RESUMO
DNA double strand break (DSB) repair through homologous recombination (HR) is crucial to maintain genome stability. DSB resection generates a single strand DNA intermediate, which is crucial for the HR process. We used a synthetic DNA structure, mimicking a resection intermediate, as a bait to identify proteins involved in this process. Among these, LC/MS analysis identified the RNA binding protein, HNRNPD. We found that HNRNPD binds chromatin, although this binding occurred independently of DNA damage. However, upon damage, HNRNPD re-localized to γH2Ax foci and its silencing impaired CHK1 S345 phosphorylation and the DNA end resection process. Indeed, HNRNPD silencing reduced: the ssDNA fraction upon camptothecin treatment; AsiSI-induced DSB resection; and RPA32 S4/8 phosphorylation. CRISPR/Cas9-mediated HNRNPD knockout impaired in vitro DNA resection and sensitized cells to camptothecin and olaparib treatment. We found that HNRNPD interacts with the heterogeneous nuclear ribonucleoprotein SAF-A previously associated with DNA damage repair. HNRNPD depletion resulted in an increased amount of RNA:DNA hybrids upon DNA damage. Both the expression of RNase H1 and RNA pol II inhibition recovered the ability to phosphorylate RPA32 S4/8 in HNRNPD knockout cells upon DNA damage, suggesting that RNA:DNA hybrid resolution likely rescues the defective DNA damage response of HNRNPD-depleted cells.
Assuntos
Cromatina/metabolismo , Genoma Humano , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/genética , Reparo de DNA por Recombinação , Proteína de Replicação A/genética , Antineoplásicos/farmacologia , Camptotecina/farmacologia , Quinase 1 do Ponto de Checagem/genética , Quinase 1 do Ponto de Checagem/metabolismo , Cromatina/efeitos dos fármacos , Cromatina/ultraestrutura , DNA/efeitos dos fármacos , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Reparo do DNA por Junção de Extremidades/efeitos dos fármacos , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Instabilidade Genômica , Células HeLa , Ribonucleoproteína Nuclear Heterogênea D0 , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/antagonistas & inibidores , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo U/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo U/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Conformação de Ácido Nucleico , Hibridização de Ácido Nucleico/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Ftalazinas/farmacologia , Piperazinas/farmacologia , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Reparo de DNA por Recombinação/efeitos dos fármacos , Proteína de Replicação A/metabolismo , Ribonuclease H/genética , Ribonuclease H/metabolismoRESUMO
RNA binding proteins (RBPs) can regulate the stability and/or translatability of messengerRNAs (mRNAs) through interactions with their 30-untranslated regions. However, individual mRNAs may be regulated simultaneously or successively by more than one RBP, as well as by Argonaute (AGO)-bound miRNAs; the coordination of these various influences on an individual mRNA is therefore complex and not well studied. In this report we examine the roles of two RBPs that bind to AU-rich elements (ARE) - AUF1 and HuR - in the stability and translation of cyclin D1 (Ccnd1) mRNA in rat myoblasts transiting the G phase of the cell cycle, and their interactions with miRNAs. Knockdown (KD) of AUF1 resulted in (1) transient upregulation of the mRNA level as well as an advancement of translation onset time (TOT) from 6 to 5 h post-serum addition, (2) loss of miRNA loading on AGO1 and AGO2 and (3) reduction in the level of AGO-1 and AGO-2 bound mRNA. In contrast, KD of HuR had no effect on the mRNA level, or on the AGO-mRNA complexes, but delayed TOT by 1 h independent of miRNA let-7. Thus the dynamics of RBP-mRNA binding and -RBP-AGO-miRNA interactions are coordinated to fine tune the expression of Ccnd1 in the G1 phase.
Assuntos
Proteínas Argonautas/genética , Ciclina D1/genética , Mioblastos/metabolismo , Proteínas de Ligação a RNA/genética , Animais , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Ribonucleoproteína Nuclear Heterogênea D0 , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/antagonistas & inibidores , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/genética , Humanos , MicroRNAs/genética , Estabilidade de RNA/genética , RNA Mensageiro/genética , RatosRESUMO
Polyamines critically regulate all mammalian cell growth and proliferation by mechanisms such as the repression of growth-inhibitory proteins, including JunD. Decreasing the levels of cellular polyamines stabilizes JunD mRNA without affecting its transcription, but the exact mechanism whereby polyamines regulate JunD mRNA degradation has not been elucidated. RNA-binding proteins HuR and AUF1 associate with labile mRNAs bearing AU-rich elements located in the 3' untranslated regions (3'-UTRs) and modulate their stability. Here, we show that JunD mRNA is a target of HuR and AUF1 and that polyamines modulate JunD mRNA degradation by altering the competitive binding of HuR and AUF1 to the JunD 3'-UTR. The depletion of cellular polyamines enhanced HuR binding to JunD mRNA and decreased the levels of JunD transcript associated with AUF1, thus stabilizing JunD mRNA. The silencing of HuR increased AUF1 binding to the JunD mRNA, decreased the abundance of HuR-JunD mRNA complexes, rendered the JunD mRNA unstable, and prevented increases in JunD mRNA and protein in polyamine-deficient cells. Conversely, increasing the cellular polyamines repressed JunD mRNA interaction with HuR and enhanced its association with AUF1, resulting in an inhibition of JunD expression. These results indicate that polyamines modulate the stability of JunD mRNA in intestinal epithelial cells through HuR and AUF1 and provide new insight into the molecular functions of cellular polyamines.
Assuntos
Antígenos de Superfície/metabolismo , Genes jun , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/metabolismo , Poliaminas/metabolismo , Estabilidade de RNA , Proteínas de Ligação a RNA/metabolismo , Regiões 3' não Traduzidas , Animais , Antígenos de Superfície/genética , Sequência de Bases , Sítios de Ligação/genética , Ligação Competitiva , Células CACO-2 , Linhagem Celular , Primers do DNA/genética , Proteínas ELAV , Proteína Semelhante a ELAV 1 , Ribonucleoproteína Nuclear Heterogênea D0 , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/antagonistas & inibidores , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/genética , Humanos , Dados de Sequência Molecular , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/genética , Ratos , Deleção de SequênciaRESUMO
AUF1/heterogeneous nuclear ribonucleoprotein D is an adenylate-uridylate-rich elements (AREs) -binding protein, which regulates the mRNA stability of many genes related to growth regulation, such as proto-oncogenes, growth factors, cytokines, and cell cycle-regulatory genes. Several studies demonstrated AUF1 involvement in the processes of apoptosis, tumorigenesis, and development by its interactions with ARE-bearing mRNAs. We report here that AUF1 may be involved in thyroid carcinoma progression. Investigations on thyroid tissues revealed that cytoplasmic expression of AUF1 in malignant tissues was increased when compared with benign thyroid tissues. In thyroid carcinoma cell lines, AUF1 was mostly detectable in the nucleus; however, in dividing cells, its increased production was also observed in the cytoplasm. We found AUF1 in complexes with ARE-bearing mRNAs, previously described to be crucial for proliferation and cell cycle of thyroid carcinoma. Total or exon-selective knockdown of AUF1 led to growth inhibition accompanied by induction of cell cycle inhibitors and decreased levels of cell cycle promoters. Our data demonstrate the existence of a complex network between AUF1 and mRNAs encoding proteins related to cell proliferation. AUF1 may control the balance between stabilizing and destabilizing effects, both of which are exerted on cell cycle machinery in thyroid carcinoma. Although we cannot exclude participation of other factors, thyroid carcinoma may recruit cytoplasmic AUF1 to disturb the stability of mRNAs encoding cyclin-dependent kinase inhibitors, leading to uncontrolled growth and progression of tumor cells. Thus, AUF1 may be considered as a new, additional marker for thyroid carcinoma.
Assuntos
Carcinoma/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/fisiologia , Neoplasias da Glândula Tireoide/genética , Biomarcadores Tumorais/antagonistas & inibidores , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Carcinoma/metabolismo , Carcinoma/patologia , Proliferação de Células , Progressão da Doença , Regulação para Baixo/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Genes cdc , Ribonucleoproteína Nuclear Heterogênea D0 , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/antagonistas & inibidores , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/metabolismo , Humanos , Ligação Proteica , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/farmacologia , Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/patologia , Células Tumorais CultivadasRESUMO
A 6-nucleotide insertion (I)/deletion (D) polymorphism in the 3'-untranslated region of the thymidylate synthase gene was shown to influence mRNA stability, but the molecular basis of this effect has not been elucidated. Here, studies of both endogenous and ectopically expressed thymidylate synthase alleles revealed that the mRNA-binding, decay-promoting protein AUF1 has higher affinity for allele D mRNA. AUF1 overexpression preferentially suppressed D allele mRNA levels, whereas AUF1 silencing selectively elevated D allele mRNA levels. Our results illustrate the functional consequences of ribonucleoprotein associations involving a polymorphic RNA sequence and uncover a novel mechanism of action for non-coding RNA polymorphisms.
Assuntos
Regiões 3' não Traduzidas/genética , Variação Genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/fisiologia , Polimorfismo Genético , Timidilato Sintase/genética , Timidilato Sintase/metabolismo , Alelos , Sequência de Bases , Linhagem Celular Tumoral , Estabilidade Enzimática/genética , Inativação Gênica , Células HeLa , Ribonucleoproteína Nuclear Heterogênea D0 , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/antagonistas & inibidores , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/biossíntese , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/genética , Humanos , Dados de Sequência Molecular , Estabilidade de RNA , RNA Mensageiro/biossíntese , RNA Mensageiro/metabolismo , Regulação para Cima/genéticaRESUMO
HT1080 cells stably expressing green fluorescent protein (GFP) linked to a 3' terminal AU-rich element (ARE) proved to be a convenient system to study the dynamics of mRNA stability, as changes in mRNA levels are reflected in increased or decreased fluorescence intensity. This study examined whether mRNA stability can be regulated by small interfering RNAs (siRNAs) targeted to AU-binding proteins (AUBPs), which in turn should reveal their intrinsic role as stabilizers or destabilizers of ARE-mRNAs. Indeed, siRNAs targeting HuR or BRF1 decreased or increased fluorescence, respectively. This effect was abolished if cells were treated with both siRNAs, thus indicating antagonistic control of ARE-mRNA stability. Unexpectedly, downregulation of all four AUF1 isoforms by targeting common exons did not affect fluorescence whereas selective downregulation of p40AUF1/p45AUF1 strongly increased fluorescence by stabilizing the GFP-ARE reporter mRNA. This observation was fully confirmed by the finding that only selective reduction of p40AUF1/p45AUF1 induced the production of GM-CSF, an endogenous target of AUF1. These data suggest that the relative levels of individual isoforms, rather than the absolute amount of AUF1, determine the net mRNA stability of ARE-containing transcripts, consistent with the differing ARE-binding capacities of the isoforms.