of the RaTV polyprotein compared to Flavivirus and Flavivirus-like members indicated that RaTV was sufficiently divergent and basal to the vertebrate Tamanavirus clade. In addition to the Mitcham strain, partial but divergent RaTV, sharing 95.64-97.39 % pairwise nucleotide identity, were also obtained from the Poole and Deal samples, indicating that RaTV is widespread in UK frog samples. Bioinformatic analyses of predicted secondary structures in the 3'UTR of RaTV showed the presence of an exoribonuclease-resistant RNA (xrRNA) structure standard in flaviviruses and TABV. To examine this biochemically, we conducted an in vitro Xrn1 digestion assay showing that RaTV probably forms a functional Xrn1-resistant xrRNA.


Assuntos
Flaviviridae , Flavivirus , Animais , Flaviviridae/genética , Rana temporaria/genética , Filogenia , RNA Viral/genética , RNA Viral/química , Flavivirus/genética , Poliproteínas/genética , Reino Unido , Genoma Viral
13.
RNA ; 27(10): 1265-1280, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34266995

RESUMO

XRN1 is a highly conserved exoribonuclease which degrades uncapped RNAs in a 5'-3' direction. Degradation of RNAs by XRN1 is important in many cellular and developmental processes and is relevant to human disease. Studies in D. melanogaster demonstrate that XRN1 can target specific RNAs, which have important consequences for developmental pathways. Osteosarcoma is a malignancy of the bone and accounts for 2% of all pediatric cancers worldwide. Five-year survival of patients has remained static since the 1970s and therefore furthering our molecular understanding of this disease is crucial. Previous work has shown a down-regulation of XRN1 in osteosarcoma cells; however, the transcripts regulated by XRN1 which might promote osteosarcoma remain elusive. Here, we confirm reduced levels of XRN1 in osteosarcoma cell lines and patient samples and identify XRN1-sensitive transcripts in human osteosarcoma cells. Using RNA-seq in XRN1-knockdown SAOS-2 cells, we show that 1178 genes are differentially regulated. Using a novel bioinformatic approach, we demonstrate that 134 transcripts show characteristics of direct post-transcriptional regulation by XRN1. Long noncoding RNAs (lncRNAs) are enriched in this group, suggesting that XRN1 normally plays an important role in controlling lncRNA expression in these cells. Among potential lncRNAs targeted by XRN1 is HOTAIR, which is known to be up-regulated in osteosarcoma and contributes to disease progression. We have also identified G-rich and GU motifs in post-transcriptionally regulated transcripts which appear to sensitize them to XRN1 degradation. Our results therefore provide significant insights into the specificity of XRN1 in human cells which are relevant to disease.


Assuntos
Neoplasias Ósseas/genética , Exorribonucleases/genética , Proteínas Associadas aos Microtúbulos/genética , Osteossarcoma/genética , RNA Longo não Codificante/genética , RNA Mensageiro/genética , RNA Neoplásico/genética , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Linhagem Celular Tumoral , Criança , Biologia Computacional , Exorribonucleases/deficiência , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Estudo de Associação Genômica Ampla , Humanos , Proteínas Associadas aos Microtúbulos/deficiência , Anotação de Sequência Molecular , Motivos de Nucleotídeos , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Processamento Pós-Transcricional do RNA , Estabilidade de RNA , RNA Longo não Codificante/metabolismo , RNA Mensageiro/metabolismo , RNA Neoplásico/metabolismo
14.
J Virol ; 96(22): e0131922, 2022 11 23.
Artigo em Inglês | MEDLINE | ID: mdl-36300942

RESUMO

Many negative-sense RNA viruses, including measles virus (MeV), are thought to carry out much of their viral replication in cytoplasmic membraneless foci known as inclusion bodies (IBs). The mechanisms by which IBs facilitate efficient viral replication remain largely unknown but may involve an intricate network of regulation at the host-virus interface. Viruses are able to modulate such interactions by a variety of strategies including adaptation of their genomes and "hijacking" of host proteins. The latter possibility broadens the molecular reservoir available for a virus to enhance its replication and/or antagonize host antiviral responses. Here, we show that the cellular 5'-3' exoribonuclease, XRN1, is a host protein hijacked by MeV. We found that upon MeV infection, XRN1 is translocated to cytoplasmic IBs where it acts in a proviral manner by preventing the accumulation of double-stranded RNA (dsRNA) within the IBs. This leads to the suppression of the dsRNA-induced innate immune responses mediated via the protein kinase R (PKR)-integrated stress response (ISR) pathway. IMPORTANCE Measles virus remains a major global health threat due to its high transmissibility and significant morbidity in children and immunocompromised individuals. Although there is an effective vaccine against MeV, a large population in the world remains without access to the vaccine, contributing to more than 7,000,000 measles cases and 60,000 measles deaths in 2020 (CDC). For negative-sense RNA viruses including MeV, one active research area is the exploration of virus-host interactions occurring at cytoplasmic IBs where viral replication takes place. In this study we present evidence suggesting a model in which MeV IBs antagonize host innate immunity by recruiting XRN1 to reduce dsRNA accumulation and subsequent PKR kinase activation/ISR induction. In the absence of XRN1, the increased dsRNA level acts as a potent activator of the antiviral PKR/ISR pathway leading to suppression of global cap-dependent mRNA translation and inhibition of viral replication.


Assuntos
Exorribonucleases , Sarampo , Proteínas Associadas aos Microtúbulos , Replicação Viral , Humanos , eIF-2 Quinase/metabolismo , Exorribonucleases/genética , Exorribonucleases/metabolismo , Sarampo/genética , Sarampo/virologia , Vírus do Sarampo/genética , Vírus do Sarampo/fisiologia , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Quinases/metabolismo , Provírus/genética , RNA de Cadeia Dupla , Corpos de Inclusão Viral
15.
RNA Biol ; 20(1): 409-418, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-37400999

RESUMO

Xrn1-resistant RNA structures are multifunctional elements employed by an increasing number of RNA viruses. One of such elements is the coremin motif, discovered in plant virus RNAs, of which the structure has been hypothesized to form a yet unelucidated pseudoknot. Recently, the coremin motif was shown to be capable of stalling not only Xrn1, but scanning ribosomes as well. Following that observation, in this study we demonstrate that the coremin motif can promote -1 ribosomal frameshifting, similar to better-characterized viral frameshifting pseudoknots. Since this function was lost in concert with substitutions that were known to disturb Xrn1-resistance, we developed a frameshifting screen for finding novel Xrn1-resistant RNAs by randomizing parts of the coremin motif. This yielded new insights into the coremin motif structure, as Xrn1-resistant variations were identified that more clearly indicate a pseudoknot interaction. In addition, we show that the Xrn1-resistant RNA of Zika virus promotes frameshifting as well, while known -1 programmed ribosomal frameshifting pseudoknots do not stall Xrn1, suggesting that promoting frameshifting is a universal characteristic of Xrn1-resistant RNAs, but that Xrn1-resistance requires more than just a frameshifting pseudoknot.


Assuntos
Infecção por Zika virus , Zika virus , Humanos , RNA Viral/metabolismo , Sequência de Bases , Conformação de Ácido Nucleico , Mudança da Fase de Leitura do Gene Ribossômico , Ribossomos/metabolismo , Zika virus/genética , Infecção por Zika virus/genética
16.
J Virol ; 95(20): e0103421, 2021 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-34346764

RESUMO

One of the many challenges faced by RNA viruses is the maintenance of their genomes during infections of host cells. Members of the family Tombusviridae are plus-strand RNA viruses with unmodified triphosphorylated genomic 5' termini. The tombusvirus Carnation Italian ringspot virus was used to investigate how it protects its RNA genome from attack by 5'-end-targeting degradation enzymes. In vivo and in vitro assays were employed to determine the role of genomic RNA structure in conferring protection from the 5'-to-3' exoribonuclease Xrn. The results revealed that (i) the CIRV RNA genome is more resistant to Xrn than its sg mRNAs, (ii) the genomic 5'-untranslated region (UTR) folds into a compact RNA structure that effectively and independently prevents Xrn access, (iii) the RNA structure limiting 5' access is formed by secondary and tertiary interactions that function cooperatively, (iv) the structure is also able to block access of RNA pyrophosphohydrolase to the genomic 5' terminus, and (v) the RNA structure does not stall an actively digesting Xrn. Based on its proficiency at impeding Xrn 5' access, we have termed this 5'-terminal structure an Xrn-evading RNA, or xeRNA. These and other findings demonstrate that the 5'UTR of the CIRV RNA genome folds into a complex structural conformation that helps to protect its unmodified 5' terminus from enzymatic decay during infections. IMPORTANCE The plus-strand RNA genomes of plant viruses in the large family Tombusviridae are not 5' capped. Here, we explored how a species in the type genus Tombusvirus protects its genomic 5' end from cellular nuclease attack. Our results revealed that the 5'-terminal sequence of the CIRV genome folds into a complex RNA structure that limits access of the 5'-to-3' exoribonuclease Xrn, thereby protecting it from processive degradation. The RNA conformation also impeded access of RNA pyrophosphohydrolase, which converts 5'-triphosphorylated RNA termini into 5'-monophosphorylated forms, the preferred substrate for Xrn. This study represents the first report of a higher-order RNA structure in an RNA plant virus genome independently conferring resistance to 5'-end-attacking cellular enzymes.


Assuntos
Regiões 5' não Traduzidas/genética , Estabilidade de RNA/genética , Tombusvirus/genética , Regiões 3' não Traduzidas/genética , Sequência de Bases/genética , Exorribonucleases , Genoma Viral/genética , Conformação de Ácido Nucleico , Biossíntese de Proteínas/genética , Estabilidade de RNA/fisiologia , Vírus de RNA/genética , RNA Mensageiro/metabolismo , RNA Viral/genética , Ribonucleases/metabolismo , Relação Estrutura-Atividade , Tombusvirus/metabolismo , Proteínas Virais/metabolismo
17.
J Virol ; 95(9)2021 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-33597210

RESUMO

Opium poppy mosaic virus (OPMV) is a recently discovered umbravirus in the family Tombusviridae OPMV has a plus-sense genomic RNA (gRNA) of 4,241 nucleotides (nt) from which replication protein p35 and p35 extension product p98, the RNA-dependent RNA polymerase (RdRp), are expressed. Movement proteins p27 (long distance) and p28 (cell to cell) are expressed from a 1,440-nt subgenomic RNA (sgRNA2). A highly conserved structure was identified just upstream from the sgRNA2 transcription start site in all umbraviruses, which includes a carmovirus consensus sequence, denoting generation by an RdRp-mediated mechanism. OPMV also has a second sgRNA of 1,554 nt (sgRNA1) that starts just downstream of a canonical exoribonuclease-resistant sequence (xrRNAD). sgRNA1 codes for a 30-kDa protein in vitro that is in frame with p28 and cannot be synthesized in other umbraviruses. Eliminating sgRNA1 or truncating the p30 open reading frame (ORF) without affecting p28 substantially reduced accumulation of OPMV gRNA, suggesting a functional role for the protein. The 652-nt 3' untranslated region of OPMV contains two 3' cap-independent translation enhancers (3' CITEs), a T-shaped structure (TSS) near its 3' end, and a Barley yellow dwarf virus-like translation element (BTE) in the central region. Only the BTE is functional in luciferase reporter constructs containing gRNA or sgRNA2 5' sequences in vivo, which differs from how umbravirus 3' CITEs were used in a previous study. Similarly to most 3' CITEs, the OPMV BTE links to the 5' end via a long-distance RNA-RNA interaction. Analysis of 14 BTEs revealed additional conserved sequences and structural features beyond the previously identified 17-nt conserved sequence.IMPORTANCEOpium poppy mosaic virus (OPMV) is an umbravirus in the family Tombusviridae We determined that OPMV accumulates two similarly sized subgenomic RNAs (sgRNAs), with the smaller known to code for proteins expressed from overlapping open reading frames. The slightly larger sgRNA1 has a 5' end just upstream from a previously predicted xrRNAD site, identifying this sgRNA as an unusually long product produced by exoribonuclease trimming. Although four umbraviruses have similar predicted xrRNAD sites, only sgRNA1 of OPMV can code for a protein that is an extension product of umbravirus ORF4. Inability to generate the sgRNA or translate this protein was associated with reduced gRNA accumulation in vivo We also characterized the OPMV BTE structure, a 3' cap-independent translation enhancer (3' CITE). Comparisons of 13 BTEs with the OPMV BTE revealed additional stretches of sequence similarity beyond the 17-nt signature sequence, as well as conserved structural features not previously recognized in these 3' CITEs.


Assuntos
Regulação Viral da Expressão Gênica , Genoma Viral , RNA Viral/genética , Tombusviridae , Proteínas Virais/genética , Regiões 3' não Traduzidas , Fases de Leitura Aberta , Biossíntese de Proteínas , Tombusviridae/genética
18.
Genes Dev ; 28(14): 1556-61, 2014 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-25030695

RESUMO

In eukaryotes and archaea, tRNA splicing generates free intron molecules. Although ∼ 600,000 introns are produced per generation in yeast, they are barely detectable in cells, indicating efficient turnover of introns. Through a genome-wide search for genes involved in tRNA biology in yeast, we uncovered the mechanism for intron turnover. This process requires healing of the 5' termini of linear introns by the tRNA ligase Rlg1 and destruction by the cytoplasmic tRNA quality control 5'-to-3' exonuclease Xrn1, which has specificity for RNAs with 5' monophosphate.


Assuntos
Citoplasma/metabolismo , Exorribonucleases/metabolismo , Íntrons , RNA de Transferência/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae , Genoma Fúngico , Mutação , Fosforilação , RNA Ligase (ATP)/genética , RNA Ligase (ATP)/metabolismo , RNA de Transferência/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
19.
Crit Rev Biochem Mol Biol ; 54(4): 385-398, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31656086

RESUMO

Transcription and RNA decay are key determinants of gene expression; these processes are typically considered as the uncoupled beginning and end of the messenger RNA (mRNA) lifecycle. Here we describe the growing number of studies demonstrating interplay between these spatially disparate processes in eukaryotes. Specifically, cells can maintain mRNA levels by buffering against changes in mRNA stability or transcription, and can also respond to virally induced accelerated decay by reducing RNA polymerase II gene expression. In addition to these global responses, there is also evidence that mRNAs containing a premature stop codon can cause transcriptional upregulation of homologous genes in a targeted fashion. In each of these systems, RNA binding proteins (RBPs), particularly those involved in mRNA degradation, are critical for cytoplasmic to nuclear communication. Although their specific mechanistic contributions are yet to be fully elucidated, differential trafficking of RBPs between subcellular compartments are likely to play a central role in regulating this gene expression feedback pathway.


Assuntos
Citoplasma/genética , Estabilidade de RNA/genética , RNA Mensageiro/genética , Transcrição Gênica , Animais , Núcleo Celular/genética , Núcleo Celular/metabolismo , Códon de Terminação/genética , Citoplasma/metabolismo , Exorribonucleases/metabolismo , Expressão Gênica , Homeostase/genética , Humanos , Infecções/genética , Proteínas Associadas aos Microtúbulos/metabolismo , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética
20.
J Biol Chem ; 295(33): 11435-11454, 2020 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-32518159

RESUMO

mRNA levels are determined by the balance between mRNA synthesis and decay. Protein factors that mediate both processes, including the 5'-3' exonuclease Xrn1, are responsible for a cross-talk between the two processes that buffers steady-state mRNA levels. However, the roles of these proteins in transcription remain elusive and controversial. Applying native elongating transcript sequencing (NET-seq) to yeast cells, we show that Xrn1 functions mainly as a transcriptional activator and that its disruption manifests as a reduction of RNA polymerase II (Pol II) occupancy downstream of transcription start sites. By combining our sequencing data and mathematical modeling of transcription, we found that Xrn1 modulates transcription initiation and elongation of its target genes. Furthermore, Pol II occupancy markedly increased near cleavage and polyadenylation sites in xrn1Δ cells, whereas its activity decreased, a characteristic feature of backtracked Pol II. We also provide indirect evidence that Xrn1 is involved in transcription termination downstream of polyadenylation sites. We noted that two additional decay factors, Dhh1 and Lsm1, seem to function similarly to Xrn1 in transcription, perhaps as a complex, and that the decay factors Ccr4 and Rpb4 also perturb transcription in other ways. Interestingly, the decay factors could differentiate between SAGA- and TFIID-dominated promoters. These two classes of genes responded differently to XRN1 deletion in mRNA synthesis and were differentially regulated by mRNA decay pathways, raising the possibility that one distinction between these two gene classes lies in the mechanisms that balance mRNA synthesis with mRNA decay.


Assuntos
Exorribonucleases/metabolismo , Regulação Fúngica da Expressão Gênica , RNA Polimerase II/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Exorribonucleases/genética , Deleção de Genes , RNA Polimerase II/genética , Estabilidade de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Sítio de Iniciação de Transcrição , Ativação Transcricional
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