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1.
Nucleic Acids Res ; 49(6): 3185-3203, 2021 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-33693809

RESUMO

Protein methylation occurs primarily on lysine and arginine, but also on some other residues, such as histidine. METTL18 is the last uncharacterized member of a group of human methyltransferases (MTases) that mainly exert lysine methylation, and here we set out to elucidate its function. We found METTL18 to be a nuclear protein that contains a functional nuclear localization signal and accumulates in nucleoli. Recombinant METTL18 methylated a single protein in nuclear extracts and in isolated ribosomes from METTL18 knockout (KO) cells, identified as 60S ribosomal protein L3 (RPL3). We also performed an RPL3 interactomics screen and identified METTL18 as the most significantly enriched MTase. We found that His-245 in RPL3 carries a 3-methylhistidine (3MH; τ-methylhistidine) modification, which was absent in METTL18 KO cells. In addition, both recombinant and endogenous METTL18 were found to be automethylated at His-154, thus further corroborating METTL18 as a histidine-specific MTase. Finally, METTL18 KO cells displayed altered pre-rRNA processing, decreased polysome formation and codon-specific changes in mRNA translation, indicating that METTL18-mediated methylation of RPL3 is important for optimal ribosome biogenesis and function. In conclusion, we have here established METTL18 as the second human histidine-specific protein MTase, and demonstrated its functional relevance.


Assuntos
Biossíntese de Proteínas , Metiltransferases de Proteína/metabolismo , RNA Ribossômico/metabolismo , Proteínas Ribossômicas/metabolismo , Motivos de Aminoácidos , Nucléolo Celular/enzimologia , Células HEK293 , Células HeLa , Histidina/metabolismo , Humanos , Sinais de Localização Nuclear , Metiltransferases de Proteína/química , Processamento Pós-Transcricional do RNA , Ribossomos/metabolismo
2.
Nucleic Acids Res ; 49(7): 4066-4084, 2021 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-33721027

RESUMO

RNA helicases play important roles in diverse aspects of RNA metabolism through their functions in remodelling ribonucleoprotein complexes (RNPs), such as pre-ribosomes. Here, we show that the DEAD box helicase Dbp3 is required for efficient processing of the U18 and U24 intron-encoded snoRNAs and 2'-O-methylation of various sites within the 25S ribosomal RNA (rRNA) sequence. Furthermore, numerous box C/D snoRNPs accumulate on pre-ribosomes in the absence of Dbp3. Many snoRNAs guiding Dbp3-dependent rRNA modifications have overlapping pre-rRNA basepairing sites and therefore form mutually exclusive interactions with pre-ribosomes. Analysis of the distribution of these snoRNAs between pre-ribosome-associated and 'free' pools demonstrated that many are almost exclusively associated with pre-ribosomal complexes. Our data suggest that retention of such snoRNPs on pre-ribosomes when Dbp3 is lacking may impede rRNA 2'-O-methylation by reducing the recycling efficiency of snoRNPs and by inhibiting snoRNP access to proximal target sites. The observation of substoichiometric rRNA modification at adjacent sites suggests that the snoRNPs guiding such modifications likely interact stochastically rather than hierarchically with their pre-rRNA target sites. Together, our data provide new insights into the dynamics of snoRNPs on pre-ribosomal complexes and the remodelling events occurring during the early stages of ribosome assembly.


Assuntos
RNA Helicases DEAD-box/metabolismo , RNA Ribossômico/metabolismo , RNA Nucleolar Pequeno/metabolismo , Ribossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Escherichia coli , Metilação , Precursores de RNA/metabolismo , Leveduras/enzimologia
3.
Nucleic Acids Res ; 49(7): 4085-4103, 2021 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-33772581

RESUMO

The most abundant cellular RNA species, ribosomal RNA (rRNA), appears to be a source of massive amounts of non-randomly generated fragments. We found rRNA fragments (rRFs) in immunoprecipitated Argonaute (Ago-IP) complexes in human and mouse cells and in small RNA sequencing datasets. In human Ago1-IP, guanine-rich rRFs were preferentially cut in single-stranded regions of mature rRNAs between pyrimidines and adenosine, and non-randomly paired with cellular transcripts in crosslinked chimeras. Numerous identical rRFs were found in the cytoplasm and nucleus in mouse Ago2-IP. We report specific interaction motifs enriched in rRF-target pairs. Locations of such motifs on rRFs were compatible with the Ago structural features and patterns of the Ago-RNA crosslinking in both species. Strikingly, many of these motifs may bind to double-stranded regions on target RNAs, suggesting a potential pathway for regulating translation by unwinding mRNAs. Occurring on either end of rRFs and matching intronic, untranslated or coding regions in targets, such interaction sites extend the concept of microRNA seed regions. Targeting both borders of certain short introns, rRFs may be involved in their biogenesis or function, facilitated by Ago. Frequently dismissed as noise, rRFs are poised to greatly enrich the known functional spectrum of small RNA regulation.


Assuntos
Proteínas Argonauta/metabolismo , RNA de Cadeia Dupla/metabolismo , RNA Ribossômico/metabolismo , RNA de Transferência/metabolismo , Motivos de Aminoácidos , Animais , Bases de Dados Genéticas , Células HEK293 , Humanos , Camundongos , Ligação Proteica
4.
Nucleic Acids Res ; 49(7): 4104-4119, 2021 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-33784398

RESUMO

Eukaryotic rRNAs and snRNAs are decorated with abundant 2'-O-methylated nucleotides (Nm) that are predominantly synthesized by box C/D snoRNA-guided enzymes. In the model plant Arabidopsis thaliana, C/D snoRNAs have been well categorized, but there is a lack of systematic mapping of Nm. Here, we applied RiboMeth-seq to profile Nm in cytoplasmic, chloroplast and mitochondrial rRNAs and snRNAs. We identified 111 Nm in cytoplasmic rRNAs and 19 Nm in snRNAs and assigned guide for majority of the detected sites using an updated snoRNA list. At least four sites are directed by guides with multiple specificities as shown in yeast. We found that C/D snoRNAs frequently form extra pairs with nearby sequences of methylation sites, potentially facilitating the substrate binding. Chloroplast and mitochondrial rRNAs contain five almost identical methylation sites, including two novel sites mediating ribosomal subunit joining. Deletion of FIB1 or FIB2 gene reduced the accumulation of C/D snoRNA and rRNA methylation with FIB1 playing a bigger role in methylation. Our data reveal the comprehensive 2'-O-methylation maps for Arabidopsis rRNAs and snRNAs and would facilitate study of their function and biosynthesis.


Assuntos
Arabidopsis/genética , RNA de Cloroplastos/metabolismo , RNA Mitocondrial/metabolismo , RNA Ribossômico/metabolismo , RNA Nucleolar Pequeno/metabolismo , Ribose/metabolismo , Metilação , Processamento Pós-Transcricional do RNA
5.
Nat Commun ; 12(1): 949, 2021 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-33574238

RESUMO

Spindlin1 is a unique multivalent epigenetic reader that facilitates ribosomal RNA transcription. In this study, we provide molecular and structural basis by which Spindlin1 acts in complex with C11orf84 to preferentially recognize non-canonical bivalent mark of trimethylated lysine 4 and lysine 9 present on the same histone H3 tail (H3K4me3K9me3). We demonstrate that C11orf84 binding stabilizes Spindlin1 and enhances its association with bivalent H3K4me3K9me3 mark. The functional analysis suggests that Spindlin1/C11orf84 complex can displace HP1 proteins from H3K4me3K9me3-enriched rDNA loci, thereby facilitating the conversion of these poised rDNA repeats from the repressed state to the active conformation, and the consequent recruitment of RNA Polymerase I for rRNA transcription. Our study uncovers a previously unappreciated mechanism of bivalent H3K4me3K9me3 recognition by Spindlin1/C11orf84 complex required for activation of rRNA transcription.


Assuntos
Histonas/metabolismo , Transcrição Genética , Ativação Transcricional , Proteínas de Ciclo Celular/metabolismo , Proliferação de Células , DNA Ribossômico/genética , DNA Ribossômico/metabolismo , Genes de RNAr , Células HEK293 , Humanos , Proteínas Associadas aos Microtúbulos/metabolismo , Fosfoproteínas/metabolismo , RNA Polimerase I , RNA Ribossômico/metabolismo
6.
Mol Biol (Mosk) ; 55(1): 20-41, 2021.
Artigo em Russo | MEDLINE | ID: mdl-33566023

RESUMO

Protein synthesis on ribosomes is considered the main process in cell life. Regulation of ribosomal protein gene expression plays an important role in the balanced synthesis of proteins and RNA in ribosomal biogenesis. This review is focused on some features of autoregulation of ribosomal protein synthesis in prokaryotes. Inhibition of the synthesis of ribosomal proteins encoded by 12 operons by mechanisms of competition , "entrapment", and retroregulation are discussed. Examples of regulation of protein synthesis by individual ribosomal proteins and their complexes are presented.


Assuntos
Escherichia coli , Biossíntese de Proteínas , Escherichia coli/genética , Óperon , RNA Ribossômico/metabolismo , Proteínas Ribossômicas/genética , Ribossomos/genética , Ribossomos/metabolismo
7.
Nat Commun ; 12(1): 793, 2021 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-33542240

RESUMO

Adenosine-to-inosine (A-to-I) editing of eukaryotic cellular RNAs is essential for protection against auto-immune disorders. Editing is carried out by ADAR1, whose innate immune response-specific cytoplasmic isoform possesses a Z-DNA binding domain (Zα) of unknown function. Zα also binds to CpG repeats in RNA, which are a hallmark of Z-RNA formation. Unexpectedly, Zα has been predicted - and in some cases even shown - to bind to specific regions within mRNA and rRNA devoid of such repeats. Here, we use NMR, circular dichroism, and other biophysical approaches to demonstrate and characterize the binding of Zα to mRNA and rRNA fragments. Our results reveal a broad range of RNA sequences that bind to Zα and adopt Z-RNA conformations. Binding is accompanied by destabilization of neighboring A-form regions which is similar in character to what has been observed for B-Z-DNA junctions. The binding of Zα to non-CpG sequences is specific, cooperative and occurs with an affinity in the low micromolar range. This work allows us to propose a model for how Zα could influence the RNA binding specificity of ADAR1.


Assuntos
Adenosina Desaminase/metabolismo , Elementos Alu/genética , Domínios Proteicos , RNA Ribossômico/metabolismo , Proteínas de Ligação a RNA/metabolismo , Adenosina Desaminase/genética , Adenosina Desaminase/isolamento & purificação , Adenosina Desaminase/ultraestrutura , Dicroísmo Circular , Imunidade Inata , Ressonância Magnética Nuclear Biomolecular , Conformação de Ácido Nucleico , Motivo de Reconhecimento de RNA , RNA Ribossômico/genética , RNA Ribossômico/imunologia , RNA Ribossômico/ultraestrutura , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/isolamento & purificação , Proteínas de Ligação a RNA/ultraestrutura , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura
8.
Nucleic Acids Res ; 49(7): e40, 2021 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-33503245

RESUMO

Generation of conditional knockout (cKO) and various gene-modified cells is laborious and time-consuming. Here, we established an all-in-one cKO system, which enables highly efficient generation of cKO cells and simultaneous gene modifications, including epitope tagging and reporter gene knock-in. We applied this system to mouse embryonic stem cells (ESCs) and generated RNA helicase Ddx1 cKO ESCs. The targeted cells displayed endogenous promoter-driven EGFP and FLAG-tagged DDX1 expression, and they were converted to Ddx1 KO via FLP recombinase. We further established TetFE ESCs, which carried a reverse tetracycline transactivator (rtTA) expression cassette and a tetracycline response element (TRE)-regulated FLPERT2 cassette in the Gt(ROSA26)Sor locus for instant and tightly regulated induction of gene KO. By utilizing TetFE Ddx1F/F ESCs, we isolated highly pure Ddx1F/F and Ddx1-/- ESCs and found that loss of Ddx1 caused rRNA processing defects, thereby activating the ribosome stress-p53 pathway. We also demonstrated cKO of various genes in ESCs and homologous recombination-non-proficient human HT1080 cells. The frequency of cKO clones was remarkably high for both cell types and reached up to 96% when EGFP-positive clones were analyzed. This all-in-one cKO system will be a powerful tool for rapid and precise analyses of gene functions.


Assuntos
RNA Helicases DEAD-box/metabolismo , Técnicas de Inativação de Genes/métodos , RNA Ribossômico/metabolismo , Animais , Linhagem Celular , Células-Tronco Embrionárias , Fibroblastos , Expressão Gênica , Técnicas de Introdução de Genes , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Processamento Pós-Transcricional do RNA , Ribossomos/metabolismo
9.
RNA ; 27(4): 411-419, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33479117

RESUMO

Ribosomes are the macromolecular machines at the heart of protein synthesis; however, their function can be modulated by a variety of additional protein factors that directly interact with them. Here, we report the cryo-EM structure of human Ebp1 (p48 isoform) bound to the human 80S ribosome at 3.3 Å resolution. Ebp1 binds in the vicinity of the peptide exit tunnel on the 80S ribosome, and this binding is enhanced upon puromycin-mediated translational inhibition. The association of Ebp1 with the 80S ribosome centers around its interaction with ribosomal proteins eL19 and uL23 and the 28S rRNA. Further analysis of the Ebp1-ribosome complex suggests that Ebp1 can rotate around its insert domain, which may enable it to assume a wide range of conformations while maintaining its interaction with the ribosome. Structurally, Ebp1 shares homology with the methionine aminopeptidase 2 family of enzymes; therefore, this inherent flexibility may also be conserved.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Biossíntese de Proteínas , RNA Ribossômico/química , Proteínas de Ligação a RNA/química , Proteínas Ribossômicas/química , Ribossomos/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sítios de Ligação , Microscopia Crioeletrônica , Humanos , Modelos Moleculares , Ligação Proteica , Biossíntese de Proteínas/efeitos dos fármacos , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Inibidores da Síntese de Proteínas/farmacologia , Puromicina/farmacologia , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , Termodinâmica
10.
RNA ; 27(4): 496-512, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33483369

RESUMO

Ribosomal RNA (rRNA) carries extensive 2'-O-methyl marks at functionally important sites. This simple chemical modification is thought to confer stability, promote RNA folding, and contribute to generate a heterogenous ribosome population with a yet-uncharacterized function. 2'-O-methylation occurs both in archaea and eukaryotes and is accomplished by the Box C/D RNP enzyme in an RNA-guided manner. Extensive and partially conflicting structural information exists for the archaeal enzyme, while no structural data is available for the eukaryotic enzyme. The yeast Box C/D RNP consists of a guide RNA, the RNA-primary binding protein Snu13, the two scaffold proteins Nop56 and Nop58, and the enzymatic module Nop1. Here we present the high-resolution structure of the eukaryotic Box C/D methyltransferase Nop1 from Saccharomyces cerevisiae bound to the amino-terminal domain of Nop56. We discuss similarities and differences between the interaction modes of the two proteins in archaea and eukaryotes and demonstrate that eukaryotic Nop56 recruits the methyltransferase to the Box C/D RNP through a protein-protein interface that differs substantially from the archaeal orthologs. This study represents a first achievement in understanding the evolution of the structure and function of these proteins from archaea to eukaryotes.


Assuntos
Proteínas Arqueais/química , Proteínas Cromossômicas não Histona/química , Proteínas Nucleares/química , Pyrococcus furiosus/genética , Ribonucleoproteínas Nucleolares Pequenas/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Sítios de Ligação , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Cristalografia por Raios X , Expressão Gênica , Metilação , Modelos Moleculares , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Pyrococcus furiosus/metabolismo , RNA Fúngico/genética , RNA Fúngico/metabolismo , RNA Guia/genética , RNA Guia/metabolismo , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , RNA Nucleolar Pequeno/genética , RNA Nucleolar Pequeno/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribonucleoproteínas Nucleares Pequenas/química , Ribonucleoproteínas Nucleares Pequenas/genética , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Ribonucleoproteínas Nucleolares Pequenas/genética , Ribonucleoproteínas Nucleolares Pequenas/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Homologia Estrutural de Proteína
11.
Nat Commun ; 12(1): 528, 2021 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-33483500

RESUMO

Ribosomal RNA (rRNA) is most highly expressed in rapidly growing bacteria and is drastically downregulated under stress conditions by the global transcriptional regulator DksA and the alarmone ppGpp. Here, we determined cryo-electron microscopy structures of the Escherichia coli RNA polymerase (RNAP) σ70 holoenzyme during rRNA promoter recognition with and without DksA/ppGpp. RNAP contacts the UP element using dimerized α subunit carboxyl-terminal domains and scrunches the template DNA with the σ finger and ß' lid to select the transcription start site favorable for rapid promoter escape. Promoter binding induces conformational change of σ domain 2 that opens a gate for DNA loading and ejects σ1.1 from the RNAP cleft to facilitate open complex formation. DksA/ppGpp binding also opens the DNA loading gate, which is not coupled to σ1.1 ejection and impedes open complex formation. These results provide a molecular basis for the exceptionally active rRNA transcription and its vulnerability to DksA/ppGpp.


Assuntos
Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Regiões Promotoras Genéticas/genética , RNA Ribossômico/genética , Transcrição Genética , Microscopia Crioeletrônica , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/metabolismo , RNA Polimerases Dirigidas por DNA/ultraestrutura , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Guanosina Tetrafosfato/metabolismo , Holoenzimas/química , Holoenzimas/metabolismo , Holoenzimas/ultraestrutura , Conformação Proteica , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Ribossômico/metabolismo , Fator sigma/química , Fator sigma/metabolismo , Fator sigma/ultraestrutura , Sítio de Iniciação de Transcrição
12.
Nucleic Acids Res ; 49(3): 1662-1687, 2021 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-33434266

RESUMO

Ribosomes are intricate molecular machines ensuring proper protein synthesis in every cell. Ribosome biogenesis is a complex process which has been intensively analyzed in bacteria and eukaryotes. In contrast, our understanding of the in vivo archaeal ribosome biogenesis pathway remains less characterized. Here, we have analyzed the in vivo role of the almost universally conserved ribosomal RNA dimethyltransferase KsgA/Dim1 homolog in archaea. Our study reveals that KsgA/Dim1-dependent 16S rRNA dimethylation is dispensable for the cellular growth of phylogenetically distant archaea. However, proteomics and functional analyses suggest that archaeal KsgA/Dim1 and its rRNA modification activity (i) influence the expression of a subset of proteins and (ii) contribute to archaeal cellular fitness and adaptation. In addition, our study reveals an unexpected KsgA/Dim1-dependent variability of rRNA modifications within the archaeal phylum. Combining structure-based functional studies across evolutionary divergent organisms, we provide evidence on how rRNA structure sequence variability (re-)shapes the KsgA/Dim1-dependent rRNA modification status. Finally, our results suggest an uncoupling between the KsgA/Dim1-dependent rRNA modification completion and its release from the nascent small ribosomal subunit. Collectively, our study provides additional understandings into principles of molecular functional adaptation, and further evolutionary and mechanistic insights into an almost universally conserved step of ribosome synthesis.


Assuntos
Archaea/enzimologia , Metiltransferases/metabolismo , RNA Arqueal/metabolismo , RNA Ribossômico/metabolismo , Archaea/genética , Movimento Celular , Crenarchaeota/enzimologia , Euryarchaeota/enzimologia , Haloferax volcanii/enzimologia , Metiltransferases/fisiologia , Biossíntese de Proteínas , RNA Arqueal/química , RNA Ribossômico/química , Subunidades Ribossômicas Menores de Arqueas/enzimologia
13.
EMBO J ; 40(2): e105699, 2021 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-33347626

RESUMO

Pathogen type 3 secretion systems (T3SS) manipulate host cell pathways by directly delivering effector proteins into host cells. In Vibrio parahaemolyticus, the leading cause of bacterial seafood-borne diarrheal disease, we showed that a T3SS effector, VgpA, localizes to the host cell nucleolus where it binds Epstein-Barr virus nuclear antigen 1-binding protein 2 (EBP2). An amino acid substitution in VgpA (VgpAL10A ) did not alter its translocation to the nucleus but abolished the effector's capacity to interact with EBP2. VgpA-EBP2 interaction led to the re-localization of c-Myc to the nucleolus and increased cellular rRNA expression and proliferation of cultured cells. The VgpA-EBP2 interaction elevated EBP2's affinity for c-Myc and prolonged the oncoprotein's half-life. Studies in infant rabbits demonstrated that VgpA is translocated into intestinal epithelial cells, where it interacts with EBP2 and leads to nucleolar re-localization of c-Myc. Moreover, the in vivo VgpA-EBP2 interaction during infection led to proliferation of intestinal cells and heightened V. parahaemolyticus' colonization and virulence. These observations suggest that direct effector stimulation of a c-Myc controlled host cell growth program can contribute to pathogenesis.


Assuntos
Proteínas de Bactérias/metabolismo , Nucléolo Celular/metabolismo , Proliferação de Células/fisiologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , Sistemas de Secreção Tipo III/metabolismo , Vibrio parahaemolyticus/metabolismo , Virulência/fisiologia , Animais , Células CACO-2 , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Células Epiteliais/metabolismo , Infecções por Vírus Epstein-Barr/metabolismo , Antígenos Nucleares do Vírus Epstein-Barr/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Herpesvirus Humano 4/patogenicidade , Humanos , RNA Ribossômico/metabolismo , Proteínas de Ligação a RNA/metabolismo , Coelhos , Vibrioses/metabolismo
14.
Methods Mol Biol ; 2192: 159-181, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33230773

RESUMO

Human mitochondria contain their own DNA (mtDNA) that encodes 13 proteins all of which are core subunits of oxidative phosphorylation (OXPHOS) complexes. To form functional complexes, these 13 components need to be correctly assembled with approximately 70 nuclear-encoded subunits that are imported following synthesis in the cytosol. How this complicated coordinated translation and assembly is choreographed is still not clear. Methods are being developed to determine whether all members of a particular complex are translated in close proximity, whether protein synthesis is clustered in submitochondrial factories, whether these align with incoming polypeptides, and if there is evidence for co-translational translation that is regulated and limited by the interaction of the incoming proteins with synthesis of their mtDNA-encoded partners. Two methods are described in this chapter to visualize the distribution of mitochondrial ribosomal RNAs in conjunction with newly synthesized mitochondrial proteins. The first combines RNA Fluorescent In Situ Hybridization (FISH) and super-resolution immunocytochemistry to pinpoint mitochondrial ribosomal RNA. The second localizes nascent translation within the mitochondrial network through non-canonical amino acid labeling, click chemistry and fluorescent microscopy.


Assuntos
Química Click/métodos , Imuno-Histoquímica/métodos , Hibridização in Situ Fluorescente/métodos , Proteínas Mitocondriais/metabolismo , Ribossomos Mitocondriais/metabolismo , RNA Mitocondrial/metabolismo , RNA Ribossômico/metabolismo , Aminoácidos/química , Linhagem Celular Tumoral , DNA Mitocondrial/genética , Humanos , Microscopia de Fluorescência/métodos , Fosforilação Oxidativa , Biossíntese de Proteínas , Proteínas Ribossômicas/metabolismo
15.
PLoS One ; 15(12): e0236850, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33315868

RESUMO

Ribosomes are the sophisticated machinery that is responsible for protein synthesis in a cell. Recently, quantitative mass spectrometry (qMS) have been successfully applied for understanding the dynamics of protein complexes. Here, we developed a highly specific and reproducible method to quantify all ribosomal proteins (r-proteins) by combining selected reaction monitoring (SRM) and isotope labeling. We optimized the SRM methods using purified ribosomes and Escherichia coli lysates and verified this approach as detecting 41 of the 54 r-proteins separately synthesized in E. coli S30 extracts. The SRM methods will enable us to utilize qMS as a highly specific analytical tool in the research of E. coli ribosomes, and this methodology have potential to accelerate the understanding of ribosome biogenesis, function, and the development of engineered ribosomes with additional functions.


Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo , Marcação por Isótopo/métodos , Espectrometria de Massas/métodos , Biossíntese de Proteínas/fisiologia , RNA Ribossômico/metabolismo , Subunidades Ribossômicas Menores de Bactérias/metabolismo
16.
J Vis Exp ; (166)2020 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-33369608

RESUMO

The bacterial Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Streptococcus pyogenes CRISPR-associated protein (Cas) system has been harnessed by researchers to study important biologically relevant problems. The unparalleled power of the CRISPR/Cas genome editing method allows researchers to precisely edit any locus of their choosing, thereby facilitating an increased understanding of gene function. Several methods for editing the C. elegans genome by CRISPR/Cas9 have been described previously. Here, we discuss and demonstrate a method which utilizes in vitro assembled ribonucleoprotein complexes and the dpy-10 co-CRISPR marker for screening. Specifically, in this article, we go through the step-by-step process of introducing premature stop codons into the C. elegans rbm-3.2 gene by homology-directed repair using this method of CRISPR/Cas9 editing. This relatively simple editing method can be used to study the function of any gene of interest and allows for the generation of homozygous-edited C. elegans by CRISPR/Cas9 editing in less than two weeks.


Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Colágeno/genética , Testes Genéticos , Ribonucleoproteínas/metabolismo , Animais , Sequência de Bases , Primers do DNA/metabolismo , Gônadas/metabolismo , Homozigoto , Microinjeções , Edição de RNA/genética , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , Mapeamento por Restrição , Ribonucleoproteínas/genética , Streptococcus pyogenes/genética
17.
Nat Commun ; 11(1): 4941, 2020 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-33009412

RESUMO

Methods to directly inhibit gene expression using small molecules hold promise for the development of new therapeutics targeting proteins that have evaded previous attempts at drug discovery. Among these, small molecules including the drug-like compound PF-06446846 (PF846) selectively inhibit the synthesis of specific proteins, by stalling translation elongation. These molecules also inhibit translation termination by an unknown mechanism. Using cryo-electron microscopy (cryo-EM) and biochemical approaches, we show that PF846 inhibits translation termination by arresting the nascent chain (NC) in the ribosome exit tunnel. The arrested NC adopts a compact α-helical conformation that induces 28 S rRNA nucleotide rearrangements that suppress the peptidyl transferase center (PTC) catalytic activity stimulated by eukaryotic release factor 1 (eRF1). These data support a mechanism of action for a small molecule targeting translation that suppresses peptidyl-tRNA hydrolysis promoted by eRF1, revealing principles of eukaryotic translation termination and laying the foundation for new therapeutic strategies.


Assuntos
Terminação Traducional da Cadeia Peptídica , Preparações Farmacêuticas/metabolismo , Linhagem Celular , Humanos , Modelos Moleculares , Mutação/genética , Conformação Proteica , RNA Ribossômico/metabolismo , Ribossomos/metabolismo , Ribossomos/ultraestrutura
18.
Science ; 370(6513): 227-231, 2020 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-33033220

RESUMO

Stem cells in plants constantly supply daughter cells to form new organs and are expected to safeguard the integrity of the cells from biological invasion. Here, we show how stem cells of the Arabidopsis shoot apical meristem and their nascent daughter cells suppress infection by cucumber mosaic virus (CMV). The stem cell regulator WUSCHEL responds to CMV infection and represses virus accumulation in the meristem central and peripheral zones. WUSCHEL inhibits viral protein synthesis by repressing the expression of plant S-adenosyl-l-methionine-dependent methyltransferases, which are involved in ribosomal RNA processing and ribosome stability. Our results reveal a conserved strategy in plants to protect stem cells against viral intrusion and provide a molecular basis for WUSCHEL-mediated broad-spectrum innate antiviral immunity in plants.


Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/imunologia , Arabidopsis/virologia , Cucumovirus , Proteínas de Homeodomínio/fisiologia , Imunidade Inata , Doenças das Plantas/virologia , Imunidade Vegetal , Proteínas de Arabidopsis/genética , Proteínas de Homeodomínio/genética , Meristema/citologia , Meristema/imunologia , Meristema/virologia , Metiltransferases/metabolismo , RNA Ribossômico/metabolismo , Células-Tronco/imunologia , Células-Tronco/virologia
19.
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
20.
Folia Biol (Praha) ; 66(3): 111-115, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33069190

RESUMO

Progenitor cells of the human erythroid and granulocytic cell lineages are characterized by the presence of several nucleoli. One of these nucleoli is larger and possesses more fibrillar centres than others. Such nucleolus is apparently dominant in respect of both size and main nucleolar function such as nucleolar-ribosomal RNA transcription. Such nucleolus is also visible in specimens using conventional visualization procedures, in contrast to smaller nucleoli. In the terminal differentiation nucleated stages of the erythroid and granulocytic development, dominant nucleoli apparently disappeared, since these cells mostly contained very small nucleoli of a similar size with one fibrillar centre. Thus, the easily visible dominant nucleoli appear to be useful markers of the progenitor cell state, such as proliferation, and differentiation potential.


Assuntos
Nucléolo Celular/fisiologia , Células Precursoras Eritroides/ultraestrutura , Células Precursoras de Granulócitos/ultraestrutura , Diferenciação Celular , Divisão Celular , Linhagem da Célula , Nucléolo Celular/ultraestrutura , Núcleo Celular/ultraestrutura , Granulócitos/ultraestrutura , Humanos , RNA Ribossômico/metabolismo
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