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1.
Nat Commun ; 12(1): 874, 2021 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-33558533

RESUMO

Base-pairing interactions mediate many intermolecular target recognition events. Even a single base-pair mismatch can cause a substantial difference in activity but how such changes influence the target search kinetics in vivo is unknown. Here, we use high-throughput sequencing and quantitative super-resolution imaging to probe the mutants of bacterial small RNA, SgrS, and their regulation of ptsG mRNA target. Mutations that disrupt binding of a chaperone protein, Hfq, and are distal to the mRNA annealing region still decrease the rate of target association, kon, and increase the dissociation rate, koff, showing that Hfq directly facilitates sRNA-mRNA annealing in vivo. Single base-pair mismatches in the annealing region reduce kon by 24-31% and increase koff by 14-25%, extending the time it takes to find and destroy the target by about a third. The effects of disrupting contiguous base-pairing are much more modest than that expected from thermodynamics, suggesting that Hfq buffers base-pair disruptions.


Assuntos
Pareamento de Bases/genética , Estabilidade de RNA , RNA Bacteriano/genética , Sequência de Bases , Escherichia coli/genética , Dosagem de Genes , Genes Reporter , Imageamento Tridimensional , Cinética , Mutação/genética , Nucleotídeos/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Tempo
2.
Curr Protoc ; 1(2): e39, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33524237

RESUMO

Synthetic messenger RNA (mRNA)-based therapeutics are an increasingly popular approach to gene and cell therapies, genome engineering, enzyme replacement therapy, and now, during the global SARS-CoV-2 pandemic, vaccine development. mRNA for such purposes can be synthesized through an enzymatic in vitro transcription (IVT) reaction and formulated for in vivo delivery. Mature mRNA requires a 5'-cap for gene expression and mRNA stability. There are two methods to add a cap in vitro: via a two-step multi-enzymatic reaction or co-transcriptionally. Co-transcriptional methods minimize reaction steps and enzymes needed to make mRNA when compared to enzymatic capping. CleanCap® AG co-transcriptional capping results in 5 mg/ml of IVT with 94% 5'-cap 1 structure. This is highly efficient compared to first-generation cap analogs, such as mCap and ARCA, that incorporate cap 0 structures at lower efficiency and reaction yield. This article describes co-transcriptional capping using TriLink Biotechnology's CleanCap® AG in IVT. © 2021 Wiley Periodicals LLC. Basic Protocol 1: IVT with CleanCap Basic Protocol 2: mRNA purification and analysis.


Assuntos
Análogos de Capuz de RNA/síntese química , RNA Mensageiro/síntese química , Humanos , Técnicas In Vitro , Biossíntese de Proteínas , Estabilidade de RNA , RNA Mensageiro/isolamento & purificação
3.
Mol Cancer ; 20(1): 33, 2021 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-33593376

RESUMO

mRNA vaccines have tremendous potential to fight against cancer and viral diseases due to superiorities in safety, efficacy and industrial production. In recent decades, we have witnessed the development of different kinds of mRNAs by sequence optimization to overcome the disadvantage of excessive mRNA immunogenicity, instability and inefficiency. Based on the immunological study, mRNA vaccines are coupled with immunologic adjuvant and various delivery strategies. Except for sequence optimization, the assistance of mRNA-delivering strategies is another method to stabilize mRNAs and improve their efficacy. The understanding of increasing the antigen reactiveness gains insight into mRNA-induced innate immunity and adaptive immunity without antibody-dependent enhancement activity. Therefore, to address the problem, scientists further exploited carrier-based mRNA vaccines (lipid-based delivery, polymer-based delivery, peptide-based delivery, virus-like replicon particle and cationic nanoemulsion), naked mRNA vaccines and dendritic cells-based mRNA vaccines. The article will discuss the molecular biology of mRNA vaccines and underlying anti-virus and anti-tumor mechanisms, with an introduction of their immunological phenomena, delivery strategies, their importance on Corona Virus Disease 2019 (COVID-19) and related clinical trials against cancer and viral diseases. Finally, we will discuss the challenge of mRNA vaccines against bacterial and parasitic diseases.


Assuntos
Vacinas Sintéticas/uso terapêutico , Animais , Técnicas de Transferência de Genes , Humanos , Imunidade Humoral , Imunoterapia , Estabilidade de RNA , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia
4.
Nat Commun ; 12(1): 770, 2021 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-33536434

RESUMO

Long non-coding RNAs (lncRNAs) contribute to the regulation of gene expression in response to intra- or extracellular signals but the underlying molecular mechanisms remain largely unexplored. Here, we identify an uncharacterized lncRNA as a central player in shaping the meiotic gene expression program in fission yeast. We report that this regulatory RNA, termed mamRNA, scaffolds the antagonistic RNA-binding proteins Mmi1 and Mei2 to ensure their reciprocal inhibition and fine tune meiotic mRNA degradation during mitotic growth. Mechanistically, mamRNA allows Mmi1 to target Mei2 for ubiquitin-mediated downregulation, and conversely enables accumulating Mei2 to impede Mmi1 activity, thereby reinforcing the mitosis to meiosis switch. These regulations also occur within a unique Mmi1-containing nuclear body, positioning mamRNA as a spatially-confined sensor of Mei2 levels. Our results thus provide a mechanistic basis for the mutual control of gametogenesis effectors and further expand our vision of the regulatory potential of lncRNAs.


Assuntos
Regulação Fúngica da Expressão Gênica , Meiose/genética , Mitose/genética , RNA Fúngico/genética , RNA Longo não Codificante/genética , Schizosaccharomyces/genética , Ligação Proteica , Estabilidade de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/genética , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo
6.
Yi Chuan ; 43(1): 4-15, 2021 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-33509770

RESUMO

During the maturation of pre-mRNAs and some lncRNAs, their 3'ends are cleaved and polyadenylated. The cleavage and polyadenylation (C/P) require the presence of a polyadenylation signal (PAS) at the RNA 3?end. Most eukaryotic genes have multiple PASs, resulting in alternative cleavage and polyadenylation (APA). APA leads to transcript isoforms with different coding potentials and/or variable 3?UTRs. The 3'UTR affects mRNA stability, translation, transportation, and cellular localization. Therefore, APA is an important mechanism of posttranscriptional gene regulation in eukaryotes. In recent years, whole genome sequencing of animals, plants and yeast has revealed that APA is pervasive in eukaryotes, and the functional consequences and regulation of APA have been studied. To date, many cis-acting regulatory elements and trans-acting factors for APA regulation have been identified. In this review, we summarize the recent advances in the functional consequences and regulation of APA and discuss the future directions, aiming to provide clues and references for future APA study.


Assuntos
Clivagem do DNA , Regulação da Expressão Gênica , Poliadenilação , Regiões 3' não Traduzidas , Animais , Plantas , Estabilidade de RNA , Leveduras
7.
Gene ; 776: 145442, 2021 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-33482283

RESUMO

Ribosome biogenesis requires the concerted activities of three nuclear RNA polymerases, (Pols) I, II, and III, to produce 25S, 18S and 5.8S ribosomal RNA (rRNA), messenger RNA (mRNA) encoding ribosomal proteins, and the 5S rRNA, respectively. The rRNA is processed and modified before being assembled with ribosomal proteins to produce a ribosome. Ribosome biogenesis requires extensive energetic investment by the cell, so it is critical that this process is tightly regulated in accord with cellular growth potential. Previous work revealed that rRNA synthesis in Saccharomyces cerevisiae is repressed prior to the cells shift from active growth (log phase) to limited/static growth (stationary phase). The mechanism(s) by which cells anticipate imminent stationary phase are unknown. In this study, we demonstrate that growing cells produce one or more compounds that accumulate in the growth medium, and that this compound induces repression of rRNA synthesis. When cells encounter this compound, rRNA synthesis is rapidly repressed. We further show that subunits of Pols I and II are degraded during the transition from log to stationary phase growth, but this degradation does not account for the observed repression of rRNA synthesis. Interestingly, repression of rRNA synthesis by spent media requires the nuclear exosome, implying that spent media stimulates rapid rRNA degradation. Together, these data suggest that yeast use quorum sensing to regulate rRNA synthesis in anticipation of high cell density in stationary phase.


Assuntos
Percepção de Quorum/genética , RNA Ribossômico/biossíntese , RNA Ribossômico/genética , Núcleo Celular/metabolismo , Precursores de RNA/genética , Estabilidade de RNA , RNA Ribossômico 5S/genética , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
8.
Science ; 371(6529)2021 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-33414189

RESUMO

Polyadenylate [poly(A)] tail addition to the 3' end of a wide range of RNAs is a highly conserved modification that plays a central role in cellular RNA function. Elements for nuclear expression (ENEs) are cis-acting RNA elements that stabilize poly(A) tails by sequestering them in RNA triplex structures. A crystal structure of a double ENE from a rice hAT transposon messenger RNA complexed with poly(A)28 at a resolution of 2.89 angstroms reveals multiple modes of interaction with poly(A), including major-groove triple helices, extended minor-groove interactions with RNA double helices, a quintuple-base motif that transitions poly(A) from minor-groove associations to major-groove triple helices, and a poly(A) 3'-end binding pocket. Our findings both expand the repertoire of motifs involved in long-range RNA interactions and provide insights into how polyadenylation can protect an RNA's extreme 3' end.


Assuntos
Poli A/química , Poliadenilação , Estabilidade de RNA , RNA Mensageiro/química , Cristalização , Conformação de Ácido Nucleico , Oryza
9.
Nat Commun ; 12(1): 177, 2021 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-33420027

RESUMO

Glioblastoma (GBM) is the most common type of adult malignant brain tumor, but its molecular mechanisms are not well understood. In addition, the knowledge of the disease-associated expression and function of YTHDF2 remains very limited. Here, we show that YTHDF2 overexpression clinically correlates with poor glioma patient prognosis. EGFR that is constitutively activated in the majority of GBM causes YTHDF2 overexpression through the EGFR/SRC/ERK pathway. EGFR/SRC/ERK signaling phosphorylates YTHDF2 serine39 and threonine381, thereby stabilizes YTHDF2 protein. YTHDF2 is required for GBM cell proliferation, invasion, and tumorigenesis. YTHDF2 facilitates m6A-dependent mRNA decay of LXRA and HIVEP2, which impacts the glioma patient survival. YTHDF2 promotes tumorigenesis of GBM cells, largely through the downregulation of LXRα and HIVEP2. Furthermore, YTHDF2 inhibits LXRα-dependent cholesterol homeostasis in GBM cells. Together, our findings extend the landscape of EGFR downstream circuit, uncover the function of YTHDF2 in GBM tumorigenesis, and highlight an essential role of RNA m6A methylation in cholesterol homeostasis.


Assuntos
Neoplasias Encefálicas/metabolismo , Colesterol/metabolismo , Receptores ErbB/metabolismo , Glioblastoma/metabolismo , Proteínas de Ligação a RNA/metabolismo , Adulto , Animais , Neoplasias Encefálicas/genética , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Transformação Celular Neoplásica/genética , Proteínas de Ligação a DNA/metabolismo , Receptores ErbB/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Glioblastoma/genética , Glioma , Humanos , Receptores X do Fígado/metabolismo , Sistema de Sinalização das MAP Quinases , Masculino , Camundongos , Fosforilação , Estabilidade de RNA , Proteínas de Ligação a RNA/genética , Transdução de Sinais , Fatores de Transcrição/metabolismo , Transcriptoma
10.
Nucleic Acids Res ; 49(2): 818-831, 2021 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-33410890

RESUMO

Codon usage bias is a universal feature of all genomes. Although codon usage has been shown to regulate mRNA and protein levels by influencing mRNA decay and transcription in eukaryotes, little or no genome-wide correlations between codon usage and mRNA levels are detected in mammalian cells, raising doubt on the significance of codon usage effect on gene expression. Here we show that gene-specific regulation reduces the genome-wide codon usage and mRNA correlations: Constitutively expressed genes exhibit much higher genome-wide correlations than differentially expressed genes from fungi to human cells. Using Drosophila S2 cells as a model system, we showed that the effect of codon usage on mRNA expression level is promoter-dependent. Regions downstream of the core promoters of differentially expressed genes can repress the codon usage effects on mRNA expression. An element in the Hsp70 promoter was identified to be necessary and sufficient for this inhibitory effect. The promoter-dependent codon usage effects on mRNA levels are regulated at the transcriptional level through modulation of histone modifications, nucleosome densities and premature termination. Together, our results demonstrate that promoters play a major role in determining whether codon usage influences gene expression and further establish the transcription-dependent codon usage effects on gene expression.


Assuntos
Uso do Códon , Drosophila melanogaster/genética , Regulação da Expressão Gênica/genética , Regiões Promotoras Genéticas/genética , Acetilação , Animais , Composição de Bases , Linhagem Celular , Cromatina/genética , Cromatina/ultraestrutura , Códon sem Sentido , Proteínas de Drosophila/fisiologia , Drosophila melanogaster/citologia , Genes Reporter , Código das Histonas , Humanos , Luciferases de Renilla/biossíntese , Luciferases de Renilla/genética , Camundongos , Neurospora crassa/genética , Nucleossomos/metabolismo , Processamento de Proteína Pós-Traducional , Estabilidade de RNA , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Saccharomyces cerevisiae/genética , Especificidade da Espécie
11.
Biomed Pharmacother ; 133: 111075, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33378974

RESUMO

N6-methyladenosine (m6A) is one of the most abundant messenger RNAs modification. Increasing evidence illustrates its critical role on gastric cancer. Here, present research focuses on the potential function of m6A methyltransferase Wilms' tumour 1-associated protein (WTAP) in gastric cancer tumorigenesis. Firstly, m6A immunoprecipitation sequencing analysis (MeRIP-Seq) analysis demonstrated the m6A profile in gastric cancer cells. Both WTAP and the m6A expression were up-regulated in gastric cancer tissue and cells. The high-expression of WTAP was closely correlated with poor prognosis of gastric cancer patients. Functional experiments illustrated that WTAP promoted the proliferation and glycolytic capacity (glucose uptake, lactate production and extracellular acidification rate) in vitro, and the knockdown of WTAP suppressed the tumor growth in vivo. Mechanistically, HK2 was identified to be the target of WTAP using MeRIP-Seq and MeRIP-qPCR. WTAP enhanced the stability of HK2 mRNA through binding with the 3'-UTR m6A site. In conclusion, our results demonstrate the oncogenic role of WTAP and its m6A-mediated regulation on gastric cancer Warburg effect, providing a novel approach and therapeutic target in gastric cancer.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Hexoquinase/metabolismo , Fatores de Processamento de RNA/metabolismo , Neoplasias Gástricas/enzimologia , Animais , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células , Estabilidade Enzimática , Feminino , Regulação Neoplásica da Expressão Gênica , Hexoquinase/genética , Humanos , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Fatores de Processamento de RNA/genética , Estabilidade de RNA , Transdução de Sinais , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Carga Tumoral
12.
Talanta ; 224: 121850, 2021 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-33379066

RESUMO

In detecting infectious diseases, such as coronavirus 2019 (COVID-19), real-time reverse-transcription polymerase chain reaction (RT-PCR) is one of the most important technologies for RNA detection and disease diagnosis. To achieve high quality assurance, appropriate positive and negative controls are critical for disease detection using RT-PCR kits. In this study, we have found that commercial kits often adopt DNAs instead of RNAs as the positive controls, which can't report the kit problems in reverse transcription, thereby increasing risk of the false negative results when testing patient samples. To face the challenge, we have proposed and developed the chemically modified RNAs, such as phosphoroselenaote and phosphorothioate RNAs (Se-RNA and S-RNA), as the controls. We have found that while demonstrating the high thermostability, biostability, chemostability and exclusivity (or specificity), both Se-RNA and S-RNA can be fine templates for reverse transcription, indicating their potentials as both positive and negative controls for RT-PCR kits.


Assuntos
/métodos , RNA Viral/análise , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , /instrumentação , DNA Viral/análise , Reações Falso-Negativas , Humanos , Estabilidade de RNA , RNA Viral/química , Kit de Reagentes para Diagnóstico , Reação em Cadeia da Polimerase em Tempo Real/instrumentação , Reação em Cadeia da Polimerase Via Transcriptase Reversa/instrumentação , /genética
13.
Nucleic Acids Res ; 49(1): 568-583, 2021 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-33332555

RESUMO

Infection with kinetoplastid parasites, including Trypanosoma brucei (T. brucei), Trypanosoma cruzi (T. cruzi) and Leishmania can cause serious disease in humans. Like other kinetoplastid species, mRNAs of these disease-causing parasites must undergo posttranscriptional editing in order to be functional. mRNA editing is directed by gRNAs, a large group of small RNAs. Similar to mRNAs, gRNAs are also precisely regulated. In T. brucei, overexpression of RNase D ribonuclease (TbRND) leads to substantial reduction in the total gRNA population and subsequent inhibition of mRNA editing. However, the mechanisms regulating gRNA binding and cleavage by TbRND are not well defined. Here, we report a thorough structural study of TbRND. Besides Apo- and NMP-bound structures, we also solved one TbRND structure in complexed with single-stranded RNA. In combination with mutagenesis and in vitro cleavage assays, our structures indicated that TbRND follows the conserved two-cation-assisted mechanism in catalysis. TbRND is a unique RND member, as it contains a ZFD domain at its C-terminus. In addition to T. brucei, our studies also advanced our understanding on the potential gRNA degradation pathway in T. cruzi, Leishmania, as well for as other disease-associated parasites expressing ZFD-containing RNDs.


Assuntos
Proteínas de Protozoários/química , Estabilidade de RNA/fisiologia , RNA Guia/metabolismo , RNA de Protozoário/metabolismo , Ribonuclease III/química , Trypanosoma brucei brucei/enzimologia , Sequência de Aminoácidos , Sequência de Bases , Cristalografia por Raios X , Regulação da Expressão Gênica , Modelos Moleculares , Conformação de Ácido Nucleico , Conformação Proteica , Domínios Proteicos , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Ribonuclease III/metabolismo , Relação Estrutura-Atividade , Especificidade por Substrato , Dedos de Zinco
14.
Mol Cell ; 81(2): 293-303.e4, 2021 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-33326748

RESUMO

Ribosome assembly is catalyzed by numerous trans-acting factors and coupled with irreversible pre-rRNA processing, driving the pathway toward mature ribosomal subunits. One decisive step early in this progression is removal of the 5' external transcribed spacer (5'-ETS), an RNA extension at the 18S rRNA that is integrated into the huge 90S pre-ribosome structure. Upon endo-nucleolytic cleavage at an internal site, A1, the 5'-ETS is separated from the 18S rRNA and degraded. Here we present biochemical and cryo-electron microscopy analyses that depict the RNA exosome, a major 3'-5' exoribonuclease complex, in a super-complex with the 90S pre-ribosome. The exosome is docked to the 90S through its co-factor Mtr4 helicase, a processive RNA duplex-dismantling helicase, which strategically positions the exosome at the base of 5'-ETS helices H9-H9', which are dislodged in our 90S-exosome structures. These findings suggest a direct role of the exosome in structural remodeling of the 90S pre-ribosome to drive eukaryotic ribosome synthesis.


Assuntos
RNA Helicases DEAD-box/química , Endorribonucleases/química , Exonucleases/química , Complexo Multienzimático de Ribonucleases do Exossomo/ultraestrutura , RNA Ribossômico 18S/química , Ribossomos/ultraestrutura , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Sítios de Ligação , Microscopia Crioeletrônica , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Endorribonucleases/genética , Endorribonucleases/metabolismo , Exonucleases/genética , Exonucleases/metabolismo , Complexo Multienzimático de Ribonucleases do Exossomo/genética , Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , Modelos Moleculares , Ligação Proteica , Biossíntese de Proteínas , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Estabilidade de RNA , RNA Ribossômico 18S/genética , RNA Ribossômico 18S/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
15.
Nat Commun ; 11(1): 5521, 2020 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-33139748

RESUMO

A grand challenge of biological chemical production is the competition between synthetic circuits and host genes for limited cellular resources. Quorum sensing (QS)-based dynamic pathway regulations provide a pathway-independent way to rebalance metabolic flux over the course of the fermentation. Most cases, however, these pathway-independent strategies only have capacity for a single QS circuit functional in one cell. Furthermore, current dynamic regulations mainly provide localized control of metabolic flux. Here, with the aid of engineering synthetic orthogonal quorum-related circuits and global mRNA decay, we report a pathway-independent dynamic resource allocation strategy, which allows us to independently controlling two different phenotypic states to globally redistribute cellular resources toward synthetic circuits. The strategy which could pathway-independently and globally self-regulate two desired cell phenotypes including growth and production phenotypes could totally eliminate the need for human supervision of the entire fermentation.


Assuntos
Escherichia coli/metabolismo , Ácidos Graxos/metabolismo , Engenharia Metabólica/métodos , Percepção de Quorum/genética , Estabilidade de RNA/genética , Biocatálise , Vias Biossintéticas/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Fermentação/genética , Regulação Bacteriana da Expressão Gênica
16.
Nat Commun ; 11(1): 5552, 2020 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-33144582

RESUMO

Ribosomes stalled during translation must be rescued to replenish the pool of translation-competent ribosomal subunits. Bacterial alternative rescue factor B (ArfB) releases nascent peptides from ribosomes stalled on mRNAs truncated at the A site, allowing ribosome recycling. Prior structural work revealed that ArfB recognizes such ribosomes by inserting its C-terminal α-helix into the vacant mRNA tunnel. In this work, we report that ArfB can efficiently recognize a wider range of mRNA substrates, including longer mRNAs that extend beyond the A-site codon. Single-particle cryo-EM unveils that ArfB employs two modes of function depending on the mRNA length. ArfB acts as a monomer to accommodate a shorter mRNA in the ribosomal A site. By contrast, longer mRNAs are displaced from the mRNA tunnel by more than 20 Å and are stabilized in the intersubunit space by dimeric ArfB. Uncovering distinct modes of ArfB function resolves conflicting biochemical and structural studies, and may lead to re-examination of other ribosome rescue pathways, whose functions depend on mRNA lengths.


Assuntos
Proteínas de Escherichia coli/metabolismo , RNA Mensageiro/metabolismo , Ribossomos/metabolismo , Biocatálise , Dimerização , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/ultraestrutura , Modelos Biológicos , Conformação Proteica , Estabilidade de RNA , RNA Mensageiro/genética , RNA Mensageiro/ultraestrutura , Subunidades Ribossômicas/metabolismo , Ribossomos/ultraestrutura
17.
Nat Commun ; 11(1): 5508, 2020 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-33139726

RESUMO

The ubiquitous redox coenzyme nicotinamide adenine dinucleotide (NAD) acts as a non-canonical cap structure on prokaryotic and eukaryotic ribonucleic acids. Here we find that in budding yeast, NAD-RNAs are abundant (>1400 species), short (<170 nt), and mostly correspond to mRNA 5'-ends. The modification percentage of transcripts is low (<5%). NAD incorporation occurs mainly during transcription initiation by RNA polymerase II, which uses distinct promoters with a YAAG core motif for this purpose. Most NAD-RNAs are 3'-truncated. At least three decapping enzymes, Rai1, Dxo1, and Npy1, guard against NAD-RNA at different cellular locations, targeting overlapping transcript populations. NAD-mRNAs are not translatable in vitro. Our work indicates that in budding yeast, most of the NAD incorporation into RNA seems to be disadvantageous to the cell, which has evolved a diverse surveillance machinery to prematurely terminate, decap and reject NAD-RNAs.


Assuntos
Endorribonucleases/metabolismo , NAD/metabolismo , Capuzes de RNA/metabolismo , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/genética , Regiões 5' não Traduzidas , Núcleo Celular/genética , Pirofosfatases/metabolismo , Estabilidade de RNA , Proteínas de Ligação a RNA/metabolismo , Ribossomos/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transcrição Genética
18.
Nat Commun ; 11(1): 5111, 2020 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-33037216

RESUMO

The nascent polypeptide exit tunnel (NPET) is a major functional center of 60S ribosomal subunits. However, little is known about how the NPET is constructed during ribosome assembly. We utilized molecular genetics, biochemistry, and cryo-electron microscopy (cryo-EM) to investigate the functions of two NPET-associated proteins, ribosomal protein uL4 and assembly factor Nog1, in NPET assembly. Structures of mutant pre-ribosomes lacking the tunnel domain of uL4 reveal a misassembled NPET, including an aberrantly flexible ribosomal RNA helix 74, resulting in at least three different blocks in 60S assembly. Structures of pre-ribosomes lacking the C-terminal extension of Nog1 demonstrate that this extension scaffolds the tunnel domain of uL4 in the NPET to help maintain stability in the core of pre-60S subunits. Our data reveal that uL4 and Nog1 work together in the maturation of ribosomal RNA helix 74, which is required to ensure proper construction of the NPET and 60S ribosomal subunits.


Assuntos
Proteínas de Ligação ao GTP/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Ribossômicas/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/metabolismo , Microscopia Crioeletrônica , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/genética , Modelos Moleculares , Mutação , Proteínas Nucleares/química , Proteínas Nucleares/genética , Domínios Proteicos , Estabilidade de RNA , RNA Ribossômico/química , RNA Ribossômico/metabolismo , Proteínas Ribossômicas/química , Proteínas Ribossômicas/genética , Subunidades Ribossômicas Maiores de Eucariotos/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética
19.
Nat Commun ; 11(1): 4956, 2020 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-33009383

RESUMO

Tet-enzyme-mediated 5-hydroxymethylation of cytosines in DNA plays a crucial role in mouse embryonic stem cells (ESCs). In RNA also, 5-hydroxymethylcytosine (5hmC) has recently been evidenced, but its physiological roles are still largely unknown. Here we show the contribution and function of this mark in mouse ESCs and differentiating embryoid bodies. Transcriptome-wide mapping in ESCs reveals hundreds of messenger RNAs marked by 5hmC at sites characterized by a defined unique consensus sequence and particular features. During differentiation a large number of transcripts, including many encoding key pluripotency-related factors (such as Eed and Jarid2), show decreased cytosine hydroxymethylation. Using Tet-knockout ESCs, we find Tet enzymes to be partly responsible for deposition of 5hmC in mRNA. A transcriptome-wide search further reveals mRNA targets to which Tet1 and Tet2 bind, at sites showing a topology similar to that of 5hmC sites. Tet-mediated RNA hydroxymethylation is found to reduce the stability of crucial pluripotency-promoting transcripts. We propose that RNA cytosine 5-hydroxymethylation by Tets is a mark of transcriptome flexibility, inextricably linked to the balance between pluripotency and lineage commitment.


Assuntos
5-Metilcitosina/análogos & derivados , Diferenciação Celular , Proteínas de Ligação a DNA/metabolismo , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , RNA/metabolismo , 5-Metilcitosina/metabolismo , Animais , Especificidade de Anticorpos/imunologia , Sequência de Bases , Corpos Embrioides/metabolismo , Camundongos , Modelos Biológicos , Células-Tronco Pluripotentes/metabolismo , Ligação Proteica , Estabilidade de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcriptoma/genética
20.
Mol Cell ; 80(2): 193-209, 2020 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-33010203

RESUMO

Understanding the genetic design principles that determine protein production remains a major challenge. Although the key principles of gene expression were discovered 50 years ago, additional factors are still being uncovered. Both protein-coding and non-coding sequences harbor elements that collectively influence the efficiency of protein production by modulating transcription, mRNA decay, and translation. The influences of many contributing elements are intertwined, which complicates a full understanding of the individual factors. In natural genes, a functional balance between these factors has been obtained in the course of evolution, whereas for genetic-engineering projects, our incomplete understanding still limits optimal design of synthetic genes. However, notable advances have recently been made, supported by high-throughput analysis of synthetic gene libraries as well as by state-of-the-art biomolecular techniques. We discuss here how these advances further strengthen understanding of the gene expression process and how they can be harnessed to optimize protein production.


Assuntos
Código Genético , Biossíntese de Proteínas/genética , Algoritmos , Animais , Biotecnologia , Humanos , Estabilidade de RNA , Transcrição Genética
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