RESUMO
BACKGROUND INFORMATION: The dual-specificity phosphatase 3 (DUSP3) regulates cell cycle progression, proliferation, senescence, and DNA repair pathways under genotoxic stress. This phosphatase interacts with HNRNPC protein suggesting an involvement in the regulation of HNRNPC-ribonucleoprotein complex stability. In this work, we investigate the impact of DUSP3 depletion on functions of HNRNPC aiming to suggest new roles for this enzyme. RESULTS: The DUSP3 knockdown results in the tyrosine hyperphosphorylation state of HNRNPC increasing its RNA binding ability. HNRNPC is present in the cytoplasm where it interacts with IRES trans-acting factors (ITAF) complex, which recruits the 40S ribosome on mRNA during protein synthesis, thus facilitating the translation of mRNAs containing IRES sequence in response to specific stimuli. In accordance with that, we found that DUSP3 is present in the 40S, monosomes and polysomes interacting with HNRNPC, just like other previously identified DUSP3 substrates/interacting partners such as PABP and NCL proteins. By downregulating DUSP3, Tyr-phosphorylated HNRNPC preferentially binds to IRES-containing mRNAs within ITAF complexes preferentially in synchronized or stressed cells, as evidenced by the higher levels of proteins such as c-MYC and XIAP, but not their mRNAs such as measured by qPCR. Under DUSP3 absence, this increased phosphorylated-HNRNPC/RNA interaction reduces HNRNPC-p53 binding in presence of RNAs releasing p53 for specialized cellular responses. Similarly, to HNRNPC, PABP physically interacts with DUSP3 in an RNA-dependent manner. CONCLUSIONS AND SIGNIFICANCE: Overall, DUSP3 can modulate cellular responses to genotoxic stimuli at the translational level by maintaining the stability of HNRNPC-ITAF complexes and regulating the intensity and specificity of RNA interactions with RRM-domain proteins.
Assuntos
Dano ao DNA , Fosfatase 3 de Especificidade Dupla , Ribonucleoproteínas Nucleares Heterogêneas Grupo C , RNA Mensageiro , Humanos , Fosfatase 3 de Especificidade Dupla/metabolismo , Fosfatase 3 de Especificidade Dupla/genética , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas/genética , Fosforilação , Biossíntese de Proteínas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo C/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo C/metabolismoRESUMO
Heterogeneous nuclear ribonucleoproteins (hnRNPs) are essential players in the regulation of gene expression. The majority of the twenty different hnRNP proteins act through the modulation of pre-mRNA splicing. Most have been shown to regulate the expression of critical genes for the progression of tumorigenic processes and were also observed to be overexpressed in several types of cancer. Moreover, these proteins were described as essential components for the maturation of some microRNAs (miRNAs). In the human genome, over 70% of miRNAs are transcribed from introns; therefore, we hypothesized that regulatory proteins involved with splicing could be important for their maturation. Increased expression of the miR-17-92 cluster has already been shown to be related to the development of many cancers, such as thyroid, lung, and lymphoma. In this article, we show that overexpression of hnRNP A1 and hnRNP C in BCPAP thyroid cancer cells directly affects the expression of miR-17-92 miRNAs. Both proteins associate with the 5'-end of this cluster, strongly precipitate miRNAs miR-17 and miR-18a and upregulate the expression of miR-92a. Upon overexpression of these hnRNPs, BCPAP cells also show increased proliferation, migration, and invasion rates, suggesting upregulation of these proteins and miRNAs is related to an enhanced tumorigenic phenotype.
Assuntos
MicroRNAs , Neoplasias da Glândula Tireoide , Ribonucleoproteína Nuclear Heterogênea A1/genética , Ribonucleoproteínas Nucleares Heterogêneas/genética , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Neoplasias da Glândula Tireoide/genéticaRESUMO
In previous works, we identified a RNA-binding protein in presynaptic terminal of squid neurons, which is likely involved in local mRNA processing. Evidences indicate this strongly basic protein, called p65, is an SDS-stable dimer protein composed of ~ 37 kDa hnRNPA/B-like subunits. The function of p65 in presynaptic regions is not well understood. In this work, we showed p65 and its subunit p37 are concentrated in RNA-enriched regions in synaptosomes. We performed in vitro binding studies with a recombinant protein and showed its propensity to selectively bind actin mRNA at the squid presynaptic terminal. Biochemical analysis using lysed synaptosomes suggested RNA integrity may affect p65 and p37 functions. Mass spectrometry analysis of oligo(dT) pull down indicated squid hnRNPA1, hnRNPA1-like 2, hnRNPA3 and ELAV-like proteins as candidates to interact with p65 and p37 forming a ribonucleoprotein complex, suggesting a role of squid hnRNPA/B-like proteins in site-specific RNA processing.
Assuntos
Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Neurônios/metabolismo , Lobo Óptico de Animais não Mamíferos/metabolismo , Terminações Pré-Sinápticas/metabolismo , Animais , Decapodiformes , Ribonucleoproteínas Nucleares Heterogêneas/genética , Sinaptossomos/metabolismoRESUMO
The serine-threonine kinase AKT/PKB is a critical regulator of various essential cellular processes, and dysregulation of AKT has been implicated in many diseases, including cancer. Despite AKT action is known to function mainly in the cytoplasm, AKT has been reported to translocate to the nucleus. However, very little is known about the mechanism required for the nuclear import of AKT as well as its function in this cellular compartment. In the present study, we characterized the presence of endogenous nuclear AKT in human melanoma cells and addressed the possible role of AKT by exploring its potential association with key interaction nuclear partners. Confocal and Western blot analyses showed that both phosphorylated and non-phosphorylated forms of AKT are present in melanoma cells nuclei. Using mass spectrometry in combination with protein-crosslinking and co-immunoprecipitation, we identified a series of putative protein partners of nuclear AKT, including heterogeneous nuclear ribonucleoprotein (hnRNP), cytoskeleton proteins ß-actin, γ-actin, ß-actin-like 2 and vimentin. Confocal microscopy and biochemical analyses validated ß-actin as a new nuclear AKT-interacting partner. Cofilin and active RNA Polymerase II, two proteins that have been described to interact and work in concert with nuclear actin in transcription regulation, were also found associated with nuclear AKT. Overall, the present study uncovered a yet unrecognized nuclear coupling of AKT and provides insights into the involvement of AKT in the interaction network of nuclear actin.
Assuntos
Actinas/genética , Núcleo Celular/genética , Melanoma/genética , Proteína Oncogênica v-akt/genética , Transporte Ativo do Núcleo Celular/genética , Núcleo Celular/metabolismo , Citoplasma/genética , Regulação Neoplásica da Expressão Gênica , Ribonucleoproteínas Nucleares Heterogêneas/genética , Humanos , Melanoma/patologia , Fosforilação , Ligação Proteica , RNA Polimerase II/genética , Transdução de Sinais/genética , Transcrição GênicaRESUMO
Osteopontin-c splicing isoform activates ovarian cancer progression features. Imbalanced expression of splicing factors from serine/arginine -rich and heterogeneous ribonucleoproteins families has been correlated with the generation of oncogenic splicing isoforms. Our goal was to investigate whether there is any association between the transcriptional patterns of these splicing factors in ovarian cells and osteopontin-c expression levels. We also aimed to investigate the occurrence of these splicing factors binding sites inside osteopontin exon 4 and adjacent introns. To test associations between osteopontin-c and splicing factors expression patterns, we used an in vitro model in which OVCAR-3 cells overexpressing osteopontin-c (OVCAR-3/OPNc++) presented higher transcriptional levels of osteopontin-c than two other ovarian carcinoma cells (TOV-112D, SKOV-3) and ovarian non-tumoral cell lines (IOSE 364 and IOSE 385). The transcriptional levels of osteopontin-c, serine/arginine-rich, and hnRNP factors were evaluated using real-time polymerase chain reaction. Human Splice Finder software was used to search for putative splicing factor binding sites in osteopontin genomic regions. OVCAR-3/OPNc++ cells presented higher transcriptional levels of hnRNP than serine/arginine-rich when compared to TOV-112D, SKOV-3, and IOSE cells. TOV-112D and SKOV-3 cells also overexpressed hnRNP in relation to serine/arginine-rich transcripts. Putative binding sites for these splicing factors have been predicted on osteopontin exon 4 and their upstream and downstream intronic regions. Our data showed that higher osteopontin-c expression levels are associated with a predominance of hnRNP in relation to serine/arginine-rich transcripts and that osteopontin exon 4 and adjacent intronic sequences contain predicted binding sites for some of these tested splicing factors. In conclusion, differential expression of these splicing factors in ovarian cancer cells could be one of the putative mechanisms leading to aberrant splicing of the osteopontin primary transcript. Future work, aiming to control ovarian cancer progression by downregulating osteopontin-c levels, could include strategies that also regulate heterogeneous ribonucleoproteins and serine/arginine-rich expression levels in order to modulate osteopontin splicing.
Assuntos
Ribonucleoproteínas Nucleares Heterogêneas/genética , Osteopontina/metabolismo , Neoplasias Ovarianas/genética , Fatores de Processamento de Serina-Arginina/genética , Processamento Alternativo , Linhagem Celular Tumoral , Feminino , Perfilação da Expressão Gênica , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Humanos , Neoplasias Ovarianas/metabolismo , Isoformas de Proteínas , Reação em Cadeia da Polimerase em Tempo Real , Fatores de Processamento de Serina-Arginina/metabolismo , TranscriptomaRESUMO
B chromosomes are dispensable elements observed in many eukaryotic species, including the African cichlid Astatotilapia latifasciata, which might have one or two B chromosomes. Although there have been many studies focused on the biology of these chromosomes, questions about the evolution, maintenance, and potential effects of these chromosomes remain. Here, we identified a variant form of the hnRNP Q-like gene inserted into the B chromosome of A. latifasciata that is characterized by a high copy number and intron-less structure. The absence of introns and presence of transposable elements with a reverse transcriptase domain flanking hnRNP Q-like sequences suggest that this gene was retroinserted into the B chromosome. RNA-Seq analysis did not show that the B variant retroinserted copies are transcriptionally active. However, RT-qPCR results showed variations in the canonical hnRNP Q-like copy expression levels among exons, tissues, sex, and B presence/absence. Although the patterns of transcription are not well understood, the exons of the B retrocopies were overexpressed, and a bias for female B+ expression was also observed. These results suggest that retroinsertion is an additional and important mechanism contributing to B chromosome formation. Furthermore, these findings indicate a bias towards female differential expression of B chromosome sequences, suggesting that B chromosomes and sex determination are somehow associated in cichlids.
Assuntos
Cromossomos , Ciclídeos/genética , Ribonucleoproteínas Nucleares Heterogêneas/genética , Mutagênese Insercional , Animais , Evolução Molecular , Duplicação Gênica , Genoma , Genômica , Transcrição GênicaRESUMO
Bombyx mori BmHRP28 and BmPSI, which belong to the family of RNA-binding proteins, have been identified binding to the female-specific exon 4 of the sex-determining gene Bmdsx pre-mRNA. However, the relationships between BmHRP28 and BmPSI still remain unclear. In this study, we carried out yeast two-hybrid (Y2H) and co-immunoprecipitation (Co-IP) analyses to address them. Y2H analysis showed that there was little or no direct binding between the BmHRP28 and BmPSI proteins. Also, the Co-IP experiments revealed that BmHRP28 and BmPSI coexisted in a multiprotein complex. Our results suggested that BmHRP28 and BmPSI form a muliprotein complex to regulate the splicing of Bmdsx pre-mRNA, but are not directly bound to each other. In an effort to find other regulatory factors in the multiprotein complex, we constructed a silkworm Y2H cDNA library of male early embryo. By Y2H screening, we identified an RNA-binding protein BmSPX, a putative component of the spliceosome, binding to BmPSI. These results indicated that BmHRP28 and BmPSI make up a spliceosome complex to regulate Bmdsx splicing and that BmSPX is another potential protein involved in this process. Our study provides some clues to better understand the mechanism of sex determination in the silkworm.
Assuntos
Bombyx/genética , Ribonucleoproteínas Nucleares Heterogêneas/genética , Hormônios de Inseto/genética , Proteínas de Insetos/genética , Proteínas de Ligação a RNA/genética , Processos de Determinação Sexual , Processamento Alternativo , Sequência de Aminoácidos , Animais , Bombyx/crescimento & desenvolvimento , Embrião não Mamífero , Epistasia Genética , Éxons , Feminino , Biblioteca Gênica , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Hormônios de Inseto/metabolismo , Proteínas de Insetos/metabolismo , Masculino , Dados de Sequência Molecular , Ligação Proteica , Precursores de RNA/genética , Precursores de RNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , Alinhamento de Sequência , Técnicas do Sistema de Duplo-HíbridoRESUMO
Eukaryotic gene expression is regulated on different levels ranging from pre-mRNA processing to translation. One of the most characterized families of RNA-binding proteins is the group of hnRNPs: heterogenous nuclear ribonucleoproteins. Members of this protein family play important roles in gene expression control and mRNAs metabolism. In the cytoplasm, several hnRNPs proteins are involved in RNA-related processes and they can be frequently found in two specialized structures, known as GW-bodies (GWbs), previously known as processing bodies: PBs, and stress granules, which may be formed in response to specific stimuli. GWbs have been early reported to be involved in the mRNA decay process, acting as a site of mRNA degradation. In a similar way, stress granules (SGs) have been described as cytoplasmic aggregates, which contain accumulated mRNAs in cells under stress conditions and present reduced or inhibited translation. Here, we characterized the hnRNP Q localization after different stress conditions. hnRNP Q is a predominantly nuclear protein that exhibits a modular organization and several RNA-related functions. Our data suggest that the nuclear localization of hnRNP Q might be modified after different treatments, such as: PMA, thapsigargin, arsenite and heat shock. Under different stress conditions, hnRNP Q can fully co-localize with the endoplasmatic reticulum specific chaperone, BiP. However, under stress, this protein only co-localizes partially with the proteins: GW182-GWbs marker protein and TIA-1 stress granule component.
Assuntos
Arsenitos/metabolismo , Grânulos Citoplasmáticos/metabolismo , Resposta ao Choque Térmico , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Acetato de Tetradecanoilforbol/metabolismo , Tapsigargina/metabolismo , Animais , Chaperona BiP do Retículo Endoplasmático , Ativação Enzimática , Proteínas de Choque Térmico HSP70/metabolismo , Células HeLa , Proteínas de Choque Térmico/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas/genética , Humanos , Isoenzimas/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas de Ligação a Poli(A)/genética , Proteínas de Ligação a Poli(A)/metabolismo , Proteína Quinase C-delta/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Estresse Fisiológico , Antígeno-1 Intracelular de Células TRESUMO
RNA binding proteins regulate gene expression at the posttranscriptional level and play important roles in embryonic development. Here, we report the cloning and expression of Samba, a Xenopus hnRNP that is maternally expressed and persists at least until tail bud stages. During gastrula stages, Samba is enriched in the dorsal regions. Subsequently, its expression is elevated only in neural and neural crest tissues. In the latter, Samba expression overlaps with that of Slug in migratory neural crest cells. Thereafter, Samba is maintained in the neural crest derivatives, as well as other neural tissues, including the anterior and posterior neural tube and the eyes. Overexpression of Samba in the animal pole leads to defects in neural crest migration and cranial cartilage development. Thus, Samba encodes a Xenopus hnRNP that is expressed early in neural and neural crest derivatives and may regulate crest cells migratory behavior.