RNA-dependent chromatin association of transcription elongation factors and Pol II CTD kinases

For transcription through chromatin, RNA polymerase (Pol) II associates with elongation factors (EFs). Here we show that many EFs crosslink to RNA emerging from transcribing Pol II in the yeast Saccharomyces cerevisiae. Most EFs crosslink preferentially to mRNAs, rather than unstable non-coding RNAs. RNA contributes to chromatin association of many EFs, including the Pol II serine 2 kinases Ctk1 and Bur1 and the histone H3 methyltransferases Set1 and Set2. The Ctk1 kinase complex binds RNA in vitro , consistent with direct EF-RNA interaction. Set1 recruitment to genes in vivo depends on its RNA recognition motifs (RRMs). These results strongly suggest that nascent RNA contributes to EF recruitment to transcribing Pol II. We propose that EF-RNA interactions facilitate assembly of the elongation complex on transcribed genes when RNA emerges from Pol II, and that loss of EF-RNA interactions upon RNA cleavage at the polyadenylation site triggers disassembly of the elongation complex. Overall design: In this study, we used photoactivatable ribonucleoside-enhanced crosslinking and immuno-precipitation (PAR-CLIP) (Hafner et al., 2010), a method that detects and maps direct protein-RNA interactions in vivo. We applied our recently optimized PAR-CLIP protocol (Baejen et al., 2014) to 14 transcription elongation factors of the yeast Saccharomyces cerevisiae. These elongation factors included Spt5, Spt6, all five subunits of the Paf1 complex, namely Cdc73, Ctr9, Leo1, Rtf1, and Paf1, the kinases Bur1 and Ctk1, and their cyclin partners Bur2 and Ctk2, respectively, and the histone methyltransferases Set1, Set2, and Dot1. To estimate background RNA binding we collected PAR-CLIP data for the transcription initiation factor TFIIB, which is recruited to promoter DNA before RNA is made. We also measured gene occupancies of EFs using ChIP-Seq and compared them with our PAR-CLIP occupancies.

对于染色质转录过程，RNA聚合酶II（RNA polymerase II，Pol II）会与延伸因子（elongation factors，EFs）结合。本研究证实，在酿酒酵母（Saccharomyces cerevisiae）中，诸多延伸因子可与正在转录的Pol II所释放出的RNA发生交联。多数延伸因子更倾向于与mRNA发生交联，而非不稳定的非编码RNA。RNA可介导诸多延伸因子与染色质的结合，其中包括Pol II丝氨酸2激酶Ctk1与Bur1，以及组蛋白H3甲基转移酶Set1与Set2。Ctk1激酶复合物在体外可结合RNA，这与延伸因子与RNA的直接相互作用相一致。Set1在体内向基因的募集依赖于其RNA识别基序（RNA recognition motifs，RRMs）。上述结果强烈表明，新生RNA可助力延伸因子向转录中的Pol II募集。我们提出如下假说：当RNA从Pol II中释放后，延伸因子与RNA的相互作用可促进延伸复合物在转录基因上的组装；而在多聚腺苷酸化位点发生RNA切割后，延伸因子与RNA的相互作用丧失，进而触发延伸复合物的解聚。
本研究的整体实验设计：本研究采用了光激活核糖核苷增强交联免疫沉淀（photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation，PAR-CLIP）技术（Hafner等人，2010年），该方法可在活体内检测并定位蛋白质与RNA的直接相互作用。我们将近期优化的PAR-CLIP实验方案（Baejen等人，2014年）应用于酿酒酵母的14种转录延伸因子。这些延伸因子包括Spt5、Spt6，Paf1复合物的全部5个亚基：Cdc73、Ctr9、Leo1、Rtf1与Paf1，激酶Bur1、Ctk1及其各自的细胞周期蛋白伴侣Bur2与Ctk2，以及组蛋白甲基转移酶Set1、Set2与Dot1。为评估RNA结合的背景水平，我们收集了转录起始因子TFIIB的PAR-CLIP数据——该因子会在RNA合成前被招募至启动子DNA区域。我们还通过染色质免疫沉淀测序（ChIP-Seq）检测了各延伸因子的基因结合占有率，并将其与PAR-CLIP检测得到的占有率进行了比较。