Abstract 1436 Specific recruitment of CID proteins via threonine-4 phosphorylation on RNA Polymerase II C-terminal Domain

The C-terminal domain (CTD) of human RNA polymerase II (Pol II) is dynamically phosphorylated at every stage of transcription, promoting, or deterring the binding of protein complexes. Within the 52 repetitive heptads composing the CTD, five of the seven residues are able to be phosphorylated. However, the class of proteins recruited by threonine-4 phosphorylation is unclear and its function in regulating transcription remains enigmatic. We performed ChIP-seq analysis of pThr4 Pol II alongside treatment with Ssu72, a pSer5-specific phosphatase, due to the contribution of pSer5 in neighboring heptads obscuring the recognition of pThr4, thus increasing our overall ChIP-signal. Our ChIP analysis shows pThr4 Pol II has strong occupancy before TSS and gradually increases towards TES sites. Using an in vitro approach, we employed label-free quantitative proteomics on a T4E CTD substrate incubated with cell lysate, owing to a lack of in vitro characterization of CTD kinases that phosphorylate threonine-4. We identified substantial overlaps of proteins that bind to pThr4 and pSer2 marks; a class of proteins containing a C-terminal interaction domain (CID) is highly enriched in both pulldowns. We solved the structure of RPRD1B, a CID protein that regulates cell cycle process, in complex with a pThr4 CTD peptide and discovered binding motifs across CID proteins allow for dual recognition of both pThr4 and pSer2. Using fluorescence polarization, we show RPRD1B as well as other CID proteins exhibit tight binding and specificity towards pThr4 and pSer2. We studied the genome-wide occupancy of RPRD1B using ChIP-seq and found it localizes mostly at the promoter region and also at the end of genes. Using RNA-seq, we explored the function of RPRD1B and pThr4 in 3' end processing and polyadenylation site usage. In conclusion, we have expanded the collection of protein complexes that are regulated via threonine-4 phosphorylation.