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1.
Nat Rev Mol Cell Biol ; 24(10): 749-769, 2023 10.
Artigo em Inglês | MEDLINE | ID: mdl-37474727

RESUMO

RNA helicases are highly conserved proteins that use nucleoside triphosphates to bind or remodel RNA, RNA-protein complexes or both. RNA helicases are classified into the DEAD-box, DEAH/RHA, Ski2-like, Upf1-like and RIG-I families, and are the largest class of enzymes active in eukaryotic RNA metabolism - virtually all aspects of gene expression and its regulation involve RNA helicases. Mutation and dysregulation of these enzymes have been linked to a multitude of diseases, including cancer and neurological disorders. In this Review, we discuss the regulation and functional mechanisms of RNA helicases and their roles in eukaryotic RNA metabolism, including in transcription regulation, pre-mRNA splicing, ribosome assembly, translation and RNA decay. We highlight intriguing models that link helicase structure, mechanisms of function (such as local strand unwinding, translocation, winching, RNA clamping and displacing RNA-binding proteins) and biological roles, including emerging connections between RNA helicases and cellular condensates formed through liquid-liquid phase separation. We also discuss associations of RNA helicases with human diseases and recent efforts towards the design of small-molecule inhibitors of these pivotal regulators of eukaryotic gene expression.


Assuntos
RNA Helicases , RNA , Humanos , RNA Helicases/genética , RNA Helicases/metabolismo , RNA/metabolismo , Células Eucarióticas/metabolismo , DNA Helicases , Saccharomyces cerevisiae/genética
2.
Mol Cell ; 81(20): 4191-4208.e8, 2021 10 21.
Artigo em Inglês | MEDLINE | ID: mdl-34686314

RESUMO

To survive, mammalian cells must adapt to environmental challenges. While the cellular response to mild stress has been widely studied, how cells respond to severe stress remains unclear. We show here that under severe hyperosmotic stress, cells enter a transient hibernation-like state in anticipation of recovery. We demonstrate this adaptive pausing response (APR) is a coordinated cellular response that limits ATP supply and consumption through mitochondrial fragmentation and widespread pausing of mRNA translation. This pausing is accomplished by ribosome stalling at translation initiation codons, which keeps mRNAs poised to resume translation upon recovery. We further show that recovery from severe stress involves ISR (integrated stress response) signaling that permits cell cycle progression, resumption of growth, and reversal of mitochondria fragmentation. Our findings indicate that cells can respond to severe stress via a hibernation-like mechanism that preserves vital elements of cellular function under harsh environmental conditions.


Assuntos
Proliferação de Células , Fibroblastos/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/biossíntese , Pressão Osmótica , Biossíntese de Proteínas , Ribossomos/metabolismo , Adaptação Fisiológica , Trifosfato de Adenosina/metabolismo , Animais , Códon de Iniciação , Fibroblastos/patologia , Células HEK293 , Humanos , Cinética , Camundongos , Mitocôndrias/genética , Mitocôndrias/patologia , Proteínas Mitocondriais/genética , Ribossomos/genética , Transdução de Sinais
3.
Nat Rev Mol Cell Biol ; 16(9): 533-44, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26285679

RESUMO

To fully understand the regulation of gene expression, it is critical to quantitatively define whether and how RNA-binding proteins (RBPs) discriminate between alternative binding sites in RNAs. Here, we describe new methods that measure protein binding to large numbers of RNA variants, and ways to analyse and interpret data obtained by these approaches, including affinity distributions and free energy landscapes. We discuss how the new methodologies and the associated concepts enable the development of inclusive, quantitative models for RNA-protein interactions that transcend the traditional binary classification of RBPs as either specific or nonspecific.


Assuntos
Proteínas de Ligação a RNA/fisiologia , RNA/fisiologia , Animais , Sequência de Bases , Humanos , Cinética , Modelos Moleculares , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica , RNA/química , Proteínas de Ligação a RNA/química , Termodinâmica
4.
Nature ; 591(7848): 152-156, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33568810

RESUMO

Gene expression in higher eukaryotic cells orchestrates interactions between thousands of RNA-binding proteins (RBPs) and tens of thousands of RNAs1. The kinetics by which RBPs bind to and dissociate from their RNA sites are critical for the coordination of cellular RNA-protein interactions2. However, these kinetic parameters have not been experimentally measured in cells. Here we show that time-resolved RNA-protein cross-linking with a pulsed femtosecond ultraviolet laser, followed by immunoprecipitation and high-throughput sequencing, allows the determination of binding and dissociation kinetics of the RBP DAZL for thousands of individual RNA-binding sites in cells. This kinetic cross-linking and immunoprecipitation (KIN-CLIP) approach reveals that DAZL resides at individual binding sites for time periods of only seconds or shorter, whereas the binding sites remain DAZL-free for markedly longer. The data also indicate that DAZL binds to many RNAs in clusters of multiple proximal sites. The effect of DAZL on mRNA levels and ribosome association correlates with the cumulative probability of DAZL binding in these clusters. Integrating kinetic data with mRNA features quantitatively connects DAZL-RNA binding to DAZL function. Our results show how kinetic parameters for RNA-protein interactions can be measured in cells, and how these data link RBP-RNA binding to the cellular function of RBPs.


Assuntos
Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , Regiões 3' não Traduzidas/genética , Animais , Sítios de Ligação/genética , Linhagem Celular , Cinética , Lasers , Camundongos , Ligação Proteica , RNA/genética , Proteínas de Ligação a RNA/genética , Ribossomos/metabolismo
5.
Development ; 150(20)2023 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-37306388

RESUMO

The eIF4E family of translation initiation factors bind 5' methylated caps and act as the limiting step for mRNA translation. The canonical eIF4E1A is required for cell viability, yet other related eIF4E families exist and are utilized in specific contexts or tissues. Here, we describe a family called Eif4e1c, for which we find roles during heart development and regeneration in zebrafish. The Eif4e1c family is present in all aquatic vertebrates but is lost in all terrestrial species. A core group of amino acids shared over 500 million years of evolution forms an interface along the protein surface, suggesting that Eif4e1c functions in a novel pathway. Deletion of eif4e1c in zebrafish caused growth deficits and impaired survival in juveniles. Mutants surviving to adulthood had fewer cardiomyocytes and reduced proliferative responses to cardiac injury. Ribosome profiling of mutant hearts demonstrated changes in translation efficiency of mRNA for genes known to regulate cardiomyocyte proliferation. Although eif4e1c is broadly expressed, its disruption had most notable impact on the heart and at juvenile stages. Our findings reveal context-dependent requirements for translation initiation regulators during heart regeneration.


Assuntos
Traumatismos Cardíacos , Miócitos Cardíacos , Animais , Peixe-Zebra/genética , Fator de Iniciação 4E em Eucariotos/genética , Proliferação de Células/genética
6.
Cell ; 145(6): 890-901, 2011 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-21663793

RESUMO

Many steps in nuclear RNA processing, surveillance, and degradation require TRAMP, a complex containing the poly(A) polymerase Trf4p, the Zn-knuckle protein Air2p, and the RNA helicase Mtr4p. TRAMP polyadenylates RNAs designated for decay or trimming by the nuclear exosome. It has been unclear how polyadenylation by TRAMP differs from polyadenylation by conventional poly(A) polymerase, which produces poly(A) tails that stabilize RNAs. Using reconstituted S. cerevisiae TRAMP, we show that TRAMP inherently suppresses poly(A) addition after only 3-4 adenosines. This poly(A) tail length restriction is controlled by Mtr4p. The helicase detects the number of 3'-terminal adenosines and, over several adenylation steps, elicits precisely tuned adjustments of ATP affinities and rate constants for adenylation and TRAMP dissociation. Our data establish Mtr4p as a critical regulator of polyadenylation by TRAMP and reveal that an RNA helicase can control the activity of another enzyme in a highly complex fashion and in response to features in RNA.


Assuntos
RNA Helicases DEAD-box/metabolismo , Poliadenilação , RNA Fúngico/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Adenosina/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Complexos Multiproteicos/metabolismo , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética
7.
Nature ; 559(7712): 130-134, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29950728

RESUMO

The conserved and essential DEAD-box RNA helicase Ded1p from yeast and its mammalian orthologue DDX3 are critical for the initiation of translation1. Mutations in DDX3 are linked to tumorigenesis2-4 and intellectual disability5, and the enzyme is targeted by a range of viruses6. How Ded1p and its orthologues engage RNAs during the initiation of translation is unknown. Here we show, by integrating transcriptome-wide analyses of translation, RNA structure and Ded1p-RNA binding, that the effects of Ded1p on the initiation of translation are connected to near-cognate initiation codons in 5' untranslated regions. Ded1p associates with the translation pre-initiation complex at the mRNA entry channel and repressing the activity of Ded1p leads to the accumulation of RNA structure in 5' untranslated regions, the initiation of translation from near-cognate start codons immediately upstream of these structures and decreased protein synthesis from the corresponding main open reading frames. The data reveal a program for the regulation of translation that links Ded1p, the activation of near-cognate start codons and mRNA structure. This program has a role in meiosis, in which a marked decrease in the levels of Ded1p is accompanied by the activation of the alternative translation initiation sites that are seen when the activity of Ded1p is repressed. Our observations indicate that Ded1p affects translation initiation by controlling the use of near-cognate initiation codons that are proximal to mRNA structure in 5' untranslated regions.


Assuntos
Regiões 5' não Traduzidas/genética , Códon de Iniciação/genética , RNA Helicases DEAD-box/metabolismo , Iniciação Traducional da Cadeia Peptídica/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Reagentes de Ligações Cruzadas/química , Subunidades Ribossômicas Menores de Eucariotos/química , Subunidades Ribossômicas Menores de Eucariotos/metabolismo
8.
RNA ; 27(4): 465-476, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33408095

RESUMO

The 3' to 5' exonuclease Pop2p (Caf1p) is part of the CCR4-NOT deadenylation complex that removes poly(A) tails from mRNAs in cells. Pop2p is structurally conserved in eukaryotes, but Saccharomyces cerevisiae Pop2p harbors noncanonical amino acids in its catalytic center. The enzymatic properties of S. cerevisiae Pop2p are not well defined. Here we characterize the RNA exonuclease activity of recombinant S. cerevisiae Pop2p. We find that S. cerevisiae Pop2p degrades RNAs via two alternative reactions pathways, one generating nucleotides with 5'-phosphates and RNA intermediates with 3'-hydroxyls, and the other generating nucleotides with 3'-phosphates and RNA intermediates with 3'-phosphates. The enzyme is not able to initiate the reaction on RNAs with a 3'-phosphate, which leads to accumulation of RNAs with 3'-phosphates that can exceed 10 nt and are resistant to further degradation by S. cerevisiae Pop2p. We further demonstrate that S. cerevisiae Pop2p degrades RNAs in three reaction phases: an initial distributive phase, a second processive phase and a third phase during which processivity gradually declines. We also show that mutations of subsets of amino acids in the catalytic center, including those previously thought to inactivate the enzyme, moderately reduce, but not eliminate activity. Only mutation of all five amino acids in the catalytic center diminishes activity of Pop2p to background levels. Collectively, our results reveal robust exonuclease activity of S. cerevisiae Pop2p with unusual enzymatic properties, characterized by alternative degradation pathways, multiple reaction phases and functional redundancy of amino acids in the catalytic core.


Assuntos
Aminoácidos/química , Estabilidade de RNA , RNA Mensageiro/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas Repressoras/metabolismo , Ribonucleases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Monofosfato de Adenosina/metabolismo , Substituição de Aminoácidos , Aminoácidos/metabolismo , Sítios de Ligação , Domínio Catalítico , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Modelos Moleculares , Mutação , Fosfatos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , RNA Mensageiro/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Repressoras/química , Proteínas Repressoras/genética , Ribonucleases/química , Ribonucleases/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Especificidade por Substrato
9.
Nat Rev Mol Cell Biol ; 12(8): 505-16, 2011 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-21779027

RESUMO

RNA helicases of the DEAD box family are present in all eukaryotic cells and in many bacteria and Archaea. These highly conserved enzymes are required for RNA metabolism from transcription to degradation and are therefore important players in gene expression. DEAD box proteins use ATP to unwind short duplex RNA in an unusual fashion and remodel RNA-protein complexes, but they can also function as ATP-dependent RNA clamps to provide nucleation centres that establish larger RNA-protein complexes. Structural, mechanistic and molecular biological studies have started to reveal how these conserved proteins can perform such diverse functions and how accessory proteins have a central role in their regulation.


Assuntos
RNA Helicases DEAD-box/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Sítios de Ligação , RNA Helicases DEAD-box/química , RNA Helicases DEAD-box/genética , Fator de Iniciação 4A em Eucariotos/metabolismo , Humanos , Modelos Biológicos , Modelos Moleculares , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Biossíntese de Proteínas , Estrutura Terciária de Proteína , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/metabolismo
10.
Methods ; 204: 376-385, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35429628

RESUMO

RNA helicases are the largest class of enzymes in eukaryotic RNA metabolism. In cells, protein cofactors regulate RNA helicase functions and impact biochemical helicase activities. Understanding how cofactors affect enzymatic activities of RNA helicases is thus critical for delineating physical roles and regulation of RNA helicases in cells. Here, we discuss approaches and conceptual considerations for the design of experiments to interrogate cofactor effects on RNA helicase activities in vitro. We outline the mechanistic frame for helicase reactions, discuss optimization of experimental setup and reaction parameters for measuring cofactor effects on RNA helicase activities, and provide basic guides to data analysis and interpretation. The described approaches are also instructive for determining the impact of small molecule inhibitors of RNA helicases.


Assuntos
RNA Helicases DEAD-box , RNA , RNA Helicases DEAD-box/química , RNA/química
11.
Cell ; 135(7): 1224-36, 2008 Dec 26.
Artigo em Inglês | MEDLINE | ID: mdl-19109894

RESUMO

Alternative splicing makes a major contribution to proteomic diversity in higher eukaryotes with approximately 70% of genes encoding two or more isoforms. In most cases, the molecular mechanisms responsible for splice site choice remain poorly understood. Here, we used a randomization-selection approach in vitro to identify sequence elements that could silence a proximal strong 5' splice site located downstream of a weakened 5' splice site. We recovered two exonic and four intronic motifs that effectively silenced the proximal 5' splice site both in vitro and in vivo. Surprisingly, silencing was only observed in the presence of the competing upstream 5' splice site. Biochemical evidence strongly suggests that the silencing motifs function by altering the U1 snRNP/5' splice site complex in a manner that impairs commitment to specific splice site pairing. The data indicate that perturbations of non-rate-limiting step(s) in splicing can lead to dramatic shifts in splice site choice.


Assuntos
Processamento Alternativo , Regulação da Expressão Gênica , Sítios de Splice de RNA , Éxons , Técnicas Genéticas , Células HeLa , Humanos , Modelos Biológicos
12.
Mol Cell ; 59(4): 541-52, 2015 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-26212457

RESUMO

Most aspects of RNA metabolism involve DEAD-box RNA helicases, enzymes that bind and remodel RNA and RNA-protein complexes in an ATP-dependent manner. Here we show that the DEAD-box helicase Ded1p oligomerizes in the cell and in vitro, and unwinds RNA as a trimer. Two protomers bind the single-stranded region of RNA substrates and load a third protomer to the duplex, which then separates the strands. ATP utilization differs between the strand-separating protomer and those bound to the single-stranded region. Binding of the eukaryotic initiation factor 4G to Ded1p interferes with oligomerization and thereby modulates unwinding activity and RNA affinity of the helicase. Our data reveal a strict division of labor between the Ded1p protomers in the oligomer. This mode of oligomerization fundamentally differs from other helicases. Oligomerization represents a previously unappreciated level of regulation for DEAD-box helicase activities.


Assuntos
RNA Helicases DEAD-box/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , Trifosfato de Adenosina/química , Biocatálise , RNA Helicases DEAD-box/fisiologia , Hidrólise , Ligação Proteica , Multimerização Proteica , Estrutura Quaternária de Proteína , Subunidades Proteicas/química , RNA de Cadeia Dupla/química , Proteínas de Saccharomyces cerevisiae/fisiologia
13.
Proc Natl Acad Sci U S A ; 117(2): 982-992, 2020 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-31879344

RESUMO

The exoribonuclease Rrp6p is critical for RNA decay in the nucleus. While Rrp6p acts on a large range of diverse substrates, it does not indiscriminately degrade all RNAs. How Rrp6p accomplishes this task is not understood. Here, we measure Rrp6p-RNA binding and degradation kinetics in vitro at single-nucleotide resolution and find an intrinsic substrate selectivity that enables Rrp6p to discriminate against specific RNAs. RNA length and the four 3'-terminal nucleotides contribute most to substrate selectivity and collectively enable Rrp6p to discriminate between different RNAs by several orders of magnitude. The most pronounced discrimination is seen against RNAs ending with CCA-3'. These RNAs correspond to 3' termini of uncharged tRNAs, which are not targeted by Rrp6p in cells. The data show that in contrast to many other proteins that use substrate selectivity to preferentially interact with specific RNAs, Rrp6p utilizes its selectivity to discriminate against specific RNAs. This ability allows Rrp6p to target diverse substrates while avoiding a subset of RNAs.


Assuntos
Exorribonucleases/metabolismo , Estabilidade de RNA , Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , Escherichia coli , Exorribonucleases/química , Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , Cinética , RNA/química , RNA de Transferência/metabolismo , Especificidade por Substrato
14.
RNA ; 26(5): 541-549, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32014999

RESUMO

The PI3K/Akt/mTOR kinase pathway is extensively deregulated in human cancers. One critical node under regulation of this signaling axis is eukaryotic initiation factor (eIF) 4F, a complex involved in the control of translation initiation rates. eIF4F-dependent addictions arise during tumor initiation and maintenance due to increased eIF4F activity-generally in response to elevated PI3K/Akt/mTOR signaling flux. There is thus much interest in exploring eIF4F as a small molecule target for the development of new anticancer drugs. The DEAD-box RNA helicase, eIF4A, is an essential subunit of eIF4F, and several potent small molecules (rocaglates, hippuristanol, pateamine A) affecting its activity have been identified and shown to demonstrate anticancer activity in vitro and in vivo in preclinical models. Recently, a number of new small molecules have been reported as having the capacity to target and inhibit eIF4A. Here, we undertook a comparative analysis of their biological activity and specificity relative to the eIF4A inhibitor, hippuristanol.


Assuntos
Antineoplásicos/química , Fator de Iniciação 4A em Eucariotos/química , Neoplasias/tratamento farmacológico , Bibliotecas de Moléculas Pequenas/química , Esteróis/química , Antineoplásicos/farmacologia , Benzofuranos/química , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Compostos de Epóxi/química , Fator de Iniciação 4A em Eucariotos/antagonistas & inibidores , Fator de Iniciação 4F em Eucariotos/antagonistas & inibidores , Fator de Iniciação 4F em Eucariotos/química , Humanos , Macrolídeos/química , Neoplasias/genética , Fosfatidilinositol 3-Quinases/genética , Biossíntese de Proteínas/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/genética , Bibliotecas de Moléculas Pequenas/farmacologia , Esteróis/farmacologia , Serina-Treonina Quinases TOR/genética , Tiazóis/química
15.
Nucleic Acids Res ; 48(14): 8063-8073, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32609821

RESUMO

The mechanism for how internal ribosome entry sites (IRESs) recruit ribosomes to initiate translation of an mRNA is not completely understood. We investigated how a 40S subunit was recruited by the cricket paralysis virus intergenic region (CrPV IGR) IRES to form a stable 40S-IRES complex. Kinetic binding studies revealed that formation of the complex between the CrPV IGR and the 40S subunit consisted of two-steps: an initial fast binding step of the IRES to the 40S ribosomal subunit, followed by a slow unimolecular reaction consistent with a conformational change that stabilized the complex. We further showed that the ribosomal protein S25 (eS25), which is required by functionally and structurally diverse IRESs, impacts both steps of the complex formation. Mutations in eS25 that reduced CrPV IGR IRES activity either decreased 40S-IRES complex formation, or increased the rate of the conformational change that was required to form a stable 40S-IRES complex. Our data are consistent with a model in which eS25 facilitates initial binding of the CrPV IGR IRES to the 40S while ensuring that the conformational change stabilizing the 40S-IRES complex does not occur prematurely.


Assuntos
Sítios Internos de Entrada Ribossomal , Proteínas Ribossômicas/metabolismo , Subunidades Ribossômicas Menores de Eucariotos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Sítios de Ligação , DNA Intergênico/genética , DNA Intergênico/metabolismo , Dicistroviridae/genética , Mutação , Ligação Proteica , Proteínas Ribossômicas/química , Proteínas Ribossômicas/genética , Subunidades Ribossômicas Menores de Eucariotos/química , Subunidades Ribossômicas Menores de Eucariotos/genética , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética
16.
Methods ; 178: 3-10, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-31494245

RESUMO

To understand the regulation of gene expression it is critical to determine how proteins interact with and discriminate between different RNAs. In this review, we discuss experimental techniques that utilize high throughput approaches to characterize the interactions of proteins with large numbers of RNAs in vitro. We describe the underlying principles for the main methods, briefly discuss their scope and limitations, and outline how insight from the techniques contributes to our understanding of specificity for RNA-protein interactions.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/tendências , Ensaios de Triagem em Larga Escala/tendências , Proteínas de Ligação a RNA/isolamento & purificação , RNA/genética , RNA/isolamento & purificação , Proteínas de Ligação a RNA/genética
18.
RNA ; 24(12): 1693-1705, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30262458

RESUMO

The DEAD-box RNA helicase Dbp2p is highly conserved in eukaryotes and has been implicated in transcription, ribosome biogenesis, mRNP assembly, nuclear export, and long noncoding RNA (lncRNA) function. It is not understood how Dbp2p performs these seemingly unrelated biological roles. An important step toward addressing this question is the determination of cellular RNA binding sites of Dbp2p. Here, we identify transcriptome-wide RNA binding sites of Dbp2p from Saccharomyces cerevisiae using UV-crosslinking, denaturing tandem affinity purification, and next generation sequencing. We find that Dbp2p crosslinks to mRNAs and ribosomal RNAs, and markedly to noncoding RNAs, including snoRNA, snRNAs, and tRNAs. In snoRNAs, Dbp2p preferentially crosslinks at sites near the 3' ends. These sites coincide with regions where RNA-DNA hybrids (R-loops) form and with binding sites of Sen1p, another RNA helicase that functions in transcription termination and 3' processing of noncoding RNAs. We show that Dbp2p interacts in an RNA-independent manner with Sen1p in vivo. Dbp2p crosslinks to tRNAs and other RNAs also at sites where R-loops form. Collectively, our data link Dbp2p to noncoding RNAs, Sen1p, and R-loops. The transcriptome-wide connection to R-loops provides a unifying theme for diverse cellular roles of Dbp2p.


Assuntos
RNA Helicases DEAD-box/química , Complexo de Endopeptidases do Proteassoma/química , RNA não Traduzido/química , Proteínas de Saccharomyces cerevisiae/química , Sítios de Ligação , RNA Helicases DEAD-box/genética , Regulação Fúngica da Expressão Gênica , Complexo de Endopeptidases do Proteassoma/genética , RNA Nuclear Pequeno/química , RNA Nuclear Pequeno/genética , RNA Nucleolar Pequeno/química , RNA Nucleolar Pequeno/genética , RNA de Transferência/química , RNA de Transferência/genética , RNA não Traduzido/genética , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
19.
Curr Genet ; 65(2): 453-456, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30483885

RESUMO

Upstream open reading frames (uORFs) in 5' UTRs of eukaryotic mRNAs are increasingly recognized as important elements that regulate cellular protein synthesis. Since uORFs can start from non-AUG codons, an enormous number of potential uORF initiation sites exists in 5'UTRs. However, only a subset of these sites is used and it has been unclear how actual start sites are selected. Studies of the DEAD-box helicase Ded1p from S. cerevisiae show that translation of uORFs with non-AUG initiation codons occurs upstream of mRNA structures that emerge with defective Ded1p. The data designate mRNA structure as important determinant for non-AUG initiation sites of uORFs. Ded1p can control this RNA structure and thereby regulate uORF translation.


Assuntos
Regiões 5' não Traduzidas , DNA Helicases/metabolismo , Fases de Leitura Aberta , RNA Mensageiro/química , RNA Mensageiro/genética , Códon , Regulação Fúngica da Expressão Gênica , Meiose/genética , Biossíntese de Proteínas , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
20.
RNA ; 23(10): 1502-1511, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28694328

RESUMO

Recognition of RNA by RNA processing enzymes and RNA binding proteins often involves cooperation between multiple subunits. However, the interdependent contributions of RNA and protein subunits to molecular recognition by ribonucleoproteins are relatively unexplored. RNase P is an endonuclease that removes 5' leaders from precursor tRNAs and functions in bacteria as a dimer formed by a catalytic RNA subunit (P RNA) and a protein subunit (C5 in E. coli). The P RNA subunit contacts the tRNA body and proximal 5' leader sequences [N(-1) and N(-2)] while C5 binds distal 5' leader sequences [N(-3) to N(-6)]. To determine whether the contacts formed by P RNA and C5 contribute independently to specificity or exhibit cooperativity or anti-cooperativity, we compared the relative kcat/Km values for all possible combinations of the six proximal 5' leader nucleotides (n = 4096) for processing by the E. coli P RNA subunit alone and by the RNase P holoenzyme. We observed that while the P RNA subunit shows specificity for 5' leader nucleotides N(-2) and N(-1), the presence of the C5 protein reduces the contribution of P RNA to specificity, but changes specificity at N(-2) and N(-3). The results reveal that the contribution of C5 protein to RNase P processing is controlled by the identity of N(-2) in the pre-tRNA 5' leader. The data also clearly show that pairing of the 5' leader with the 3' ACCA of tRNA acts as an anti-determinant for RNase P cleavage. Comparative analysis of genomically encoded E. coli tRNAs reveals that both anti-determinants are subject to negative selection in vivo.


Assuntos
Proteínas de Escherichia coli/metabolismo , Precursores de RNA/metabolismo , RNA de Transferência/metabolismo , Ribonuclease P/metabolismo , Proteínas de Escherichia coli/química , Nucleotídeos/química , Nucleotídeos/metabolismo , Precursores de RNA/química , RNA de Transferência/química , RNA de Transferência de Metionina/química , RNA de Transferência de Metionina/metabolismo , Ribonuclease P/química , Especificidade por Substrato
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