An actin-ribonucleoprotein interaction is involved in transcription by RNA polymerase II
An actin-ribonucleoprotein interaction is involved in transcription by RNA polymerase II
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DOI：
10.1073/pnas.1131933100
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发表时间：
2003-05-27
影响因子：
11.1
通讯作者：
Visa, N
中科院分区：
文献类型：
--
作者：
Percipalle, P;Fomproix, N;Visa, N
为了确定肌动蛋白在细胞核中的功能，我们试图利用DNase I亲和层析来鉴定双翅目摇蚊的核肌动蛋白结合蛋白。我们鉴定了RNA结合蛋白hrp65是一种肌动蛋白结合蛋白，并表明hrp65-2亚型的C端序列在体外能够直接与肌动蛋白相互作用。体内交联和免疫共沉淀实验表明，活细胞中也存在hrp65和肌动蛋白。此外，在体内给予对应于hrp65-2 C末端序列的竞争多肽中断了肌动蛋白-hrp65-2的相互作用，并导致特异性和急剧的转录减少，根据Puff回归和Bromo-UTP掺入减少的判断。我们的结果表明，大部分RNA聚合酶II基因的转录都涉及肌动蛋白机制，并提示肌动蛋白-hrp65-2相互作用是维持细胞正常转录活性所必需的。此外，免疫电子显微镜实验和核连续分析表明，肌动蛋白-hrp65-2复合体在转录延伸中发挥作用。
To determine the function of actin in the cell nucleus, we sought to identify nuclear actin-binding proteins in the dipteran Chironomus tentans using DNase I-affinity chromatography. We identified the RNA-binding protein hrp65 as an actin-binding protein and showed that the C-terminal sequence of the hrp65-2 isoform is able to interact directly with actin in vitro. In vivo crosslinking and coimmunoprecipitation experiments indicated that hrp65 and actin are also associated in the living cell. Moreover, in vivo administration of a competing peptide corresponding to the C-terminal sequence of hrp65-2 disrupted the actin-hrp65-2 interaction and caused a specific and drastic reduction of transcription as judged by puff regression and diminished bromo-UTP incorporation. Our results indicate that an actin-based mechanism is implicated in the transcription of most if not all RNA polymerase II genes and suggest that an actin-hrp65-2 interaction is required to maintain the normal transcriptional activity of the cell. Furthermore, immunoelectron microscopy experiments and nuclear run-on assays suggest that the actin-hrp65-2 complex plays a role in transcription elongation.