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. 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.