RESUMO
Telomeres are nucleoprotein structures at the end of chromosomes. The telomerase complex, constituted of the catalytic subunit TERT, the RNA matrix hTR and several cofactors, including the H/ACA box ribonucleoproteins Dyskerin, NOP10, GAR1, NAF1 and NHP2, regulates telomere length. In humans, inherited defects in telomere length maintenance are responsible for a wide spectrum of clinical premature aging manifestations including pulmonary fibrosis (PF), dyskeratosis congenita (DC), bone marrow failure and predisposition to cancer. NHP2 mutations have been so far reported only in two patients with DC. Here, we report the first case of Høyeraal-Hreidarsson syndrome, the severe form of DC, caused by biallelic missense mutations in NHP2. Additionally, we identified three unrelated patients with PF carrying NHP2 heterozygous mutations. Strikingly, one of these patients acquired a somatic mutation in the promoter of TERT that likely conferred a selective advantage in a subset of blood cells. Finally, we demonstrate that a functional deficit of human NHP2 affects ribosomal RNA biogenesis. Together, our results broaden the functional consequences and clinical spectrum of NHP2 deficiency.
Assuntos
Disceratose Congênita/patologia , Retardo do Crescimento Fetal/patologia , Deficiência Intelectual/patologia , Microcefalia/patologia , Mutação , Proteínas Nucleares/deficiência , Proteínas Nucleares/genética , Fibrose Pulmonar/patologia , RNA Ribossômico/biossíntese , Ribonucleoproteínas Nucleares Pequenas/deficiência , Ribonucleoproteínas Nucleares Pequenas/genética , Idoso , Sequência de Aminoácidos , Disceratose Congênita/etiologia , Feminino , Retardo do Crescimento Fetal/etiologia , Humanos , Recém-Nascido , Deficiência Intelectual/etiologia , Masculino , Microcefalia/etiologia , Pessoa de Meia-Idade , Proteínas Nucleares/química , Linhagem , Regiões Promotoras Genéticas , Fibrose Pulmonar/etiologia , Ribonucleoproteínas Nucleares Pequenas/química , Homologia de Sequência , Telomerase/genética , Transcrição GênicaRESUMO
Splicing is an essential step in eukaryotic gene expression. While the majority of introns is excised by the U2-dependent, or major class, spliceosome, the appropriate expression of a very small subset of genes depends on U12-dependent, or minor class, splicing. The U11/U12 65K protein (hereafter 65K), encoded by RNPC3, is one of seven proteins that are unique to the U12-dependent spliceosome, and previous studies including our own have established that it plays a role in plant and vertebrate development. To pinpoint the impact of 65K loss during mammalian development and in adulthood, we generated germline and conditional Rnpc3-deficient mice. Homozygous Rnpc3-/- embryos died prior to blastocyst implantation, whereas Rnpc3+/- mice were born at the expected frequency, achieved sexual maturity, and exhibited a completely normal lifespan. Systemic recombination of conditional Rnpc3 alleles in adult (Rnpc3lox/lox ) mice caused rapid weight loss, leukopenia, and degeneration of the epithelial lining of the entire gastrointestinal tract, the latter due to increased cell death and a reduction in cell proliferation. Accompanying this, we observed a loss of both 65K and the pro-proliferative phospho-ERK1/2 proteins from the stem/progenitor cells at the base of intestinal crypts. RT-PCR analysis of RNA extracted from purified preparations of intestinal epithelial cells with recombined Rnpc3lox alleles revealed increased frequency of U12-type intron retention in all transcripts tested. Our study, using a novel conditional mouse model of Rnpc3 deficiency, establishes that U12-dependent splicing is not only important during development but is indispensable throughout life.
Assuntos
Splicing de RNA/genética , Proteínas de Ligação a RNA/genética , Ribonucleoproteínas Nucleares Pequenas/genética , Alelos , Animais , Trato Gastrointestinal/metabolismo , Humanos , Íntrons/genética , Camundongos , RNA Nuclear Pequeno/síntese química , RNA Nuclear Pequeno/genética , Proteínas de Ligação a RNA/química , Ribonucleoproteínas Nucleares Pequenas/química , Ribonucleoproteínas Nucleares Pequenas/deficiência , Spliceossomos/química , Spliceossomos/genéticaRESUMO
How pseudouridylation (Ψ), the most common and evolutionarily conserved modification of rRNA, regulates ribosome activity is poorly understood. Medically, Ψ is important because the rRNA Ψ synthase, DKC1, is mutated in X-linked dyskeratosis congenita (X-DC) and Hoyeraal-Hreidarsson (HH) syndrome. Here, we characterize ribosomes isolated from a yeast strain in which Cbf5p, the yeast homolog of DKC1, is catalytically impaired through a D95A mutation (cbf5-D95A). Ribosomes from cbf5-D95A cells display decreased affinities for tRNA binding to the A and P sites as well as the cricket paralysis virus internal ribosome entry site (IRES), which interacts with both the P and the E sites of the ribosome. This biochemical impairment in ribosome activity manifests as decreased translational fidelity and IRES-dependent translational initiation, which are also evident in mouse and human cells deficient for DKC1 activity. These findings uncover specific roles for Ψ modification in ribosome-ligand interactions that are conserved in yeast, mouse, and humans.
Assuntos
Proteínas de Ciclo Celular/deficiência , Disceratose Congênita/genética , Retardo do Crescimento Fetal/genética , Hidroliases/deficiência , Hidroliases/metabolismo , Deficiência Intelectual/genética , Microcefalia/genética , Proteínas Associadas aos Microtúbulos/deficiência , Proteínas Nucleares/deficiência , RNA Ribossômico/metabolismo , RNA de Transferência/metabolismo , Ribonucleoproteínas Nucleares Pequenas/deficiência , Saccharomyces cerevisiae/genética , Animais , Sítios de Ligação , Proteínas de Ciclo Celular/genética , Disceratose Congênita/enzimologia , Retardo do Crescimento Fetal/enzimologia , Genes Reporter , Humanos , Hidroliases/genética , Deficiência Intelectual/enzimologia , Luciferases/análise , Camundongos , Microcefalia/enzimologia , Proteínas Associadas aos Microtúbulos/genética , Mutação , Proteínas Nucleares/genética , Plasmídeos , Biossíntese de Proteínas , RNA Ribossômico/química , RNA Ribossômico/genética , RNA de Transferência/química , RNA de Transferência/genética , Ribonucleoproteínas Nucleares Pequenas/genética , Ribossomos/química , Ribossomos/metabolismo , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/genética , Homologia de Sequência de Aminoácidos , Transdução GenéticaRESUMO
Alternative splicing of pre-messenger RNAs diversifies gene products in eukaryotes and is guided by factors that enable spliceosomes to recognize particular splice sites. Here we report that alternative splicing of Saccharomyces cerevisiae SRC1 pre-mRNA is promoted by the conserved ubiquitin-like protein Hub1. Structural and biochemical data show that Hub1 binds non-covalently to a conserved element termed HIND, which is present in the spliceosomal protein Snu66 in yeast and mammals, and Prp38 in plants. Hub1 binding mildly alters spliceosomal protein interactions and barely affects general splicing in S. cerevisiae. However, spliceosomes that lack Hub1, or are defective in Hub1-HIND interaction, cannot use certain non-canonical 5' splice sites and are defective in alternative SRC1 splicing. Hub1 confers alternative splicing not only when bound to HIND, but also when experimentally fused to Snu66, Prp38, or even the core splicing factor Prp8. Our study indicates a novel mechanism for splice site utilization that is guided by non-covalent modification of the spliceosome by an unconventional ubiquitin-like modifier.
Assuntos
Processamento Alternativo , Regulação Fúngica da Expressão Gênica , Ligases/metabolismo , Sítios de Splice de RNA/genética , RNA Fúngico/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Linhagem Celular , Deleção de Genes , Humanos , Ligases/deficiência , Ligases/genética , Proteínas de Membrana/genética , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Nucleares/genética , Ligação Proteica , Conformação Proteica , RNA Fúngico/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribonucleoproteína Nuclear Pequena U4-U6/deficiência , Ribonucleoproteína Nuclear Pequena U4-U6/genética , Ribonucleoproteína Nuclear Pequena U5/deficiência , Ribonucleoproteína Nuclear Pequena U5/genética , Ribonucleoproteínas Nucleares Pequenas/química , Ribonucleoproteínas Nucleares Pequenas/deficiência , Ribonucleoproteínas Nucleares Pequenas/genética , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Schizosaccharomyces/química , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Spliceossomos/química , Spliceossomos/metabolismo , Complexos Ubiquitina-Proteína Ligase/deficiência , Complexos Ubiquitina-Proteína Ligase/genética , Complexos Ubiquitina-Proteína Ligase/metabolismo , UbiquitinasRESUMO
Conversion of promoter-proximally paused RNA polymerase II (RNAPII) into elongating polymerase by the positive transcription elongation factor b (P-TEFb) is a central regulatory step of mRNA synthesis. The activity of P-TEFb is controlled mainly by the 7SK small nuclear ribonucleoprotein (snRNP), which sequesters active P-TEFb into inactive 7SK/P-TEFb snRNP. Here we demonstrate that under normal culture conditions, the lack of 7SK snRNP has only minor impacts on global RNAPII transcription without detectable consequences on cell proliferation. However, upon ultraviolet (UV)-light-induced DNA damage, cells lacking 7SK have a defective transcriptional response and reduced viability. Both UV-induced release of "lesion-scanning" polymerases and activation of key early-responsive genes are compromised in the absence of 7SK. Proper induction of 7SK-dependent UV-responsive genes requires P-TEFb activity directly mobilized from the nucleoplasmic 7SK/P-TEFb snRNP. Our data demonstrate that the primary function of the 7SK/P-TEFb snRNP is to orchestrate the proper transcriptional response to stress.
Assuntos
Leucócitos/efeitos da radiação , Fator B de Elongação Transcricional Positiva/genética , RNA Polimerase II/genética , Ribonucleoproteínas Nucleares Pequenas/genética , Transcrição Gênica/efeitos da radiação , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Proliferação de Células/efeitos da radiação , Sobrevivência Celular , Cromatina/química , Cromatina/metabolismo , Cromatina/efeitos da radiação , Dano ao DNA , Deleção de Genes , Regulação da Expressão Gênica , Humanos , Leucócitos/citologia , Leucócitos/metabolismo , Fator B de Elongação Transcricional Positiva/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , RNA Polimerase II/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas Nucleares Pequenas/deficiência , Estresse Fisiológico/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Raios UltravioletaRESUMO
BACKGROUND: Decapping of mRNA is an important step in the regulation of mRNA turnover and therefore of gene expression, which is a key process controlling development and homeostasis of all organisms. It has been shown that EDC3 plays a role in mRNA decapping, however its function is not well understood. Previously, we have associated a homozygous variant in EDC3 with autosomal recessive intellectual disability. Here, we investigate the functional role of EDC3. METHODS: We performed transcriptome analyses in patients' samples. In addition, we established an EDC3 loss-of-function model using siRNA-based knockdown in the human neuroblastoma cell line SKNBE and carried out RNA sequencing. Integrative bioinformatics analyses were performed to identify EDC3-dependent candidate genes and/or pathways. RESULTS: Our analyses revealed that 235 genes were differentially expressed in patients versus controls. In addition, AU-rich element (ARE)-containing mRNAs, whose degradation in humans has been suggested to involve EDC3, had higher fold changes than non-ARE-containing genes. The analysis of RNA sequencing data from the EDC3 in vitro loss-of-function model confirmed the higher fold changes of ARE-containing mRNAs compared to non-ARE-containing mRNAs and further showed an upregulation of long non-coding and coding RNAs. In total, 764 genes were differentially expressed. Integrative bioinformatics analyses of these genes identified dysregulated candidate pathways, including pathways related to synapses/coated vesicles and DNA replication/cell cycle. CONCLUSION: Our data support the involvement of EDC3 in mRNA decay, including ARE-containing mRNAs, and suggest that EDC3 might be preferentially involved in the degradation of long coding and non-coding RNAs. Furthermore, our results associate ECD3 loss-of-function with synapses-related pathways. Collectively, our data provide novel information that might help elucidate the molecular mechanisms underlying the association of intellectual disability with the dysregulation of mRNA degradation.
Assuntos
Biologia Computacional , Deficiência Intelectual/metabolismo , Estabilidade de RNA , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Regulação para Baixo , Sequência Rica em GC , Técnicas de Silenciamento de Genes , Redes Reguladoras de Genes , Humanos , Deficiência Intelectual/genética , RNA Longo não Codificante/genética , Ribonucleoproteínas Nucleares Pequenas/deficiência , Ribonucleoproteínas Nucleares Pequenas/genética , Sinapses/metabolismoRESUMO
Small nuclear ribonucleoproteins (snRNPs) are particles present only in eukaryotic cells. They are involved in a large variety of RNA maturation processes, most notably in pre-mRNA splicing. Several of the proteins typically found in snRNPs contain a sequence signature, the Sm domain, conserved from yeast to mammals. By using a promoter trap strategy to target actively transcribed loci in murine embryonic stem cells, a new murine gene encoding an Sm motif-containing protein was identified. Database searches revealed that it is the mouse orthologue of Lsm4p, a protein found in yeast and human cells and putatively associated with U6 snRNA. Introduction of the geo reporter gene cassette under the control of the murine Lsm4 (mLsm4) endogenous promoter showed that the gene was ubiquitously transcribed in embryonic and adult tissues. The insertion of the geo cassette disrupted the mLsm4 allele, and homozygosity for the mutation led to a recessive embryonic lethal phenotype. mLsm4-null zygotes survived to the blastocyst stages, implanted into the uterus, but died shortly thereafter. The early death of mLsm4p-null mice suggests that the role of mLsm4p in splicing is essential and cannot be compensated by other Lsm proteins.
Assuntos
Implantação do Embrião , Regiões Promotoras Genéticas , RNA Nuclear Pequeno/metabolismo , Ribonucleoproteínas Nucleares Pequenas/genética , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Deleção de Sequência , Sequência de Aminoácidos , Animais , Linhagem Celular , Clonagem Molecular , Desenvolvimento Embrionário e Fetal , Feminino , Morte Fetal , Regulação da Expressão Gênica no Desenvolvimento , Genes Reporter , Humanos , Camundongos , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/biossíntese , Mapeamento por Restrição , Ribonucleoproteínas Nucleares Pequenas/deficiência , Saccharomyces cerevisiae/genética , Schizosaccharomyces/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Células-Tronco/fisiologia , Transcrição Gênica , TransfecçãoRESUMO
Piwi proteins and their associated Piwi-interacting RNAs (piRNAs) are implicated in transposon silencing in the mouse germ line. There is currently little information on additional proteins in the murine Piwi complex and how they might regulate the entry of transcripts that accumulate as piRNAs in the Piwi ribonucleoprotein (piRNP). We isolated Mili-containing complexes from adult mouse testes and identified Tudor domain-containing protein-1 (Tdrd1) as a factor specifically associated with the Mili piRNP throughout spermatogenesis. Complex formation is promoted by the recognition of symmetrically dimethylated arginines at the N terminus of Mili by the tudor domains of Tdrd1. Similar to a Mili mutant, mice lacking Tdrd1 show derepression of L1 transposons accompanied by a loss of DNA methylation at their regulatory elements and delocalization of Miwi2 from the nucleus to the cytoplasm. Finally, we show that Mili piRNPs devoid of Tdrd1 accept the entry of abundant cellular transcripts into the piRNA pathway and accumulate piRNAs with a profile that is drastically different from that of the wild type. Our data suggest that Tdrd1 ensures the entry of correct transcripts into the normal piRNA pool.