Centro Andaluz de Biología Molecular y Medicina Regenerativa CABIMER, Universidad de Sevilla-CSIC, Sevilla, Spain.
EMBO J. 2011 May 18;30(10):1953-64. doi: 10.1038/emboj.2011.109. Epub 2011 Apr 8.
To clarify the role of a number of mRNA processing factors in transcription elongation, we developed an in vivo assay for direct analysis of elongation on chromatin. The assay relies on two substrates containing two G-less cassettes separated by either a long and GC-rich or a short and GC-poor DNA sequence (G-less-based run-on (GLRO) assay). We demonstrate that PAF, THSC/TREX-2, SAGA, the exosome component Rrp6 and two subunits of cleavage factor IA (Rna14 and Rna15) are required for efficient transcription elongation, in contrast to some results obtained using other assays. Next, we undertook a mutant screen and found out that the Nup84 nucleoporin complex is also required for transcription elongation, as confirmed by the GLRO assay and RNA polymerase II chromatin immunoprecipitations. Therefore, in addition to showing that the GLRO assay is a sensitive and reliable method for the analysis of elongation in vivo, this study provides evidence for a new role of the Nup84 complex and a number of mRNA processing factors in transcription elongation that supports a connection of pre-mRNA processing and nuclear export with transcription elongation.
为了阐明许多 mRNA 加工因子在转录延伸中的作用，我们开发了一种用于直接分析染色质上延伸的体内测定法。该测定法依赖于两种底物，它们包含两个 G -less 盒，由长且富含 GC 或短且 GC 贫乏的 DNA 序列隔开（G-less 基启动子延伸（GLRO）测定法）。我们证明 PAF、THSC/TREX-2、SAGA、外切体成分 Rrp6 和切割因子 IA 的两个亚基（Rna14 和 Rna15）对于有效的转录延伸是必需的，这与使用其他测定法获得的一些结果相反。接下来，我们进行了突变筛选，发现 Nup84 核孔复合体也需要转录延伸，这通过 GLRO 测定法和 RNA 聚合酶 II 染色质免疫沉淀得到证实。因此，除了表明 GLRO 测定法是一种用于分析体内延伸的灵敏可靠的方法外，本研究还为 Nup84 复合体和许多 mRNA 加工因子在转录延伸中的新作用提供了证据，这支持了前体 mRNA 加工和核输出与转录延伸的联系。
