Research Projects

The carboxy terminal domain (CTD) of the catalytic or Rpb1 subunit of RNA Polymerase II (RNAPII) is composed of tandem heptad sequences of Tyr1Ser2Pro3Thr4Ser5Pro6Ser7. The dynamic phosphorylation and dephosphorylation of CTD helps facilitate the transcription of all mRNA and the majority of non-coding RNA. During transcription initiation, the phosphorylation of Ser5 (Ser5P) helps recruiting enzymes required for the capping of nascent transcripts. Subsequently, the Ser2 is phosphorylated which helps recruiting the complexes required for transcription elongation and termination. The Ser7P is present throughout the transcription cycle and shown to play a role in the processing and termination of snRNA transcription in yeast and humans. Nevertheless, the role of Ser7P in the mRNA transcription remains unclear. This is despite the fact that the persistent Ser7P does not support growth and highly transcribed mRNA genes show high levels of this phosphorylation mark. We observed over-expression of Rsp5 HECT type E3 ubiquitin ligase due to Ser7 mutation in the CTD, where cells are found to be highly sensitive to UV irradiation. The human homolog of Rsp5, known as Nedd4, is reported to be over-expressed in various cancers, contributing to tumor development and progression. Rsp5 is known to form a physical and functional complex with RNAPII recognizes a wide variety of substrates and implicated in the biogenesis of mRNA, DNA damage response, regulation of lipid biosynthesis, protein degradation, endocytosis, sorting and trafficking etc. Rsp5 is composed of an N-terminal C2 domain, WW domains, and a C-terminal catalytic HECT domain. The WW domains are involved in diverse protein-protein interactions and recognize PY motifs (P/PXY, LPXY) in the Rsp5 substrates and adaptors. Rsp5 plays an important role in the RNAPII transcription by removing stalled RNAPII complexes and allowing repair of lesions. Here, Rsp5 interacts with CTD and ubiquitinate the Rpb1, facilitating the degradation of RNAPII. The mode of interaction between Rsp5 and RNAPII-CTD is not known. Given that the interaction occurs with the stalled RNAPII during transcription elongation, the possibility of combinatorial phosphorylation of all three serine residues cannot be ruled out. In contrast to all previous findings where Ser phosphorylation is desired for the recruitment of proteins, our findings suggest that the dephosphorylation of Ser7 is essential not only for the upregulation and recruitment of Rsp5 but also sets the first example where the abnormal expression of an E3 ligase is directly regulated by a residue of RNAPII-CTD. Therefore, given the multifaceted role of Rsp5 in cellular processes and the association of its overexpression with various diseases, it is crucial to comprehend not only the Rsp5-RNAPII interaction but also see how the cellular homeostasis of Rsp5 and RNAPII is affected by Ser7P, as well as identify the substrate of the upregulated Rsp5 to identify its diverse function. We shall carry out the experiments where the expression of Rsp5 will be regulated in the cell having native and Ser7 mutant RNAPII-CTD and see the effect of the expression of Rsp5 on the level of Rpb1 in normal and in DNA damage condition. The substrates of the upregulated Rsp5 in the Ser7 mutant cells will be identified using the ubiquitin activated interaction traps technique. The change in cellular protein abundance due to Ser7 mutation will be identified through differential proteomics. To understand the molecular mechanism by which Ser7 of RNAPII-CTD influences the interaction with Rsp5, a multi-step computational strategy will be employed. The fundamental understanding of Rsp5-RNAPII interactions and function is very crucial because it not only impacts the wide range of cellular process but its dysfunction has been strongly linked to the cancer progression, cardiovascular conditions, neurological disorders etc., making them important target for research.