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1.
Elife ; 72018 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-29985129

RESUMO

The endoplasmic reticulum (ER) protein folding capacity is balanced with the protein folding burden to prevent accumulation of un- or misfolded proteins. The ER membrane-resident kinase/RNase Ire1 maintains ER protein homeostasis through two fundamentally distinct processes. First, Ire1 can initiate a transcriptional response through a non-conventional mRNA splicing reaction to increase the ER folding capacity. Second, Ire1 can decrease the ER folding burden through selective mRNA decay. In Saccharomyces cerevisiae and Schizosaccharomyces pombe, the two Ire1 functions have been evolutionarily separated. Here, we show that the respective Ire1 orthologs have become specialized for their functional outputs by divergence of their RNase specificities. In addition, RNA structural features separate the splicing substrates from the decay substrates. Using these insights, we engineered an S. pombe Ire1 cleavage substrate into a splicing substrate, which confers S. pombe with both Ire1 functional outputs.


Assuntos
Estresse do Retículo Endoplasmático/genética , Engenharia Genética , Splicing de RNA/genética , Saccharomyces cerevisiae/genética , Schizosaccharomyces/genética , Sequência de Aminoácidos , Sequência de Bases , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Conformação de Ácido Nucleico , Domínios Proteicos , Multimerização Proteica , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribonucleases/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/metabolismo , Especificidade por Substrato
2.
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
3.
Nat Methods ; 14(1): 75-82, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27819661

RESUMO

Coupling of structure-specific in vivo chemical modification to next-generation sequencing is transforming RNA secondary structure studies in living cells. The dominant strategy for detecting in vivo chemical modifications uses reverse transcriptase truncation products, which introduce biases and necessitate population-average assessments of RNA structure. Here we present dimethyl sulfate (DMS) mutational profiling with sequencing (DMS-MaPseq), which encodes DMS modifications as mismatches using a thermostable group II intron reverse transcriptase. DMS-MaPseq yields a high signal-to-noise ratio, can report multiple structural features per molecule, and allows both genome-wide studies and focused in vivo investigations of even low-abundance RNAs. We apply DMS-MaPseq for the first analysis of RNA structure within an animal tissue and to identify a functional structure involved in noncanonical translation initiation. Additionally, we use DMS-MaPseq to compare the in vivo structure of pre-mRNAs with their mature isoforms. These applications illustrate DMS-MaPseq's capacity to dramatically expand in vivo analysis of RNA structure.


Assuntos
Genoma Humano , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Proteínas de Ligação a RNA/genética , RNA/química , RNA/genética , Ésteres do Ácido Sulfúrico/química , Biologia Computacional , Células HEK293 , Humanos , Mutação/genética , Conformação de Ácido Nucleico , Biossíntese de Proteínas , Análise de Sequência de RNA
4.
Nature ; 505(7485): 701-5, 2014 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-24336214

RESUMO

RNA has a dual role as an informational molecule and a direct effector of biological tasks. The latter function is enabled by RNA's ability to adopt complex secondary and tertiary folds and thus has motivated extensive computational and experimental efforts for determining RNA structures. Existing approaches for evaluating RNA structure have been largely limited to in vitro systems, yet the thermodynamic forces which drive RNA folding in vitro may not be sufficient to predict stable RNA structures in vivo. Indeed, the presence of RNA-binding proteins and ATP-dependent helicases can influence which structures are present inside cells. Here we present an approach for globally monitoring RNA structure in native conditions in vivo with single-nucleotide precision. This method is based on in vivo modification with dimethyl sulphate (DMS), which reacts with unpaired adenine and cytosine residues, followed by deep sequencing to monitor modifications. Our data from yeast and mammalian cells are in excellent agreement with known messenger RNA structures and with the high-resolution crystal structure of the Saccharomyces cerevisiae ribosome. Comparison between in vivo and in vitro data reveals that in rapidly dividing cells there are vastly fewer structured mRNA regions in vivo than in vitro. Even thermostable RNA structures are often denatured in cells, highlighting the importance of cellular processes in regulating RNA structure. Indeed, analysis of mRNA structure under ATP-depleted conditions in yeast shows that energy-dependent processes strongly contribute to the predominantly unfolded state of mRNAs inside cells. Our studies broadly enable the functional analysis of physiological RNA structures and reveal that, in contrast to the Anfinsen view of protein folding whereby the structure formed is the most thermodynamically favourable, thermodynamics have an incomplete role in determining mRNA structure in vivo.


Assuntos
Genoma Fúngico/genética , Conformação de Ácido Nucleico , Dobramento de RNA , Estabilidade de RNA , RNA Mensageiro/química , RNA Mensageiro/genética , Saccharomyces cerevisiae/genética , Fibroblastos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Células K562 , Desnaturação de Ácido Nucleico , Dobramento de RNA/genética , Estabilidade de RNA/genética , RNA Fúngico/química , RNA Fúngico/genética , RNA Fúngico/metabolismo , RNA Mensageiro/metabolismo , Ésteres do Ácido Sulfúrico/química , Termodinâmica
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