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1.
Mol Cell ; 62(1): 34-46, 2016 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-27058786

RESUMO

Studying cancer metabolism gives insight into tumorigenic survival mechanisms and susceptibilities. In melanoma, we identify HEXIM1, a transcription elongation regulator, as a melanoma tumor suppressor that responds to nucleotide stress. HEXIM1 expression is low in melanoma. Its overexpression in a zebrafish melanoma model suppresses cancer formation, while its inactivation accelerates tumor onset in vivo. Knockdown of HEXIM1 rescues zebrafish neural crest defects and human melanoma proliferation defects that arise from nucleotide depletion. Under nucleotide stress, HEXIM1 is induced to form an inhibitory complex with P-TEFb, the kinase that initiates transcription elongation, to inhibit elongation at tumorigenic genes. The resulting alteration in gene expression also causes anti-tumorigenic RNAs to bind to and be stabilized by HEXIM1. HEXIM1 plays an important role in inhibiting cancer cell-specific gene transcription while also facilitating anti-cancer gene expression. Our study reveals an important role for HEXIM1 in coupling nucleotide metabolism with transcriptional regulation in melanoma.


Assuntos
Melanoma/metabolismo , Fator B de Elongação Transcricional Positiva/genética , Pirimidinas/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Animais , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Humanos , Melanoma/genética , Melanoma/patologia , Melanoma Experimental , Proteínas Oncogênicas/genética , Fatores de Transcrição , Transcrição Gênica , Proteínas Supressoras de Tumor/genética , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
2.
Mol Cell ; 59(4): 576-87, 2015 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-26257281

RESUMO

The Cdk7 subunit of TFIIH phosphorylates RNA polymerase II (Pol II) during initiation, and, while recent studies show that inhibition of human Cdk7 negatively influences transcription, the mechanisms involved are unclear. Using in vitro transcription with nuclear extract, we demonstrate that THZ1, a covalent Cdk7 inhibitor, causes defects in Pol II phosphorylation, co-transcriptional capping, promoter proximal pausing, and productive elongation. THZ1 does not affect initiation but blocks essentially all Pol II large subunit C-terminal domain (CTD) phosphorylation. We found that guanylylation of nascent RNAs is length dependent and modulated by a THZ1-sensitive factor present in nuclear extract. THZ1 impacts pausing through a capping-independent block of DSIF and NELF loading. The P-TEFb-dependent transition into productive elongation was also inhibited by THZ1, likely due to loss of DSIF. Capping and pausing were also reduced in THZ1-treated cells. Our results provide mechanistic insights into THZ1 action and how Cdk7 broadly influences transcription and capping.


Assuntos
Antineoplásicos/química , Quinases Ciclina-Dependentes/química , Fenilenodiaminas/química , Pirimidinas/química , Iniciação da Transcrição Genética , Antineoplásicos/farmacologia , Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/metabolismo , Células HeLa , Humanos , Cinética , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Fenilenodiaminas/farmacologia , Fosforilação , Regiões Promotoras Genéticas , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , Pirimidinas/farmacologia , RNA Polimerase II/química , Processamento Pós-Transcricional do RNA , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Fatores de Elongação da Transcrição/química , Fatores de Elongação da Transcrição/metabolismo , Quinase Ativadora de Quinase Dependente de Ciclina
3.
Nucleic Acids Res ; 45(11): 6864-6880, 2017 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-28431135

RESUMO

The 7SK small nuclear ribonucleoprotein (snRNP) plays a central role in RNA polymerase II elongation control by regulating the availability of active P-TEFb. We optimized conditions for analyzing 7SK RNA by SHAPE and demonstrated a hysteretic effect of magnesium on 7SK folding dynamics including a 7SK GAUC motif switch. We also found evidence that the 5΄ end pairs alternatively with two different regions of 7SK giving rise to open and closed forms that dictate the state of the 7SK motif. We then used recombinant P-TEFb, HEXIM1, LARP7 and MEPCE to reconstruct a functional 7SK snRNP in vitro. Stably associated P-TEFb was highly inhibited, but could still be released and activated by HIV-1 Tat. Notably, P-TEFb association with both in vitro-reconstituted and cellular snRNPs led to similar changes in SHAPE reactivities, confirming that 7SK undergoes a P-TEFb-dependent structural change. We determined that the xRRM of LARP7 binds to the 3΄ stem loop of 7SK and inhibits the methyltransferase activity of MEPCE through a C-terminal MEPCE interaction domain (MID). Inhibition of MEPCE is dependent on the structure of the 3΄ stem loop and the closed form of 7SK RNA. This study provides important insights into intramolecular interactions within the 7SK snRNP.


Assuntos
Ribonucleoproteínas Nucleares Pequenas/química , Humanos , Sequências Repetidas Invertidas , Magnésio/química , Metiltransferases/química , Conformação de Ácido Nucleico , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Estrutura Quaternária de Proteína , RNA Longo não Codificante/química , Ribonucleoproteínas/química
4.
Nucleic Acids Res ; 40(12): 5283-97, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22379134

RESUMO

Regulation of the positive transcription elongation factor, P-TEFb, plays a major role in controlling mammalian transcription and this is accomplished in part by controlled release of P-TEFb from the 7SK snRNP that sequesters the kinase in an inactive state. We demonstrate here that a similar P-TEFb control system exists in Drosophila. We show that an RNA previously suggested to be a 7SK homolog is, in fact, associated with P-TEFb, through the action of a homolog of the human HEXIM1/2 proteins (dHEXIM). In addition, a Drosophila La related protein (now called dLARP7) is shown to be the functional homolog of human LARP7. The Drosophila 7SK snRNP (d7SK snRNP) responded to treatment of cells with P-TEFb inhibitors and to nuclease treatment of cell lysates by releasing P-TEFb. Supporting a critical role for the d7SK snRNP in Drosophila development, dLARP7 and dHEXIM were found to be ubiquitously expressed throughout embryos and tissues at all stages. Importantly, knockdown of dHEXIM was embryonic lethal, and reduction of dHEXIM in specific tissues led to serious developmental defects. Our results suggest that regulation of P-TEFb by the d7SK snRNP is essential for the growth and differentiation of tissues required during Drosophila development.


Assuntos
Proteínas de Drosophila/fisiologia , Drosophila melanogaster/embriologia , Drosophila melanogaster/crescimento & desenvolvimento , Fator B de Elongação Transcricional Positiva/metabolismo , Proteínas de Ligação a RNA/fisiologia , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Desenvolvimento Embrionário/genética , Dados de Sequência Molecular , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas Nucleares Pequenas/genética
5.
Wiley Interdiscip Rev RNA ; 3(1): 92-103, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-21853533

RESUMO

The human 7SK small nuclear RNA (snRNA) is an abundant noncoding RNA whose function has been conserved in evolution from invertebrates to humans. It is transcribed by RNA polymerase III (RNAPIII) and is located in the nucleus. Together with associated cellular proteins, 7SK snRNA regulates the activity of the positive transcription elongation factor b (P-TEFb). In humans, this regulation is accomplished by the recruitment of P-TEFb by the 7SK snRNA-binding proteins, hexamethylene bisacetamide (HMBA)-induced mRNA 1/2 (HEXIM1 or HEXIM2), which inhibit the kinase activity of P-TEFb. P-TEFb regulates the transition of promoter proximally paused RNA polymerase II (RNAPII) into productive elongation, thereby, allowing efficient mRNA production. The protein composition of the 7SK small nuclear ribonucleoprotein (snRNP) is regulated dynamically. While the Lupus antigen (La)-related protein 7 (LARP7) is a constitutive component, the methylphosphate capping enzyme (MePCE) associates secondarily to phosphorylate the 5' end of 7SK snRNA. The release of active P-TEFb is closely followed by release of HEXIM proteins and both are replaced by heterogeneous nuclear ribonucleoproteins (hnRNPs). The released P-TEFb activates the expression of most cellular and viral genes. Regulated release of P-TEFb determines the expression pattern of many of the genes that respond to environmental stimuli and regulate growth, proliferation, and differentiation of cells.


Assuntos
Fator B de Elongação Transcricional Positiva/genética , RNA Nuclear Pequeno/genética , RNA não Traduzido/genética , Transcrição Gênica , Eucariotos/genética , Humanos , Motivos de Nucleotídeos , RNA Nuclear Pequeno/química , RNA não Traduzido/química
6.
J Mol Biol ; 406(1): 9-17, 2011 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-21167178

RESUMO

Eukaryotic proliferating cell nuclear antigen (PCNA) is a replication accessory protein that functions in DNA replication, repair, and recombination. The various functions of PCNA are regulated by posttranslational modifications including mono-ubiquitylation, which promotes translesion synthesis, and sumoylation, which inhibits recombination. To understand how SUMO modification regulates PCNA, we generated a split SUMO-modified PCNA protein and showed that it supports cell viability and stimulates DNA polymerase δ activity. We then determined its X-ray crystal structure and found that SUMO occupies a position on the back face of the PCNA ring, which is distinct from the position occupied by ubiquitin in the structure of ubiquitin-modified PCNA. We propose that the back of PCNA has evolved to be a site of regulation that can be easily modified without disrupting ongoing reactions on the front of PCNA, such as normal DNA replication. Moreover, these modifications likely allow PCNA to function as a tool belt, whereby proteins can be recruited to the replication machinery via the back of PCNA and be held in reserve until needed.


Assuntos
Antígenos Nucleares/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/química , Sumoilação , Antígenos Nucleares/genética , Antígenos Nucleares/metabolismo , Sobrevivência Celular , Cristalografia por Raios X , DNA Polimerase III/análise , Antígeno Nuclear de Célula em Proliferação , Conformação Proteica , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo
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