RESUMEN
Transcription of interleukin-2 (IL-2), a pivotal cytokine in the mammalian immune response, is induced by NFAT and AP-1 transcriptional activators in stimulated T cells. NFATc2 and cJun drive high levels of synergistic human IL-2 transcription, which requires a unique interaction between the C-terminal activation domain of NFATc2 and cJun homodimers. Here we studied the mechanism by which this interaction contributes to synergistic activation of IL-2 transcription. We found that NFATc2 can recruit cJun homodimers to the -45 NFAT element, which lacks a neighboring AP-1 site. The bZip domain of cJun is sufficient to interact with the C-terminal activation domain of NFATc2 in the absence of DNA and this interaction is inhibited by AP-1 DNA. When the -45 NFAT site was replaced by either an NFAT/AP-1 composite site or a single AP-1 site the specificity for cJun homodimers in synergistically activating IL-2 transcription was lost, and cJun/cFos heterodimers strongly activated transcription. These studies support a model in which IL-2 transcriptional synergy is mediated by the unique recruitment of a cJun homodimer to the -45 NFAT site by NFATc2, where it acts as a co-activator for IL-2 transcription.
Asunto(s)
Interleucina-2/genética , Factores de Transcripción NFATC/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , Transcripción Genética , Sitios de Unión/genética , Humanos , Células Jurkat , Luciferasas/genética , Luciferasas/metabolismo , Modelos Genéticos , Mutación , Factores de Transcripción NFATC/química , Factores de Transcripción NFATC/genética , Regiones Promotoras Genéticas/genética , Unión Proteica , Multimerización de Proteína , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Proto-Oncogénicas c-fos/metabolismo , Proteínas Proto-Oncogénicas c-jun/química , Proteínas Proto-Oncogénicas c-jun/genética , Factor de Transcripción AP-1/genética , Factor de Transcripción AP-1/metabolismo , Activación Transcripcional , TransfecciónRESUMEN
RNA polymerase II (Pol II) is a well-characterized DNA-dependent RNA polymerase, which has also been reported to have RNA-dependent RNA polymerase (RdRP) activity. Natural cellular RNA substrates of mammalian Pol II, however, have not been identified and the cellular function of the Pol II RdRP activity is unknown. We found that Pol II can use a non-coding RNA, B2 RNA, as both a substrate and a template for its RdRP activity. Pol II extends B2 RNA by 18 nt on its 3'-end in an internally templated reaction. The RNA product resulting from extension of B2 RNA by the Pol II RdRP can be removed from Pol II by a factor present in nuclear extracts. Treatment of cells with α-amanitin or actinomycin D revealed that extension of B2 RNA by Pol II destabilizes the RNA. Our studies provide compelling evidence that mammalian Pol II acts as an RdRP to control the stability of a cellular RNA by extending its 3'-end.
Asunto(s)
ARN Polimerasa II/fisiología , Estabilidad del ARN , ARN no Traducido/genética , ARN no Traducido/metabolismo , Elongación de la Transcripción Genética , Animales , Secuencia de Bases , Células HeLa , Humanos , Ratones , Modelos Biológicos , Datos de Secuencia Molecular , Células 3T3 NIH , Conformación de Ácido Nucleico , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , Estabilidad del ARN/genética , Estabilidad del ARN/fisiología , ARN Polimerasa Dependiente del ARN/genética , ARN Polimerasa Dependiente del ARN/metabolismo , ARN Polimerasa Dependiente del ARN/fisiología , Transcripción Genética/genéticaRESUMEN
At eukaryotic promoters, multi-faceted protein-protein and protein-DNA interactions can result in synergistic transcriptional activation. NFAT and AP-1 proteins induce interleukin-2 (IL-2) transcription in stimulated T cells, but the contributions of individual members of these activator families to synergistically activating IL-2 transcription is not known. To investigate the combinatorial regulation of IL-2 transcription we tested the ability of different combinations of NFATc2, NFATc1, cJun, and cFos to synergistically activate transcription from the IL-2 promoter. We found that NFATc2 and cJun are exclusive in their ability to synergistically activate human IL-2 transcription. Protein-protein interaction assays revealed that in the absence of DNA, NFATc2, but not NFATc1, bound directly to cJun/cJun dimers, but not to cFos/cJun heterodimers. A region of NFATc2 C-terminal of the DNA binding domain was necessary and sufficient for interaction with cJun in the absence of DNA, and this same region of NFATc2 was required for the synergistic activation of IL-2 transcription in T cells. Moreover, expression of this C-terminal region of NFATc2 specifically repressed the synergistic activation of IL-2 transcription. These studies show that a previously unidentified interaction between human NFATc2 and cJun is necessary for synergistic activation of IL-2 transcription in T cells.
Asunto(s)
Interleucina-2/genética , Factores de Transcripción NFATC/química , Factores de Transcripción NFATC/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , Secuencia de Bases , Sitios de Unión , Cartilla de ADN/genética , Humanos , Células Jurkat , Modelos Biológicos , Factores de Transcripción NFATC/genética , Regiones Promotoras Genéticas , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Proto-Oncogénicas c-fos/metabolismo , Proteínas Proto-Oncogénicas c-jun/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismo , Factor de Transcripción AP-1/metabolismo , Activación TranscripcionalRESUMEN
To better understand the mechanism of steps in early transcription by RNA polymerase II (pol II), we investigated the molecular determinants of transcript slipping within complexes assembled on promoters containing a pre-melted transcription bubble from -9 to +3. Transcript slippage occurs when an RNA transcript contains a repetitive sequence that allows the transcript to slip back and pair with the template strand of the DNA at a new register before transcription continues. We established the contributions of individual transcription factors, DNA elements, and RNA length to slipping on a heteroduplex template using a highly purified human pol II transcription system. We found that transcripts slip at a very defined point in the transcription reaction, after pol II completes phosphodiester bond synthesis at register +5. This point is set by the position of the polymerase active site on the DNA template, as opposed to the length of the transcript, as well as by a repetitive CUCU sequence that must occur from +2 to +5. Interestingly, slipping at this juncture is induced by TATA-binding protein and transcription factor IIB and requires a TATA box but not a transcription factor IIB recognition sequence. We propose a model in which transcribing complexes, upon completing phosphodiester bond synthesis at register +5, enter one of two branches in which they either complete productive synthesis of the transcript or undergo multiple rounds of transcript slipping.
Asunto(s)
ARN Polimerasa II/metabolismo , Proteína de Unión a TATA-Box/metabolismo , Factor de Transcripción TFIIB/metabolismo , Transcripción Genética/genética , Secuencia de Bases , Dominio Catalítico , ADN/genética , ADN/metabolismo , Esterificación , Humanos , Datos de Secuencia Molecular , ARN Polimerasa II/genética , Proteína de Unión a TATA-Box/genética , Moldes Genéticos , Factores de Tiempo , Factor de Transcripción TFIIB/genéticaRESUMEN
Noncoding RNAs (ncRNAs) have recently been discovered to regulate mRNA transcription in trans, a role traditionally reserved for proteins. The breadth of ncRNAs as transacting transcriptional regulators and the diversity of signals to which they respond are only now becoming recognized. Here we show that human Alu RNA, transcribed from short interspersed elements (SINEs), is a transacting transcriptional repressor during the cellular heat shock response. Alu RNA blocks transcription by binding RNA polymerase II (Pol II) and entering complexes at promoters in vitro and in human cells. Transcriptional repression by Alu RNA involves two loosely structured domains that are modular, a property reminiscent of classical protein transcriptional regulators. Two other SINE RNAs, human scAlu RNA and mouse B1 RNA, also bind Pol II but do not repress transcription in vitro. These studies provide an explanation for why mouse cells harbor two major classes of SINEs, whereas human cells contain only one.