Hidden in plain sight: masking rna pol ii phosphorylation via proline isomerization during gene expression
Prolines are unique amongst aminoacids given that they naturally exist in two isomeric states: CIS and TRANS. The transition between these states is slow but can be catalyzed by the activity of proline isomerases such as PIN1. Recent in vitro work suggests that PIN1 can alter the phosphorylation dynamics of the C-terminal domain (CTD) of RNA polymerase II (Pol II) by inhibiting phosphatase recognition. Given Pol II is solely responsible for transcribing all protein-coding genes and that CTD phosphorylation dictates the timing of RNA co-transcriptional processes, these observations suggest a crucial role for CTD proline isomerization in gene expression. Importantly, mutations in PIN1 are associated with cancer progression but a direct role for proline isomerization has remained understudied given the technical limitations imposed by its complex enzymology, such as the inability of differentiating CIS and TRANS isomers using Mass Spectrometry. In this project, I aim to functionally dissect the role of proline isomerization during Pol II transcription using rigorous biochemical, cellular and genomic techniques. Specifically, I will exploit new technological advances in peptide synthesis to permanently “lock” prolines in CIS or TRANS and identify novel isomer-specific interacting factors. I will systematically examine the consequences of CTD proline mutations and of rapid depletion of PIN1 in human cells, focusing on CTD-dependent co-transcriptional RNA processes such as splicing and poly-A–dependent 3’ termination. At the basic research level, my results will provide unprecedented resolution to the dynamics of Pol II phosphorylation, which underlies regulation of gene expression in multicellular organisms. Translationally, given that various cancers hijack the transcriptional programmes of the cell, this mechanistic understanding of CTD proline isomerization will better equip future clinical studies interested in the yet-to-be-characterized role of PIN1 in oncogenesis.