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1.
Life Sci Alliance ; 7(8)2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38858088

RESUMO

The signal recognition particle is essential for targeting transmembrane and secreted proteins to the endoplasmic reticulum. Remarkably, because they work together in the cytoplasm, the SRP and ribosomes are assembled in the same biomolecular condensate: the nucleolus. How important is the nucleolus for SRP assembly is not known. Using quantitative proteomics, we have investigated the interactomes of SRP components. We reveal that SRP proteins are associated with scores of nucleolar proteins important for ribosome biogenesis and nucleolar structure. Having monitored the subcellular distribution of SRP proteins upon controlled nucleolar disruption, we conclude that an intact organelle is required for their proper localization. Lastly, we have detected two SRP proteins in Cajal bodies, which indicates that previously undocumented steps of SRP assembly may occur in these bodies. This work highlights the importance of a structurally and functionally intact nucleolus for efficient SRP production and suggests that the biogenesis of SRP and ribosomes may be coordinated in the nucleolus by common assembly factors.


Assuntos
Nucléolo Celular , Proteômica , Ribossomos , Partícula de Reconhecimento de Sinal , Partícula de Reconhecimento de Sinal/metabolismo , Nucléolo Celular/metabolismo , Ribossomos/metabolismo , Humanos , Proteômica/métodos , Proteínas Nucleares/metabolismo , Corpos Enovelados/metabolismo , Células HeLa , Retículo Endoplasmático/metabolismo
2.
Nat Genet ; 55(12): 2160-2174, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-38049665

RESUMO

Whole-genome sequencing of longitudinal tumor pairs representing transformation of follicular lymphoma to high-grade B cell lymphoma with MYC and BCL2 rearrangements (double-hit lymphoma) identified coding and noncoding genomic alterations acquired during lymphoma progression. Many of these transformation-associated alterations recurrently and focally occur at topologically associating domain resident regulatory DNA elements, including H3K4me3 promoter marks located within H3K27ac super-enhancer clusters in B cell non-Hodgkin lymphoma. One region found to undergo recurrent alteration upon transformation overlaps a super-enhancer affecting the expression of the PAX5/ZCCHC7 gene pair. ZCCHC7 encodes a subunit of the Trf4/5-Air1/2-Mtr4 polyadenylation-like complex and demonstrated copy number gain, chromosomal translocation and enhancer retargeting-mediated transcriptional upregulation upon lymphoma transformation. Consequently, lymphoma cells demonstrate nucleolar dysregulation via altered noncoding 5.8S ribosomal RNA processing. We find that a noncoding mutation acquired during lymphoma progression affects noncoding rRNA processing, thereby rewiring protein synthesis leading to oncogenic changes in the lymphoma proteome.


Assuntos
Linfoma de Células B , Linfoma , Humanos , Mutação , Linfoma de Células B/genética , Linfoma de Células B/patologia , Translocação Genética/genética , Linfoma/genética , Sequências Reguladoras de Ácido Nucleico
3.
J Extracell Vesicles ; 12(10): e12365, 2023 10.
Artigo em Inglês | MEDLINE | ID: mdl-37807017

RESUMO

Formation of extracellular vesicles (EVs) has emerged as a novel paradigm in cell-to-cell communication in health and disease. EVs are notably produced during cell death but it had remained unclear whether different modalities of regulated cell death (RCD) influence the biogenesis and composition of EVs. To this end, we performed a comparative analysis of steady-state (ssEVs) and cell death-associated EVs (cdEVs) following TNF-induced necroptosis (necEVs), anti-Fas-induced apoptosis (apoEVs), and ML162-induced ferroptosis (ferEVs) using the same cell line. For each RCD condition, we determined the biophysical and biochemical characteristics of the cell death-associated EVs (cdEVs), the protein cargo, and the presence of methylated ribosomal RNA. We found that the global protein content of all cdEVs was increased compared to steady-state EVs. Qualitatively, the isolated exosomal ssEVs and cdEVs, contained a largely overlapping protein cargo including some quantitative differences in particular proteins. All cdEVs were enriched for proteins involved in RNA splicing and nuclear export, and showed distinctive rRNA methylation patterns compared to ssEVs. Interestingly, necEVs and apoEVs, but strikingly not ferEVs, showed enrichment of proteins involved in ribosome biogenesis. Altogether, our work documents quantitative and qualitative differences between ssEVs and cdEVs.


Assuntos
Vesículas Extracelulares , Ferroptose , Vesículas Extracelulares/metabolismo , Necroptose , Proteínas/metabolismo , Apoptose
4.
RNA Biol ; 20(1): 652-665, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-37635368

RESUMO

Ribosomal RNAs are decorated by numerous post-transcriptional modifications whose exact roles in ribosome biogenesis, function, and human pathophysiology remain largely unknown. Here, we report a targeted direct rRNA sequencing approach involving a substrate selection step and demonstrate its suitability to identify differential modification sites in combination with the JACUSA2 software. We compared JACUSA2 to other tools designed for RNA modification detection and show that JACUSA2 outperforms other software with regard to detection of base modifications such as methylation, acetylation and aminocarboxypropylation. To illustrate its widespread usability, we applied our method to a collection of CRISPR-Cas9 engineered colon carcinoma cells lacking specific enzymatic activities responsible for particular rRNA modifications and systematically compared them to isogenic wild-type RNAs. Besides the numerous 2'-O methylated riboses and pseudouridylated residues, our approach was suitable to reliably identify differential base methylation and acetylation events. Importantly, our method does not require any prior knowledge of modification sites or the need to train complex models. We further report for the first time detection of human rRNA modifications by direct RNA-sequencing on Flongle flow cells, the smallest-scale nanopore flow cell available to date. The use of these smaller flow cells reduces RNA input requirements, making our workflow suitable for the analysis of samples with limited availability and clinical work.


Assuntos
Nanoporos , RNA , Humanos , RNA/genética , Ribossomos/genética , RNA Ribossômico/genética , Processamento Pós-Transcricional do RNA
5.
Animals (Basel) ; 12(18)2022 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-36139214

RESUMO

Consistent inter-individual variation in the propensity to perform different tasks (animal personality) can contribute significantly to the success of group-living organisms. The distribution of different personalities in a group influences collective actions and therefore how these organisms interact with their environment. However, we have little understanding of the proximate mechanisms underlying animal personality in animal groups, and research on this theme has often been biased towards organisms with advanced social systems. The goal of this study is to investigate the mechanistic basis for personality variation during collective behaviour in a species with rudimentary societies: the American cockroach. We thus use an approach which combines experimental classification of individuals into behavioural phenotypes ('bold' and 'shy' individuals) with comparative gene expression. Our analyses reveal differences in gene expression between behavioural phenotypes and suggest that social context may modulate gene expression related to bold/shy characteristics. We also discuss how cockroaches could be a valuable model for the study of genetic mechanisms underlying the early steps in the evolution of social behaviour and social complexity. This study provides a first step towards a better understanding of the molecular mechanisms associated with differences in boldness and behavioural plasticity in these organisms.

6.
Blood ; 139(21): 3111-3126, 2022 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-35213692

RESUMO

The congenital bone marrow failure syndrome Diamond-Blackfan anemia (DBA) is typically associated with variants in ribosomal protein (RP) genes impairing erythroid cell development. Here we report multiple individuals with biallelic HEATR3 variants exhibiting bone marrow failure, short stature, facial and acromelic dysmorphic features, and intellectual disability. These variants destabilize a protein whose yeast homolog is known to synchronize the nuclear import of RPs uL5 (RPL11) and uL18 (RPL5), which are both critical for producing ribosomal subunits and for stabilizing the p53 tumor suppressor when ribosome biogenesis is compromised. Expression of HEATR3 variants or repression of HEATR3 expression in primary cells, cell lines of various origins, and yeast models impairs growth, differentiation, pre-ribosomal RNA processing, and ribosomal subunit formation reminiscent of DBA models of large subunit RP gene variants. Consistent with a role of HEATR3 in RP import, HEATR3-depleted cells or patient-derived fibroblasts display reduced nuclear accumulation of uL18. Hematopoietic progenitor cells expressing HEATR3 variants or small-hairpin RNAs knocking down HEATR3 synthesis reveal abnormal acceleration of erythrocyte maturation coupled to severe proliferation defects that are independent of p53 activation. Our study uncovers a new pathophysiological mechanism leading to DBA driven by biallelic HEATR3 variants and the destabilization of a nuclear import protein important for ribosome biogenesis.


Assuntos
Anemia de Diamond-Blackfan , Proteínas , Transporte Ativo do Núcleo Celular/genética , Anemia de Diamond-Blackfan/metabolismo , Humanos , Mutação , Proteínas/genética , Proteínas/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
7.
PLoS Genet ; 18(1): e1010012, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-35041640

RESUMO

Ribosomes are essential nanomachines responsible for protein production. Although ribosomes are present in every living cell, ribosome biogenesis dysfunction diseases, called ribosomopathies, impact particular tissues specifically. Here, we evaluate the importance of the box C/D snoRNA-associated ribosomal RNA methyltransferase fibrillarin (Fbl) in the early embryonic development of Xenopus laevis. We report that in developing embryos, the neural plate, neural crest cells (NCCs), and NCC derivatives are rich in fbl transcripts. Fbl knockdown leads to striking morphological defects affecting the eyes and craniofacial skeleton, due to lack of NCC survival caused by massive p53-dependent apoptosis. Fbl is required for efficient pre-rRNA processing and 18S rRNA production, which explains the early developmental defects. Using RiboMethSeq, we systematically reinvestigated ribosomal RNA 2'-O methylation in X. laevis, confirming all 89 previously mapped sites and identifying 15 novel putative positions in 18S and 28S rRNA. Twenty-three positions, including 10 of the new ones, were validated orthogonally by low dNTP primer extension. Bioinformatic screening of the X. laevis transcriptome revealed candidate box C/D snoRNAs for all methylated positions. Mapping of 2'-O methylation at six developmental stages in individual embryos indicated a trend towards reduced methylation at specific positions during development. We conclude that fibrillarin knockdown in early Xenopus embryos causes reduced production of functional ribosomal subunits, thus impairing NCC formation and migration.


Assuntos
Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Precursores de RNA/metabolismo , RNA Ribossômico 18S/metabolismo , RNA Ribossômico 28S/metabolismo , Xenopus laevis/crescimento & desenvolvimento , Animais , Olho/crescimento & desenvolvimento , Olho/metabolismo , Técnicas de Silenciamento de Genes , Metilação , Crista Neural/crescimento & desenvolvimento , Crista Neural/metabolismo , Placa Neural/crescimento & desenvolvimento , Placa Neural/metabolismo , Processamento Pós-Transcricional do RNA , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevis/genética
8.
Mol Cell ; 82(2): 404-419.e9, 2022 01 20.
Artigo em Inglês | MEDLINE | ID: mdl-34798057

RESUMO

The epitranscriptome has emerged as a new fundamental layer of control of gene expression. Nevertheless, the determination of the transcriptome-wide occupancy and function of RNA modifications remains challenging. Here we have developed Rho-seq, an integrated pipeline detecting a range of modifications through differential modification-dependent rhodamine labeling. Using Rho-seq, we confirm that the reduction of uridine to dihydrouridine (D) by the Dus reductase enzymes targets tRNAs in E. coli and fission yeast. We find that the D modification is also present on fission yeast mRNAs, particularly those encoding cytoskeleton-related proteins, which is supported by large-scale proteome analyses and ribosome profiling. We show that the α-tubulin encoding mRNA nda2 undergoes Dus3-dependent dihydrouridylation, which affects its translation. The absence of the modification on nda2 mRNA strongly impacts meiotic chromosome segregation, resulting in low gamete viability. Applying Rho-seq to human cells revealed that tubulin mRNA dihydrouridylation is evolutionarily conserved.


Assuntos
Segregação de Cromossomos , Escherichia coli/genética , Meiose , Processamento Pós-Transcricional do RNA , RNA Bacteriano/genética , RNA Fúngico/genética , RNA Mensageiro/genética , Schizosaccharomyces/genética , Uridina/metabolismo , Cromossomos Bacterianos , Cromossomos Fúngicos , Cromossomos Humanos , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Evolução Molecular , Células HCT116 , Humanos , Oxirredução , RNA Bacteriano/metabolismo , RNA Fúngico/metabolismo , RNA Mensageiro/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/metabolismo , Análise de Sequência de RNA , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo
9.
Cell Death Dis ; 12(12): 1139, 2021 12 08.
Artigo em Inglês | MEDLINE | ID: mdl-34880223

RESUMO

Transcriptional and cellular-stress surveillance deficits are hallmarks of Huntington's disease (HD), a fatal autosomal-dominant neurodegenerative disorder caused by a pathological expansion of CAG repeats in the Huntingtin (HTT) gene. The nucleolus, a dynamic nuclear biomolecular condensate and the site of ribosomal RNA (rRNA) transcription, is implicated in the cellular stress response and in protein quality control. While the exact pathomechanisms of HD are still unclear, the impact of nucleolar dysfunction on HD pathophysiology in vivo remains elusive. Here we identified aberrant maturation of rRNA and decreased translational rate in association with human mutant Huntingtin (mHTT) expression. The protein nucleophosmin 1 (NPM1), important for nucleolar integrity and rRNA maturation, loses its prominent nucleolar localization. Genetic disruption of nucleolar integrity in vulnerable striatal neurons of the R6/2 HD mouse model decreases the distribution of mHTT in a disperse state in the nucleus, exacerbating motor deficits. We confirmed NPM1 delocalization in the gradually progressing zQ175 knock-in HD mouse model: in the striatum at a presymptomatic stage and in the skeletal muscle at an early symptomatic stage. In Huntington's patient skeletal muscle biopsies, we found a selective redistribution of NPM1, similar to that in the zQ175 model. Taken together, our study demonstrates that nucleolar integrity regulates the formation of mHTT inclusions in vivo, and identifies NPM1 as a novel, readily detectable peripheral histopathological marker of HD progression.


Assuntos
Doença de Huntington , Animais , Corpo Estriado/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Humanos , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Doença de Huntington/metabolismo , Camundongos , Neurônios/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo
10.
Nat Commun ; 12(1): 6648, 2021 11 17.
Artigo em Inglês | MEDLINE | ID: mdl-34789764

RESUMO

The U6 snRNA, the core catalytic component of the spliceosome, is extensively modified post-transcriptionally, with 2'-O-methylation being most common. However, how U6 2'-O-methylation is regulated remains largely unknown. Here we report that TFIP11, the human homolog of the yeast spliceosome disassembly factor Ntr1, localizes to nucleoli and Cajal Bodies and is essential for the 2'-O-methylation of U6. Mechanistically, we demonstrate that TFIP11 knockdown reduces the association of U6 snRNA with fibrillarin and associated snoRNAs, therefore altering U6 2'-O-methylation. We show U6 snRNA hypomethylation is associated with changes in assembly of the U4/U6.U5 tri-snRNP leading to defects in spliceosome assembly and alterations in splicing fidelity. Strikingly, this function of TFIP11 is independent of the RNA helicase DHX15, its known partner in yeast. In sum, our study demonstrates an unrecognized function for TFIP11 in U6 snRNP modification and U4/U6.U5 tri-snRNP assembly, identifying TFIP11 as a critical spliceosome assembly regulator.


Assuntos
Fatores de Processamento de RNA/metabolismo , Splicing de RNA/fisiologia , RNA Nuclear Pequeno/metabolismo , Ribonucleoproteína Nuclear Pequena U4-U6/metabolismo , Ribonucleoproteína Nuclear Pequena U5/metabolismo , Nucléolo Celular/metabolismo , Sobrevivência Celular , Corpos Enovelados/metabolismo , Células HeLa , Humanos , Metilação , Mitose , Proteínas Nucleares/metabolismo , Salpicos Nucleares/metabolismo , Ligação Proteica , Estabilidade Proteica , Precursores de RNA/metabolismo , Fatores de Processamento de RNA/genética , RNA Nucleolar Pequeno/metabolismo , Spliceossomos/metabolismo
11.
Nature ; 600(7889): 536-542, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34819669

RESUMO

The cell is a multi-scale structure with modular organization across at least four orders of magnitude1. Two central approaches for mapping this structure-protein fluorescent imaging and protein biophysical association-each generate extensive datasets, but of distinct qualities and resolutions that are typically treated separately2,3. Here we integrate immunofluorescence images in the Human Protein Atlas4 with affinity purifications in BioPlex5 to create a unified hierarchical map of human cell architecture. Integration is achieved by configuring each approach as a general measure of protein distance, then calibrating the two measures using machine learning. The map, known as the multi-scale integrated cell (MuSIC 1.0), resolves 69 subcellular systems, of which approximately half are to our knowledge undocumented. Accordingly, we perform 134 additional affinity purifications and validate subunit associations for the majority of systems. The map reveals a pre-ribosomal RNA processing assembly and accessory factors, which we show govern rRNA maturation, and functional roles for SRRM1 and FAM120C in chromatin and RPS3A in splicing. By integration across scales, MuSIC increases the resolution of imaging while giving protein interactions a spatial dimension, paving the way to incorporate diverse types of data in proteome-wide cell maps.


Assuntos
Cromossomos , Proteoma , Antígenos Nucleares/genética , Antígenos Nucleares/metabolismo , Cromatina/genética , Cromossomos/metabolismo , Humanos , Proteínas Associadas à Matriz Nuclear/metabolismo , Proteoma/metabolismo , RNA Ribossômico , Proteínas de Ligação a RNA/genética
12.
Genes Dev ; 35(15-16): 1123-1141, 2021 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-34301768

RESUMO

Spliceosomal small nuclear RNAs (snRNAs) are modified by small Cajal body (CB)-specific ribonucleoproteins (scaRNPs) to ensure snRNP biogenesis and pre-mRNA splicing. However, the function and subcellular site of snRNA modification are largely unknown. We show that CB localization of the protein Nopp140 is essential for concentration of scaRNPs in that nuclear condensate; and that phosphorylation by casein kinase 2 (CK2) at ∼80 serines targets Nopp140 to CBs. Transiting through CBs, snRNAs are apparently modified by scaRNPs. Indeed, Nopp140 knockdown-mediated release of scaRNPs from CBs severely compromises 2'-O-methylation of spliceosomal snRNAs, identifying CBs as the site of scaRNP catalysis. Additionally, alternative splicing patterns change indicating that these modifications in U1, U2, U5, and U12 snRNAs safeguard splicing fidelity. Given the importance of CK2 in this pathway, compromised splicing could underlie the mode of action of small molecule CK2 inhibitors currently considered for therapy in cholangiocarcinoma, hematological malignancies, and COVID-19.


Assuntos
Células Intersticiais de Cajal/metabolismo , Metilação , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Splicing de RNA , RNA Nuclear Pequeno/metabolismo , Caseína Quinase II/antagonistas & inibidores , Caseína Quinase II/metabolismo , Colangiocarcinoma/tratamento farmacológico , Neoplasias Hematológicas/tratamento farmacológico , Humanos , Fosforilação , RNA Nuclear Pequeno/química , Ribonucleoproteínas/metabolismo , Spliceossomos/genética , Tratamento Farmacológico da COVID-19
13.
bioRxiv ; 2021 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-33948588

RESUMO

Spliceosomal small nuclear RNAs (snRNAs) are modified by small Cajal body (CB) specific ribonucleoproteins (scaRNPs) to ensure snRNP biogenesis and pre-mRNA splicing. However, the function and subcellular site of snRNA modification are largely unknown. We show that CB localization of the protein Nopp140 is essential for concentration of scaRNPs in that nuclear condensate; and that phosphorylation by casein kinase 2 (CK2) at some 80 serines targets Nopp140 to CBs. Transiting through CBs, snRNAs are apparently modified by scaRNPs. Indeed, Nopp140 knockdown-mediated release of scaRNPs from CBs severely compromises 2'-O-methylation of spliceosomal snRNAs, identifying CBs as the site of scaRNP catalysis. Additionally, alternative splicing patterns change indicating that these modifications in U1, U2, U5, and U12 snRNAs safeguard splicing fidelity. Given the importance of CK2 in this pathway, compromised splicing could underlie the mode of action of small molecule CK2 inhibitors currently considered for therapy in cholangiocarcinoma, hematological malignancies, and COVID-19.

14.
Elife ; 92020 12 08.
Artigo em Inglês | MEDLINE | ID: mdl-33289480

RESUMO

Our knowledge about the repertoire of ribosomal RNA modifications and the enzymes responsible for installing them is constantly expanding. Previously, we reported that NSUN-5 is responsible for depositing m5C at position C2381 on the 26S rRNA in Caenorhabditis elegans. Here, we show that NSUN-1 is writing the second known 26S rRNA m5C at position C2982. Depletion of nsun-1 or nsun-5 improved thermotolerance and slightly increased locomotion at midlife, however, only soma-specific knockdown of nsun-1 extended lifespan. Moreover, soma-specific knockdown of nsun-1 reduced body size and impaired fecundity, suggesting non-cell-autonomous effects. While ribosome biogenesis and global protein synthesis were unaffected by nsun-1 depletion, translation of specific mRNAs was remodeled leading to reduced production of collagens, loss of structural integrity of the cuticle, and impaired barrier function. We conclude that loss of a single enzyme required for rRNA methylation has profound and highly specific effects on organismal development and physiology.


Assuntos
Envelhecimento/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Longevidade/fisiologia , Metiltransferases/metabolismo , Animais , Caenorhabditis elegans , Feminino , Fertilidade/fisiologia , Oogênese/fisiologia , Processamento Pós-Transcricional do RNA/fisiologia
15.
Nucleic Acids Res ; 48(21): 12310-12325, 2020 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-33166396

RESUMO

The Mtq2-Trm112 methyltransferase modifies the eukaryotic translation termination factor eRF1 on the glutamine side chain of a universally conserved GGQ motif that is essential for release of newly synthesized peptides. Although this modification is found in the three domains of life, its exact role in eukaryotes remains unknown. As the deletion of MTQ2 leads to severe growth impairment in yeast, we have investigated its role further and tested its putative involvement in ribosome biogenesis. We found that Mtq2 is associated with nuclear 60S subunit precursors, and we demonstrate that its catalytic activity is required for nucleolar release of pre-60S and for efficient production of mature 5.8S and 25S rRNAs. Thus, we identify Mtq2 as a novel ribosome assembly factor important for large ribosomal subunit formation. We propose that Mtq2-Trm112 might modify eRF1 in the nucleus as part of a quality control mechanism aimed at proof-reading the peptidyl transferase center, where it will subsequently bind during translation termination.


Assuntos
Regulação Fúngica da Expressão Gênica , Metiltransferases/genética , Biogênese de Organelas , Fatores de Terminação de Peptídeos/genética , Subunidades Ribossômicas Maiores de Eucariotos/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , tRNA Metiltransferases/genética , Sítios de Ligação , Biocatálise , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Metiltransferases/química , Metiltransferases/metabolismo , Modelos Moleculares , Terminação Traducional da Cadeia Peptídica , Fatores de Terminação de Peptídeos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , RNA Ribossômico/biossíntese , RNA Ribossômico/genética , RNA Ribossômico 5,8S/biossíntese , RNA Ribossômico 5,8S/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidade por Substrato , tRNA Metiltransferases/química , tRNA Metiltransferases/metabolismo
16.
Nucleic Acids Res ; 48(19): e110, 2020 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-32976574

RESUMO

Developing methods for accurate detection of RNA modifications remains a major challenge in epitranscriptomics. Next-generation sequencing-based mapping approaches have recently emerged but, often, they are not quantitative and lack specificity. Pseudouridine (ψ), produced by uridine isomerization, is one of the most abundant RNA modification. ψ mapping classically involves derivatization with soluble carbodiimide (CMCT), which is prone to variation making this approach only semi-quantitative. Here, we developed 'HydraPsiSeq', a novel quantitative ψ mapping technique relying on specific protection from hydrazine/aniline cleavage. HydraPsiSeq is quantitative because the obtained signal directly reflects pseudouridine level. Furthermore, normalization to natural unmodified RNA and/or to synthetic in vitro transcripts allows absolute measurements of modification levels. HydraPsiSeq requires minute amounts of RNA (as low as 10-50 ng), making it compatible with high-throughput profiling of diverse biological and clinical samples. Exploring the potential of HydraPsiSeq, we profiled human rRNAs, revealing strong variations in pseudouridylation levels at ∼20-25 positions out of total 104 sites. We also observed the dynamics of rRNA pseudouridylation throughout chondrogenic differentiation of human bone marrow stem cells. In conclusion, HydraPsiSeq is a robust approach for the systematic mapping and accurate quantification of pseudouridines in RNAs with applications in disease, aging, development, differentiation and/or stress response.


Assuntos
Pseudouridina/isolamento & purificação , RNA Mensageiro , RNA Ribossômico , RNA de Transferência , Análise de Sequência de RNA/métodos , Células Cultivadas , Humanos , Células-Tronco Mesenquimais , Saccharomyces cerevisiae/genética
17.
Am J Hum Genet ; 106(5): 694-706, 2020 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-32359472

RESUMO

How mutations in the non-coding U8 snoRNA cause the neurological disorder leukoencephalopathy with calcifications and cysts (LCC) is poorly understood. Here, we report the generation of a mutant U8 animal model for interrogating LCC-associated pathology. Mutant U8 zebrafish exhibit defective central nervous system development, a disturbance of ribosomal RNA (rRNA) biogenesis and tp53 activation, which monitors ribosome biogenesis. Further, we demonstrate that fibroblasts from individuals with LCC are defective in rRNA processing. Human precursor-U8 (pre-U8) containing a 3' extension rescued mutant U8 zebrafish, and this result indicates conserved biological function. Analysis of LCC-associated U8 mutations in zebrafish revealed that one null and one functional allele contribute to LCC. We show that mutations in three nucleotides at the 5' end of pre-U8 alter the processing of the 3' extension, and we identify a previously unknown base-pairing interaction between the 5' end and the 3' extension of human pre-U8. Indeed, LCC-associated mutations in any one of seven nucleotides in the 5' end and 3' extension alter the processing of pre-U8, and these mutations are present on a single allele in almost all individuals with LCC identified to date. Given genetic data indicating that bi-allelic null U8 alleles are likely incompatible with human development, and that LCC is not caused by haploinsufficiency, the identification of hypomorphic misprocessing mutations that mediate viable embryogenesis furthers our understanding of LCC molecular pathology and cerebral vascular homeostasis.


Assuntos
Alelos , Calcinose/genética , Cistos do Sistema Nervoso Central/genética , Cistos/genética , Leucoencefalopatias/genética , Mutação , RNA Nucleolar Pequeno/genética , Peixe-Zebra/genética , Animais , Sequência de Bases , Calcinose/patologia , Cistos do Sistema Nervoso Central/patologia , Sequência Conservada , Modelos Animais de Doenças , Desenvolvimento Embrionário/genética , Humanos , Leucoencefalopatias/patologia , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Peixe-Zebra/embriologia , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
18.
EMBO Rep ; 21(7): e49443, 2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32350990

RESUMO

RNA modifications have recently emerged as an important layer of gene regulation. N6-methyladenosine (m6 A) is the most prominent modification on eukaryotic messenger RNA and has also been found on noncoding RNA, including ribosomal and small nuclear RNA. Recently, several m6 A methyltransferases were identified, uncovering the specificity of m6 A deposition by structurally distinct enzymes. In order to discover additional m6 A enzymes, we performed an RNAi screen to deplete annotated orthologs of human methyltransferase-like proteins (METTLs) in Drosophila cells and identified CG9666, the ortholog of human METTL5. We show that CG9666 is required for specific deposition of m6 A on 18S ribosomal RNA via direct interaction with the Drosophila ortholog of human TRMT112, CG12975. Depletion of CG9666 yields a subsequent loss of the 18S rRNA m6 A modification, which lies in the vicinity of the ribosome decoding center; however, this does not compromise rRNA maturation. Instead, a loss of CG9666-mediated m6 A impacts fly behavior, providing an underlying molecular mechanism for the reported human phenotype in intellectual disability. Thus, our work expands the repertoire of m6 A methyltransferases, demonstrates the specialization of these enzymes, and further addresses the significance of ribosomal RNA modifications in gene expression and animal behavior.


Assuntos
Drosophila , Metiltransferases , Adenosina , Animais , Drosophila/genética , Humanos , Metiltransferases/genética , RNA Ribossômico , RNA Ribossômico 18S/genética , Caminhada
19.
Nucleic Acids Res ; 48(7): 3848-3868, 2020 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-31996908

RESUMO

U3 snoRNA and the associated Rrp9/U3-55K protein are essential for 18S rRNA production by the SSU-processome complex. U3 and Rrp9 are required for early pre-rRNA cleavages at sites A0, A1 and A2, but the mechanism remains unclear. Substitution of Arg 289 in Rrp9 to Ala (R289A) specifically reduced cleavage at sites A1 and A2. Surprisingly, R289 is located on the surface of the Rrp9 ß-propeller structure opposite to U3 snoRNA. To understand this, we first characterized the protein-protein interaction network of Rrp9 within the SSU-processome. This identified a direct interaction between the Rrp9 ß-propeller domain and Rrp36, the strength of which was reduced by the R289A substitution, implicating this interaction in the observed processing phenotype. The Rrp9 R289A mutation also showed strong synergistic negative interactions with mutations in U3 that destabilize the U3/pre-rRNA base-pair interactions or reduce the length of their linking segments. We propose that the Rrp9 ß-propeller and U3/pre-rRNA binding cooperate in the structure or stability of the SSU-processome. Additionally, our analysis of U3 variants gave insights into the function of individual segments of the 5'-terminal 72-nt sequence of U3. We interpret these data in the light of recently reported SSU-processome structures.


Assuntos
Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA , RNA Ribossômico 18S/metabolismo , RNA Nucleolar Pequeno/química , Ribonucleoproteínas Nucleolares Pequenas/química , Ribonucleoproteínas Nucleolares Pequenas/metabolismo , Mutação , Proteínas Nucleares/metabolismo , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas , RNA Nucleolar Pequeno/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas Nucleolares Pequenas/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
20.
Proc Natl Acad Sci U S A ; 116(35): 17330-17335, 2019 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-31399547

RESUMO

The nucleolus is a prominent nuclear condensate that plays a central role in ribosome biogenesis by facilitating the transcription and processing of nascent ribosomal RNA (rRNA). A number of studies have highlighted the active viscoelastic nature of the nucleolus, whose material properties and phase behavior are a consequence of underlying molecular interactions. However, the ways in which the material properties of the nucleolus impact its function in rRNA biogenesis are not understood. Here we utilize the Cry2olig optogenetic system to modulate the viscoelastic properties of the nucleolus. We show that above a threshold concentration of Cry2olig protein, the nucleolus can be gelled into a tightly linked, low mobility meshwork. Gelled nucleoli no longer coalesce and relax into spheres but nonetheless permit continued internal molecular mobility of small proteins. These changes in nucleolar material properties manifest in specific alterations in rRNA processing steps, including a buildup of larger rRNA precursors and a depletion of smaller rRNA precursors. We propose that the flux of processed rRNA may be actively tuned by the cell through modulating nucleolar material properties, which suggests the potential of materials-based approaches for therapeutic intervention in ribosomopathies.


Assuntos
Nucléolo Celular/metabolismo , Processamento Pós-Transcricional do RNA/fisiologia , RNA Ribossômico/metabolismo , Animais , Camundongos , Células NIH 3T3 , Optogenética , Xenopus laevis
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