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1.
Nat Commun ; 12(1): 5044, 2021 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-34413298

RESUMO

Indirect somatic genetic rescue (SGR) of a germline mutation is thought to be rare in inherited Mendelian disorders. Here, we establish that acquired mutations in the EIF6 gene are a frequent mechanism of SGR in Shwachman-Diamond syndrome (SDS), a leukemia predisposition disorder caused by a germline defect in ribosome assembly. Biallelic mutations in the SBDS or EFL1 genes in SDS impair release of the anti-association factor eIF6 from the 60S ribosomal subunit, a key step in the translational activation of ribosomes. Here, we identify diverse mosaic somatic genetic events (point mutations, interstitial deletion, reciprocal chromosomal translocation) in SDS hematopoietic cells that reduce eIF6 expression or disrupt its interaction with the 60S subunit, thereby conferring a selective advantage over non-modified cells. SDS-related somatic EIF6 missense mutations that reduce eIF6 dosage or eIF6 binding to the 60S subunit suppress the defects in ribosome assembly and protein synthesis across multiple SBDS-deficient species including yeast, Dictyostelium and Drosophila. Our data suggest that SGR is a universal phenomenon that may influence the clinical evolution of diverse Mendelian disorders and support eIF6 suppressor mimics as a therapeutic strategy in SDS.


Assuntos
Mutação , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Ribossomos/genética , Ribossomos/patologia , Síndrome de Shwachman-Diamond/genética , Síndrome de Shwachman-Diamond/patologia , Adolescente , Adulto , Animais , Fenômenos Biológicos , Células Cultivadas , Criança , Pré-Escolar , Dictyostelium , Drosophila , Fatores de Iniciação em Eucariotos/genética , Fatores de Iniciação em Eucariotos/metabolismo , Células Germinativas , Humanos , Lactente , Simulação de Dinâmica Molecular , Fatores de Alongamento de Peptídeos/genética , Fatores de Alongamento de Peptídeos/metabolismo , Ligação Proteica , Biossíntese de Proteínas , Proteínas/genética , Proteínas/metabolismo , Ribonucleoproteína Nuclear Pequena U5/genética , Ribonucleoproteína Nuclear Pequena U5/metabolismo , Ribossomos/metabolismo , Saccharomyces cerevisiae , Homologia de Sequência de Aminoácidos , Síndrome de Shwachman-Diamond/metabolismo , Adulto Jovem
2.
Int J Mol Sci ; 22(16)2021 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-34445686

RESUMO

Quinoin is a type 1 ribosome-inactivating protein (RIP) we previously isolated from the seeds of pseudocereal quinoa (Chenopodium quinoa) and is known as a functional food for its beneficial effects on human health. As the presence of RIPs in edible plants could be potentially risky, here we further characterised biochemically the protein (complete amino acid sequence, homologies/differences with other RIPs and three-dimensional homology modeling) and explored its possible defensive role against pathogens. Quinoin consists of 254 amino acid residues, without cysteinyl residues. As demonstrated by similarities and homology modeling, quinoin preserves the amino acid residues of the active site (Tyr75, Tyr122, Glu177, Arg180, Phe181 and Trp206; quinoin numbering) and the RIP-fold characteristic of RIPs. The polypeptide chain of quinoin contains two N-glycosylation sites at Asn115 and Asp231, the second of which appears to be linked to sugars. Moreover, by comparative MALDI-TOF tryptic peptide mapping, two differently glycosylated forms of quinoin, named pre-quinoin-1 and pre-quinoin-2 (~0.11 mg/100 g and ~0.85 mg/100 g of seeds, respectively) were characterised. Finally, quinoin possesses: (i) strong antiviral activity, both in vitro and in vivo towards Tobacco Necrosis Virus (TNV); (ii) a growth inhibition effect on the bacterial pathogens of plants; and (iii) a slight antifungal effect against two Cryphonectria parasitica strains.


Assuntos
Chenopodium quinoa/enzimologia , Saporinas/metabolismo , Sequência de Aminoácidos/genética , Chenopodium quinoa/metabolismo , Proteínas de Plantas/metabolismo , Inibidores da Síntese de Proteínas/farmacologia , Ribossomos/metabolismo , Saporinas/fisiologia , Sementes/enzimologia , Homologia de Sequência de Aminoácidos
3.
Nat Commun ; 12(1): 5094, 2021 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-34429433

RESUMO

Ribosome profiling measures genome-wide translation dynamics at sub-codon resolution. Cycloheximide (CHX), a widely used translation inhibitor to arrest ribosomes in these experiments, has been shown to induce biases in yeast, questioning its use. However, whether such biases are present in datasets of other organisms including humans is unknown. Here we compare different CHX-treatment conditions in human cells and yeast in parallel experiments using an optimized protocol. We find that human ribosomes are not susceptible to conformational restrictions by CHX, nor does it distort gene-level measurements of ribosome occupancy, measured decoding speed or the translational ramp. Furthermore, CHX-induced codon-specific biases on ribosome occupancy are not detectable in human cells or other model organisms. This shows that reported biases of CHX are species-specific and that CHX does not affect the outcome of ribosome profiling experiments in most settings. Our findings provide a solid framework to conduct and analyze ribosome profiling experiments.


Assuntos
Cicloeximida/farmacologia , Ribossomos/química , Ribossomos/efeitos dos fármacos , Ribossomos/metabolismo , Animais , Viés , Códon/metabolismo , Células HEK293 , Humanos , Camundongos , Biossíntese de Proteínas/efeitos dos fármacos , Inibidores da Síntese de Proteínas/farmacologia , Saccharomyces cerevisiae/metabolismo , Especificidade da Espécie
4.
Nat Commun ; 12(1): 4723, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34354064

RESUMO

Translational riboswitches are cis-acting RNA regulators that modulate the expression of genes during translation initiation. Their mechanism is considered as an RNA-only gene-regulatory system inducing a ligand-dependent shift of the population of functional ON- and OFF-states. The interaction of riboswitches with the translation machinery remained unexplored. For the adenine-sensing riboswitch from Vibrio vulnificus we show that ligand binding alone is not sufficient for switching to a translational ON-state but the interaction of the riboswitch with the 30S ribosome is indispensable. Only the synergy of binding of adenine and of 30S ribosome, in particular protein rS1, induces complete opening of the translation initiation region. Our investigation thus unravels the intricate dynamic network involving RNA regulator, ligand inducer and ribosome protein modulator during translation initiation.


Assuntos
Biossíntese de Proteínas , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , Riboswitch/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Modelos Moleculares , Conformação de Ácido Nucleico , Conformação Proteica , RNA Bacteriano/química , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , Subunidades Ribossômicas Menores de Bactérias/química , Subunidades Ribossômicas Menores de Bactérias/genética , Subunidades Ribossômicas Menores de Bactérias/metabolismo , Ribossomos/química , Vibrio vulnificus/genética , Vibrio vulnificus/metabolismo
5.
Nat Commun ; 12(1): 4696, 2021 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-34349113

RESUMO

Productive ribosomal RNA (rRNA) compaction during ribosome assembly necessitates establishing correct tertiary contacts between distant secondary structure elements. Here, we quantify the response of the yeast proteome to low temperature (LT), a condition where aberrant mis-paired RNA folding intermediates accumulate. We show that, at LT, yeast cells globally boost production of their ribosome assembly machinery. We find that the LT-induced assembly factor, Puf6, binds to the nascent catalytic RNA-rich subunit interface within the 60S pre-ribosome, at a site that eventually loads the nuclear export apparatus. Ensemble Förster resonance energy transfer studies show that Puf6 mimics the role of Mg2+ to usher a unique long-range tertiary contact to compact rRNA. At LT, puf6 mutants accumulate 60S pre-ribosomes in the nucleus, thus unveiling Puf6-mediated rRNA compaction as a critical temperature-regulated rescue mechanism that counters rRNA misfolding to prime export competence.


Assuntos
Núcleo Celular/metabolismo , Proteínas de Ligação a RNA/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transporte Ativo do Núcleo Celular , Temperatura Baixa , GTP Fosfo-Hidrolases/metabolismo , Mutação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteoma/metabolismo , Dobramento de RNA , Precursores de RNA/química , Precursores de RNA/metabolismo , RNA Ribossômico/química , RNA Ribossômico/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Subunidades Ribossômicas Maiores de Eucariotos/química , Ribossomos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética
6.
Nat Commun ; 12(1): 4851, 2021 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-34381047

RESUMO

Pathogens are thought to use host molecular cues to control when to initiate life-cycle transitions, but these signals are mostly unknown, particularly for the parasitic disease malaria caused by Plasmodium falciparum. The chemokine CXCL10 is present at high levels in fatal cases of cerebral malaria patients, but is reduced in patients who survive and do not have complications. Here we show a Pf 'decision-sensing-system' controlled by CXCL10 concentration. High CXCL10 expression prompts P. falciparum to initiate a survival strategy via growth acceleration. Remarkably, P. falciparum inhibits CXCL10 synthesis in monocytes by disrupting the association of host ribosomes with CXCL10 transcripts. The underlying inhibition cascade involves RNA cargo delivery into monocytes that triggers RIG-I, which leads to HUR1 binding to an AU-rich domain of the CXCL10 3'UTR. These data indicate that when the parasite can no longer keep CXCL10 at low levels, it can exploit the chemokine as a cue to shift tactics and escape.


Assuntos
Quimiocina CXCL10/metabolismo , Malária Falciparum/parasitologia , Plasmodium falciparum/fisiologia , Regiões 3' não Traduzidas , Quimiocina CXCL10/genética , Proteína DEAD-box 58/metabolismo , Proteína Semelhante a ELAV 1/metabolismo , Vesículas Extracelulares/metabolismo , Interações Hospedeiro-Parasita , Humanos , Estágios do Ciclo de Vida , Malária Falciparum/imunologia , Monócitos/metabolismo , Plasmodium falciparum/crescimento & desenvolvimento , Plasmodium falciparum/metabolismo , Biossíntese de Proteínas , RNA de Protozoário/metabolismo , Receptores Imunológicos/metabolismo , Ribossomos/metabolismo , Células THP-1
7.
Nat Commun ; 12(1): 4909, 2021 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-34389707

RESUMO

In bacteria, trans-translation is the main rescue system, freeing ribosomes stalled on defective messenger RNAs. This mechanism is driven by small protein B (SmpB) and transfer-messenger RNA (tmRNA), a hybrid RNA known to have both a tRNA-like and an mRNA-like domain. Here we present four cryo-EM structures of the ribosome during trans-translation at resolutions from 3.0 to 3.4 Å. These include the high-resolution structure of the whole pre-accommodated state, as well as structures of the accommodated state, the translocated state, and a translocation intermediate. Together, they shed light on the movements of the tmRNA-SmpB complex in the ribosome, from its delivery by the elongation factor EF-Tu to its passage through the ribosomal A and P sites after the opening of the B1 bridges. Additionally, we describe the interactions between the tmRNA-SmpB complex and the ribosome. These explain why the process does not interfere with canonical translation.


Assuntos
Proteínas de Escherichia coli/genética , Escherichia coli/genética , Biossíntese de Proteínas/genética , RNA Bacteriano/genética , Proteínas de Ligação a RNA/genética , Ribossomos/genética , Sítios de Ligação/genética , Microscopia Crioeletrônica , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Conformação de Ácido Nucleico , Ligação Proteica , Domínios Proteicos , RNA Bacteriano/química , RNA Bacteriano/metabolismo , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Transferência/química , RNA de Transferência/genética , RNA de Transferência/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Ribossomos/metabolismo , Ribossomos/ultraestrutura
8.
Mol Cell ; 81(16): 3310-3322.e6, 2021 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-34416138

RESUMO

Amino acid starvation is sensed by Escherichia coli RelA and Bacillus subtilis Rel through monitoring the aminoacylation status of ribosomal A-site tRNA. These enzymes are positively regulated by their product-the alarmone nucleotide (p)ppGpp-through an unknown mechanism. The (p)ppGpp-synthetic activity of Rel/RelA is controlled via auto-inhibition by the hydrolase/pseudo-hydrolase (HD/pseudo-HD) domain within the enzymatic N-terminal domain region (NTD). We localize the allosteric pppGpp site to the interface between the SYNTH and pseudo-HD/HD domains, with the alarmone stimulating Rel/RelA by exploiting intra-NTD autoinhibition dynamics. We show that without stimulation by pppGpp, starved ribosomes cannot efficiently activate Rel/RelA. Compromised activation by pppGpp ablates Rel/RelA function in vivo, suggesting that regulation by the second messenger (p)ppGpp is necessary for mounting an acute starvation response via coordinated enzymatic activity of individual Rel/RelA molecules. Control by (p)ppGpp is lacking in the E. coli (p)ppGpp synthetase SpoT, thus explaining its weak synthetase activity.


Assuntos
Regulação Alostérica/genética , Proteínas de Escherichia coli/genética , GTP Pirofosfoquinase/genética , Guanosina Pentafosfato/genética , Pirofosfatases/genética , Aminoácidos/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Domínio Catalítico/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Hidrolases/genética , Ribossomos/genética , Ribossomos/metabolismo , Inanição/genética , Inanição/metabolismo
9.
Science ; 373(6557): 876-882, 2021 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-34413231

RESUMO

Translation termination, which liberates a nascent polypeptide from the ribosome specifically at stop codons, must occur accurately and rapidly. We established single-molecule fluorescence assays to track the dynamics of ribosomes and two requisite release factors (eRF1 and eRF3) throughout termination using an in vitro-reconstituted yeast translation system. We found that the two eukaryotic release factors bound together to recognize stop codons rapidly and elicit termination through a tightly regulated, multistep process that resembles transfer RNA selection during translation elongation. Because the release factors are conserved from yeast to humans, the molecular events that underlie yeast translation termination are likely broadly fundamental to eukaryotic protein synthesis.


Assuntos
Terminação Traducional da Cadeia Peptídica , Fatores de Terminação de Peptídeos/metabolismo , Ribossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Códon de Terminação , Transferência Ressonante de Energia de Fluorescência , Ligação Proteica , Biossíntese de Proteínas , Saccharomyces cerevisiae/metabolismo , Imagem Individual de Molécula
10.
Mol Cell ; 81(16): 3243-3245, 2021 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-34416137

RESUMO

Orellana et al. (2021) and Dai et al. (2021) demonstrate that increased m7G modification of a subset of tRNAs by the METTL1/WDR4 complex stabilizes these mRNAs against decay, increases translation efficiency, reduces ribosome pausing, is associated with poor survival in human cancers, and is directly transforming.


Assuntos
Neoplasias , Processamento Pós-Transcricional do RNA , Proteínas de Ligação ao GTP/metabolismo , Humanos , Neoplasias/genética , Neoplasias/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo , Ribossomos/genética , Ribossomos/metabolismo
11.
Int J Mol Sci ; 22(14)2021 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-34299038

RESUMO

Ribosomal RNA is a major component of the ribosome. This RNA plays a crucial role in ribosome functioning by ensuring the formation of the peptide bond between amino acids and the accurate decoding of the genetic code. The rRNA carries many chemical modifications that participate in its maturation, the formation of the ribosome and its functioning. In this review, we present the different modifications and how they are deposited on the rRNA. We also describe the most recent results showing that the modified positions are not 100% modified, which creates a heterogeneous population of ribosomes. This gave rise to the concept of specialized ribosomes that we discuss. The knowledge accumulated in the yeast Saccharomyces cerevisiae is very helpful to better understand the role of rRNA modifications in humans, especially in ribosomopathies.


Assuntos
Modelos Biológicos , Processamento Pós-Transcricional do RNA , RNA Fúngico/genética , RNA Ribossômico/genética , Ribossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Humanos , Ribossomos/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética
12.
Nat Commun ; 12(1): 4358, 2021 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-34272367

RESUMO

Premature termination codons (PTCs) prevent translation of a full-length protein and trigger nonsense-mediated mRNA decay (NMD). Nonsense suppression (also termed readthrough) therapy restores protein function by selectively suppressing translation termination at PTCs. Poor efficacy of current readthrough agents prompted us to search for better compounds. An NMD-sensitive NanoLuc readthrough reporter was used to screen 771,345 compounds. Among the 180 compounds identified with readthrough activity, SRI-37240 and its more potent derivative SRI-41315, induce a prolonged pause at stop codons and suppress PTCs associated with cystic fibrosis in immortalized and primary human bronchial epithelial cells, restoring CFTR expression and function. SRI-41315 suppresses PTCs by reducing the abundance of the termination factor eRF1. SRI-41315 also potentiates aminoglycoside-mediated readthrough, leading to synergistic increases in CFTR activity. Combining readthrough agents that target distinct components of the translation machinery is a promising treatment strategy for diseases caused by PTCs.


Assuntos
Códon sem Sentido/antagonistas & inibidores , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Células Epiteliais/efeitos dos fármacos , Degradação do RNAm Mediada por Códon sem Sentido , Terminação Traducional da Cadeia Peptídica/efeitos dos fármacos , Fatores de Terminação de Peptídeos/metabolismo , Aminoglicosídeos/metabolismo , Códon sem Sentido/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Células Epiteliais/metabolismo , Genes Reporter , Gentamicinas/farmacologia , Células HEK293 , Humanos , Microssomos Hepáticos/efeitos dos fármacos , Fatores de Terminação de Peptídeos/genética , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Interferência de RNA , Ribossomos/metabolismo , Relação Estrutura-Atividade
13.
ACS Chem Biol ; 16(8): 1469-1481, 2021 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-34328734

RESUMO

The programmed -1 ribosomal frameshifting element (PFSE) of SARS-CoV-2 is a well conserved structured RNA found in all coronaviruses' genomes. By adopting a pseudoknot structure in the presence of the ribosome, the PFSE promotes a ribosomal frameshifting event near the stop codon of the first open reading frame Orf1a during translation of the polyprotein pp1a. Frameshifting results in continuation of pp1a via a new open reading frame, Orf1b, that produces the longer pp1ab polyprotein. Polyproteins pp1a and pp1ab produce nonstructural proteins NSPs 1-10 and NSPs 1-16, respectively, which contribute vital functions during the viral life cycle and must be present in the proper stoichiometry. Both drugs and sequence alterations that affect the stability of the -1 programmed ribosomal frameshifting element disrupt the stoichiometry of the NSPs produced, which compromise viral replication. For this reason, the -1 programmed frameshifting element is considered a promising drug target. Using chaperone assisted RNA crystallography, we successfully crystallized and solved the three-dimensional structure of the PFSE. We observe a three-stem H-type pseudoknot structure with the three stems stacked in a vertical orientation stabilized by two triple base pairs at the stem 1/stem 2 and stem 1/stem 3 junctions. This structure provides a new conformation of PFSE distinct from the bent conformations inferred from midresolution cryo-EM models and provides a high-resolution framework for mechanistic investigations and structure-based drug design.


Assuntos
Cristalografia/métodos , Mudança da Fase de Leitura do Gene Ribossômico/fisiologia , Chaperonas Moleculares , RNA Viral/metabolismo , SARS-CoV-2/metabolismo , Humanos , Modelos Moleculares , Conformação de Ácido Nucleico , RNA Viral/genética , Ribossomos/metabolismo , SARS-CoV-2/genética , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/metabolismo , Replicação Viral/fisiologia
14.
Methods Mol Biol ; 2314: 151-166, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34235651

RESUMO

Zinc starvation in Mycobacterium smegmatis and Mycobacterium tuberculosis induces ribosome remodeling and hibernation. Remodeling involves replacement of C+ ribosomal (r-) proteins containing the zinc-binding CXXC motif with their C- paralogues without the motif. Hibernation is characterized by binding of mycobacterial-specific protein Y (Mpy) to 70S C- ribosomes, stabilizing the ribosome in an inactive state that is also resistant to kanamycin and streptomycin. We observed that ribosome remodeling and hibernation occur at two different concentrations of cellular zinc. Here, we describe the methods to purify hibernating and active forms of C- ribosomes from zinc-starved mycobacteria, along with purification of C+ ribosomes from zinc-rich mycobacterial cells. In vitro analysis of these distinct types of ribosomes will facilitate screening of small molecule inhibitors of ribosome hibernation for improved therapeutics against mycobacterial infections.


Assuntos
Proteínas de Bactérias/metabolismo , Mycobacterium smegmatis/crescimento & desenvolvimento , Mycobacterium tuberculosis/crescimento & desenvolvimento , Polirribossomos/metabolismo , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo , Zinco/deficiência , Farmacorresistência Bacteriana , Mycobacterium smegmatis/efeitos dos fármacos , Mycobacterium smegmatis/metabolismo , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/metabolismo , RNA Ribossômico/metabolismo
15.
Chimia (Aarau) ; 75(6): 543-547, 2021 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-34233822

RESUMO

Nature has evolved a remarkable array of biosynthetic enzymes that install diverse chemistries into natural products (NPs), bestowing them with a range of important biological properties that are of considerable therapeutic value. This is epitomized by the ribosomally synthesized and post-translationally modified peptides (RiPPs), a class of peptide natural products that undergo extensive post-translational modifications to produce structurally diverse bioactive peptides. In this review, we provide an overview of our research into the proteusin RiPP family, describing characterized members and the maturation enzymes responsible for their unique chemical structures and biological activities. The diverse enzymology identified in the first two proteusin pathways highlights the enormous potential of the RiPP class for new lead structures and novel pharmacophore-installing maturases as biocatalytic tools for drug discovery efforts.


Assuntos
Produtos Biológicos , Bactérias/metabolismo , Produtos Biológicos/metabolismo , Produtos Biológicos/farmacologia , Peptídeos , Processamento de Proteína Pós-Traducional , Ribossomos/metabolismo
16.
Int J Mol Sci ; 22(11)2021 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-34200244

RESUMO

Ribosome biogenesis is a highly coordinated and complex process that requires numerous assembly factors that ensure prompt and flawless maturation of ribosomal subunits. Despite the increasing amount of data collected, the exact role of most assembly factors and mechanistic details of their operation remain unclear, mainly due to the shortage of high-resolution structural information. Here, using cryo-electron microscopy, we characterized 30S ribosomal particles isolated from an Escherichia coli strain with a deleted gene for the RbfA factor. The cryo-EM maps for pre-30S subunits were divided into six classes corresponding to consecutive assembly intermediates: from the particles with a completely unresolved head domain and unfolded central pseudoknot to almost mature 30S subunits with well-resolved body, platform, and head domains and partially distorted helix 44. The structures of two predominant 30S intermediates belonging to most populated classes obtained at 2.7 Å resolutions indicate that RbfA acts at two distinctive 30S assembly stages: early formation of the central pseudoknot including folding of the head, and positioning of helix 44 in the decoding center at a later stage. Additionally, it was shown that the formation of the central pseudoknot may promote stabilization of the head domain, likely through the RbfA-dependent maturation of the neck helix 28. An update to the model of factor-dependent 30S maturation is proposed, suggesting that RfbA is involved in most of the subunit assembly process.


Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/fisiologia , Proteínas Ribossômicas/metabolismo , Subunidades Ribossômicas Menores de Bactérias/fisiologia , Ribossomos/metabolismo , Sítios de Ligação , Microscopia Crioeletrônica/métodos , Proteínas de Escherichia coli/genética , Modelos Moleculares , Ligação Proteica , Proteínas Ribossômicas/genética , Subunidades Ribossômicas Menores de Bactérias/ultraestrutura , Ribossomos/ultraestrutura
17.
Int J Mol Sci ; 22(11)2021 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-34200446

RESUMO

Ribosome biogenesis is essential for plants to successfully acclimate to low temperature. Without dedicated steps supervising the 60S large subunits (LSUs) maturation in the cytosol, e.g., Rei-like (REIL) factors, plants fail to accumulate dry weight and fail to grow at suboptimal low temperatures. Around REIL, the final 60S cytosolic maturation steps include proofreading and assembly of functional ribosomal centers such as the polypeptide exit tunnel and the P-Stalk, respectively. In consequence, these ribosomal substructures and their assembly, especially during low temperatures, might be changed and provoke the need for dedicated quality controls. To test this, we blocked ribosome maturation during cold acclimation using two independent reil double mutant genotypes and tested changes in their ribosomal proteomes. Additionally, we normalized our mutant datasets using as a blank the cold responsiveness of a wild-type Arabidopsis genotype. This allowed us to neglect any reil-specific effects that may happen due to the presence or absence of the factor during LSU cytosolic maturation, thus allowing us to test for cold-induced changes that happen in the early nucleolar biogenesis. As a result, we report that cold acclimation triggers a reprogramming in the structural ribosomal proteome. The reprogramming alters the abundance of specific RP families and/or paralogs in non-translational LSU and translational polysome fractions, a phenomenon known as substoichiometry. Next, we tested whether the cold-substoichiometry was spatially confined to specific regions of the complex. In terms of RP proteoforms, we report that remodeling of ribosomes after a cold stimulus is significantly constrained to the polypeptide exit tunnel (PET), i.e., REIL factor binding and functional site. In terms of RP transcripts, cold acclimation induces changes in RP families or paralogs that are significantly constrained to the P-Stalk and the ribosomal head. The three modulated substructures represent possible targets of mechanisms that may constrain translation by controlled ribosome heterogeneity. We propose that non-random ribosome heterogeneity controlled by specialized biogenesis mechanisms may contribute to a preferential or ultimately even rigorous selection of transcripts needed for rapid proteome shifts and successful acclimation.


Assuntos
Aclimatação , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Temperatura Baixa , Proteoma/metabolismo , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteoma/análise , Proteínas Ribossômicas/genética , Ribossomos/genética
18.
Int J Mol Sci ; 22(12)2021 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-34201240

RESUMO

Infectious viroid clones consist of dimeric cDNAs used to generate transcripts which mimic the longer-than-unit replication intermediates. These transcripts can be either generated in vitro or produced in vivo by agro-inoculation. We have designed a new plasmid, which allows both inoculation methods, and we have compared them by infecting Solanum lycopersicum and Solanum melongena with clones of Citrus exocortis virod (CEVd), Tomato chlorotic dwarf viroid (TCDVd), and Potato spindle tuber viroid (PSTVd). Our results showed more uniform and severe symptoms in agro-inoculated plants. Viroid accumulation and the proportion of circular and linear forms were different depending on the host and the inoculation method and did not correlate with the symptoms, which correlated with an increase in PR1 induction, accumulation of the defensive signal molecules salicylic (SA) and gentisic (GA) acids, and ribosomal stress in tomato plants. The alteration in ribosome biogenesis was evidenced by both the upregulation of the tomato ribosomal stress marker SlNAC082 and the impairment in 18S rRNA processing, pointing out ribosomal stress as a novel signature of the pathogenesis of nuclear-replicating viroids. In conclusion, this updated binary vector has turned out to be an efficient and reproducible method that will facilitate the studies of viroid-host interactions.


Assuntos
Lycopersicon esculentum/virologia , Doenças das Plantas/virologia , Plasmídeos/administração & dosagem , RNA Viral/genética , Ribossomos/metabolismo , Viroides/classificação , Viroides/isolamento & purificação , Lycopersicon esculentum/genética , Lycopersicon esculentum/metabolismo , Doenças das Plantas/genética , Plasmídeos/genética , Ribossomos/genética , Viroides/patogenicidade
19.
Int J Mol Sci ; 22(13)2021 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-34201772

RESUMO

Nucleolar stress occurs when ribosome production or function declines. Nucleolar stress in stem cells or progenitor cells often leads to disease states called ribosomopathies. Drosophila offers a robust system to explore how nucleolar stress causes cell cycle arrest, apoptosis, or autophagy depending on the cell type. We provide an overview of nucleolar stress in Drosophila by depleting nucleolar phosphoprotein of 140 kDa (Nopp140), a ribosome biogenesis factor (RBF) in nucleoli and Cajal bodies (CBs). The depletion of Nopp140 in eye imaginal disc cells generates eye deformities reminiscent of craniofacial deformities associated with the Treacher Collins syndrome (TCS), a human ribosomopathy. We show the activation of c-Jun N-terminal Kinase (JNK) in Drosophila larvae homozygous for a Nopp140 gene deletion. JNK is known to induce the expression of the pro-apoptotic Hid protein and autophagy factors Atg1, Atg18.1, and Atg8a; thus, JNK is a central regulator in Drosophila nucleolar stress. Ribosome abundance declines upon Nopp140 loss, but unusual cytoplasmic granules accumulate that resemble Processing (P) bodies based on marker proteins, Decapping Protein 1 (DCP1) and Maternal expression at 31B (Me31B). Wild type brain neuroblasts (NBs) express copious amounts of endogenous coilin, but coilin levels decline upon nucleolar stress in most NB types relative to the Mushroom body (MB) NBs. MB NBs exhibit resilience against nucleolar stress as they maintain normal coilin, Deadpan, and EdU labeling levels.


Assuntos
Nucléolo Celular/genética , Corpos Enovelados/patologia , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Ligação a RNA/genética , Estresse Fisiológico , Animais , Sistemas CRISPR-Cas , Corpos Enovelados/genética , Proteínas de Drosophila/antagonistas & inibidores , Drosophila melanogaster/fisiologia , Larva/genética , Larva/crescimento & desenvolvimento , Fosfoproteínas , Proteínas de Ligação a RNA/antagonistas & inibidores , Ribossomos/genética , Ribossomos/metabolismo
20.
Int J Mol Sci ; 22(12)2021 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-34204305

RESUMO

The SARS-CoV-2 Spike glycoprotein (S protein) acquired a unique new 4 amino acid -PRRA- insertion sequence at amino acid residues (aa) 681-684 that forms a new furin cleavage site in S protein as well as several new glycosylation sites. We studied various statistical properties of the -PRRA- insertion at the RNA level (CCUCGGCGGGCA). The nucleotide composition and codon usage of this sequence are different from the rest of the SARS-CoV-2 genome. One of such features is two tandem CGG codons, although the CGG codon is the rarest codon in the SARS-CoV-2 genome. This suggests that the insertion sequence could cause ribosome pausing as the result of these rare codons. Due to population variants, the Nextstrain divergence measure of the CCU codon is extremely large. We cannot exclude that this divergence might affect host immune responses/effectiveness of SARS-CoV-2 vaccines, possibilities awaiting further investigation. Our experimental studies show that the expression level of original RNA sequence "wildtype" spike protein is much lower than for codon-optimized spike protein in all studied cell lines. Interestingly, the original spike sequence produces a higher titer of pseudoviral particles and a higher level of infection. Further mutagenesis experiments suggest that this dual-effect insert, comprised of a combination of overlapping translation pausing and furin sites, has allowed SARS-CoV-2 to infect its new host (human) more readily. This underlines the importance of ribosome pausing to allow efficient regulation of protein expression and also of cotranslational subdomain folding.


Assuntos
RNA Viral/metabolismo , Ribossomos/metabolismo , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/genética , Animais , Sequência de Bases , Células COS , COVID-19/patologia , COVID-19/virologia , Chlorocebus aethiops , Uso do Códon , Células HEK293 , Humanos , Mutagênese , SARS-CoV-2/isolamento & purificação , Alinhamento de Sequência , Glicoproteína da Espícula de Coronavírus/metabolismo
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