RESUMO
One of the earliest steps in metazoan pre-mRNA splicing involves binding of U2 snRNP auxiliary factor (U2AF) 65 KDa subunit to the polypyrimidine (Py) tract and of the 35 KDa subunit to the invariant AG dinucleotide at the intron 3' end. Here we use in vitro and in vivo depletion, as well as reconstitution assays using purified components, to identify hnRNP A1 as an RNA binding protein that allows U2AF to discriminate between pyrimidine-rich RNA sequences followed or not by a 3' splice site AG. Biochemical and NMR data indicate that hnRNP A1 forms a ternary complex with the U2AF heterodimer on AG-containing/uridine-rich RNAs, while it displaces U2AF from non-AG-containing/uridine-rich RNAs, an activity that requires the glycine-rich domain of hnRNP A1. Consistent with the functional relevance of this activity for splicing, proofreading assays reveal a role for hnRNP A1 in U2AF-mediated recruitment of U2 snRNP to the pre-mRNA.
Assuntos
Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/química , Proteínas Nucleares/química , Sítios de Splice de RNA , Ribonucleoproteínas/química , Composição de Bases , Sequência de Bases , Extratos Celulares , Cromatografia de Afinidade , Células HeLa , Ribonucleoproteína Nuclear Heterogênea A1 , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/isolamento & purificação , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/metabolismo , Humanos , Substâncias Macromoleculares/química , Proteínas Nucleares/isolamento & purificação , Proteínas Nucleares/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Precursores de RNA/química , Precursores de RNA/metabolismo , Splicing de RNA , Ribonucleoproteínas/isolamento & purificação , Ribonucleoproteínas/metabolismo , Spliceossomos/química , Fator de Processamento U2AF , Especificidade por SubstratoRESUMO
Small nucleolar and small Cajal body RNAs (snoRNAs and scaRNAs) of the H/ACA box and C/D box type are generated by exonucleolytic shortening of longer precursors. Removal of the last few nucleotides at the 3' end is known to be a distinct step. We report that, in human cells, knock-down of the poly(A) specific ribonuclease (PARN), previously implicated only in mRNA metabolism, causes the accumulation of oligoadenylated processing intermediates of H/ACA box but not C/D box RNAs. In agreement with a role of PARN in snoRNA and scaRNA processing, the enzyme is concentrated in nucleoli and Cajal bodies. Oligo(A) tails are attached to a short stub of intron sequence remaining beyond the mature 3' end of the snoRNAs. The noncanonical poly(A) polymerase PAPD5 is responsible for addition of the oligo(A) tails. We suggest that deadenylation is coupled to clean 3' end trimming, which might serve to enhance snoRNA stability.
Assuntos
Exorribonucleases/metabolismo , RNA Nucleotidiltransferases/metabolismo , RNA Nucleolar Pequeno/química , RNA Nucleolar Pequeno/metabolismo , Sequência de Bases , Catálise , Nucléolo Celular/metabolismo , Corpos Enovelados/metabolismo , Exorribonucleases/genética , Complexo Multienzimático de Ribonucleases do Exossomo , Humanos , Proteínas Nucleares/metabolismo , Motivos de Nucleotídeos , Poliadenilação , Transporte Proteico , Edição de RNARESUMO
The Lusitanian (Microtus lusitanicus) and the Mediterranean (Microtus duodecimcostatus) pine voles are recently diverged sister species endemic of the Iberian Peninsula that can be identified with ecological and morphological characters, but in areas where the 2 species co-occur, species designation may be difficult. Genetic discrimination between M. lusitanicus and M. duodecimcostatus has not been achieved yet possibly because of their estimated recent split and an evolutionary history that includes inter-species gene flow. Following our previous observations on exons 5-7 of the p53 gene, here we analyze the potential use of the p53 genomic region as a discrimination marker of these species by extending our analyses to several kb upstream and downstream of the p53 gene and characterizing the degree of genetic differentiation in 7 markers within this region. Additionally, we fully sequenced the P53 protein of both species. We observed: (i) generally high differentiation in this region; (ii) M. duodecimcostatus showed in general higher values of nucleotide and haplotype diversities; (iii) the concatenated phylogenetic tree separates the 2 species; (iv) the 2 P53 proteins only differ in 1 amino acid; (v) 4 of the markers, 2 in p53, one in Atp1b2, and another in Wrap53, contain species-specific genetic variation thus allowing a reliable discrimination between specimens from both species, irrespective of sampling location or introgression status. We provide additional data on the putative role of p53 in the evolution of these species and present researchers with a fast and cost-effective resource for M. lusitanicus and M. duodecimcostatus identification.
Assuntos
Arvicolinae , Proteína Supressora de Tumor p53 , Animais , Arvicolinae/genética , Filogenia , Proteína Supressora de Tumor p53/genética , Especificidade da Espécie , Variação GenéticaRESUMO
Hepatitis delta virus (HDV) is the agent responsible for the most severe form of human viral hepatitis. The HDV genome consists of a single-stranded circular RNA molecule that encodes for one single protein, the delta antigen. Given its simplicity, HDV must make use of several host cellular proteins to accomplish its life cycle processes, including transcription, replication, post-transcriptional, and post-translational modifications. Consequently, identification of the interactions established between HDV components and host proteins assumes a pivotal interest in the search of novel therapeutic targets. Here, we used the yeast three-hybrid system to screen a human liver cDNA library to identify host proteins that interact with the HDV genomic RNA. One of the identified proteins corresponded to the splicing factor SF3B155, a component of the U2snRNP complex that is essential for the early recognition of 3' splice sites in the pre-mRNAs of human genes. We show that the interaction between the HDV genomic RNA and SF3B155 occurs in vivo and that the expression of HDV promotes changes in splicing of human genes whose alternative splicing is SF3B155-dependent. We further show that expression of HDV triggers alterations in several constitutive and alternative splicing events in the tumor suppressor RBM5 transcript, with consequent reduction of its protein levels. This is the first description that HDV expression promotes changes in the splicing of human genes, and we suggest that the HDV-induced alternative splicing changes, through SF3B155 sequester, may contribute for the early progression to hepatocellular carcinoma characteristic of HDV-infected patients.
Assuntos
Ciclo Celular/genética , Genes cdc , Hepatite D/genética , Vírus Delta da Hepatite/fisiologia , Fosfoproteínas/genética , Precursores de RNA/genética , Fatores de Processamento de RNA/genética , Splicing de RNA/genética , Carcinoma Hepatocelular/virologia , Transformação Celular Neoplásica/genética , Cocarcinogênese/genética , Coinfecção/genética , Humanos , Neoplasias Hepáticas/virologia , Fator de Transcrição STAT3/biossíntese , Fator de Transcrição STAT3/genéticaRESUMO
After being released from transcription sites, messenger ribonucleoprotein particles (mRNPs) must reach the nuclear pore complexes in order to be translocated to the cytoplasm. Whether the intranuclear movement of mRNPs results largely from Brownian motion or involves molecular motors remains unknown. Here we have used quantitative photobleaching techniques to monitor the intranuclear mobility of protein components of mRNPs tagged with GFP. The results show that the diffusion coefficients of the poly(A)-binding protein II (PABP2) and the export factor TAP are significantly reduced when these proteins are bound to mRNP complexes, as compared with nonbound proteins. The data further show that the mobility of wild-type PABP2 and TAP, but not of a point mutant variant of PABP2 that fails to bind to RNA, is significantly reduced when cells are ATP depleted or incubated at 22 degrees C. Energy depletion has only minor effects on the intranuclear mobility of a 2,000-kD dextran (which corresponds approximately in size to 40S mRNP particles), suggesting that the reduced mobility of PABP2 and TAP is not caused by a general alteration of the nuclear environment. Taken together, the data suggest that the mobility of mRNPs in the living cell nucleus involves a combination of passive diffusion and ATP-dependent processes.
Assuntos
Trifosfato de Adenosina/fisiologia , Núcleo Celular/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transporte Ativo do Núcleo Celular , Trifosfato de Adenosina/metabolismo , Anticorpos Monoclonais/imunologia , Ligação Competitiva , Dactinomicina/farmacologia , Dextranos/farmacologia , Proteínas de Fluorescência Verde , Células HeLa , Humanos , Cinética , Proteínas Luminescentes/metabolismo , Fotodegradação , Mutação Puntual , Estrutura Terciária de Proteína , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Sensibilidade e Especificidade , Temperatura , Fatores de Tempo , Células Tumorais CultivadasRESUMO
Nuclear bodies represent a heterogeneous class of nuclear structures. Herein, we describe that a subset of nuclear bodies is highly enriched in components of the ubiquitin-proteasome pathway of proteolysis. We coined the term clastosome (from the Greek klastos, broken and soma, body) to refer to this type of nuclear body. Clastosomes contain a high concentration of 1) ubiquitin conjugates, 2) the proteolytically active 20S core and the 19S regulatory complexes of the 26S proteasome, and 3) protein substrates of the proteasome. Although detected in a variety of cell types, clastosomes are scarce under normal conditions; however, they become more abundant when proteasomal activity is stimulated. In contrast, clastosomes disappear when cells are treated with proteasome inhibitors. Protein substrates of the proteasome that are found concentrated in clastosomes include the short-lived transcription factors c-Fos and c-Jun, adenovirus E1A proteins, and the PML protein. We propose that clastosomes are sites where proteolysis of a variety of protein substrates is taking place.
Assuntos
Estruturas do Núcleo Celular/metabolismo , Cisteína Endopeptidases/metabolismo , Complexos Multienzimáticos/metabolismo , Proteínas Nucleares/metabolismo , Subunidades Proteicas/metabolismo , Ubiquitina/metabolismo , Animais , Animais Recém-Nascidos , Estruturas do Núcleo Celular/química , Estruturas do Núcleo Celular/ultraestrutura , Células Cultivadas , Inibidores Enzimáticos/metabolismo , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Imuno-Histoquímica , Masculino , Complexos Multienzimáticos/antagonistas & inibidores , Proteínas de Neoplasias/metabolismo , Proteína da Leucemia Promielocítica , Complexo de Endopeptidases do Proteassoma , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-jun/genética , Proteínas Proto-Oncogênicas c-jun/metabolismo , Ratos , Ratos Sprague-Dawley , Células de Schwann/citologia , Células de Schwann/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Supressoras de TumorRESUMO
AIM: To further characterize the structure and nucleic acid binding properties of the 195 amino acid small delta antigen, S-HDAg, a study was made of a truncated form of S-HDAg, comprising amino acids 61-195 (∆60HDAg), thus lacking the domain considered necessary for dimerization and higher order multimerization. METHODS: Circular dichroism, and nuclear magnetic resonance experiments were used to assess the structure of ∆60HDAg. Nucleic acid binding properties were investigated by gel retardation assays. RESULTS: Results showed that the truncated ∆60HDAg protein is intrinsically disordered but compact, whereas the RNA binding domain, comprising residues 94-146, adopts a dynamic helical conformation. We also found that ∆60HDAg fails to multimerize but still contains nucleic acid binding activity, indicating that multimerization is not essential for nucleic acid binding. Moreover, in agreement with what has been previously reported for full-length protein, no apparent specificity was found for the truncated protein regarding nucleic acid binding. CONCLUSION: Taken together these results allowed concluding that ∆60HDAg is intrinsically disordered but compact; ∆60HDAg is not a multimer but is still capable of nucleic acid binding albeit without apparent specificity.
RESUMO
Hepatitis delta virus (HDV) is the etiologic agent of the most severe form of virus hepatitis in humans. Sharing some structural and functional properties with plant viroids, the HDV RNA contains a single open reading frame coding for the only virus protein, the Delta antigen. A number of unique features, including ribozyme activity, RNA editing, rolling-circle RNA replication, and redirection for a RNA template of host DNA-dependent RNA polymerase II, make this small pathogen an excellent model to study virus-cell interactions and RNA biology. Treatment options for chronic hepatitis Delta are scarce and ineffective. The disease burden is perhaps largely underestimated making the search for new, specific drugs, targets, and treatment strategies an important public health challenge. In this review we address the main features of virus structure, replication, and interaction with the host. Virus pathogenicity and current treatment options are discussed in the light of recent developments.
RESUMO
Genomic instability at loci with tandem arrays of simple repeats is the cause for many neurological, neurodegenerative and neuromuscular diseases. When located in coding regions, disease-associated expansions of trinucleotide repeats are translated into homopolymeric amino acid stretches of glutamine or alanine. Polyalanine expansions in the poly(A)-binding protein nuclear 1 (PABPN1) gene causes oculopharyngeal muscular dystrophy (OPMD). To gain novel insight into the molecular pathophysiology of OPMD, we studied the interaction of cellular proteins with normal and expanded PABPN1. Pull-down assays show that heat shock proteins including Hsp70, and type I arginine methyl transferases (PRMT1 and PRMT3) associate preferentially with expanded PABPN1. Immunofluorescence microscopy further reveals accumulation of these proteins at intranuclear inclusions in muscle from OPMD patients. Recombinant PABPN1 with expanded polyalanine stretches binds Hsp70 with higher affinity, and data from molecular simulations suggest that expansions of the PABPN1 polyalanine tract result in transition from a disordered, flexible conformation to a stable helical secondary structure. Taken together, our results suggest that the pathological mutation in the PABPN1 gene alters the protein conformation and induces a preferential interaction with type I PRMTs and Hsp70 chaperones. This in turn causes sequestration in intranuclear inclusions, possibly leading to a progressive cellular defect in arginine methylation and chaperone activity.
Assuntos
Proteínas de Choque Térmico HSP70/metabolismo , Distrofia Muscular Oculofaríngea/metabolismo , Peptídeos/metabolismo , Proteínas de Ligação a Poli(A)/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Proteínas Repressoras/metabolismo , Animais , Linhagem Celular , Eletroforese em Gel de Poliacrilamida , Humanos , Espectrometria de Massas , Microscopia de Fluorescência , Modelos Moleculares , Proteínas de Ligação a Poli(A)/químicaRESUMO
A broad range of degenerative diseases is associated with intracellular inclusions formed by toxic, aggregation-prone mutant proteins. Intranuclear inclusions constitute a pathological hallmark of oculopharyngeal muscular dystrophy (OPMD), a dominantly inherited disease caused by (GCG) repeat expansions in the gene that encodes for nuclear poly(A) binding protein (PABPN1). The mutation results in an extended polyalanine stretch that has been proposed to induce protein aggregation and formation of intranuclear inclusions. Here we show that normal PABPN1 is inherently aggregation-prone when exogenously expressed in either HeLa or myogenic C2 cells. Similar deposits of insoluble PABPN1 are formed by variant forms of the protein containing either a polyalanine expansion or a complete deletion of the polyalanine tract, indicating that the mutation responsible for OPMD is not essential for formation of PABPN1 inclusions. In contrast, interfering with any of the protein domains required for stimulation of poly(A) polymerase prevents the formation of inclusions. Most surprisingly, photobleaching experiments reveal that both normal and expanded PABPN1 molecules are not irreversibly sequestered into aggregates, but rather move rapidly in and out of the inclusions. These findings have important implications for the interpretation of OPMD model systems based on exogenous expression of PABPN1.