Post translational modifications such as phosphorylation, can alter the substrate-binding specificity of proteins. This fact has been the basis of the “code” encountered during RNA synthesis, since phosphorylation of key aminoacids in the largest protein subunit of the RNA polymerase II complex (RNAPII) alters the recruitment of protein binding partners. This project introduced the structural regulation of RNA polymerase II activity via a process known as proline isomerization. Proline is unique amongst aminoacids since it can exist in two different isomeric states: cis or trans. Interestingly, proline isomerization can have drastic ramifications on the “code” of transcription, since removal of phosphorylation marks on RNAPII seems to be affected by the presence of PIN1, the only known protein capable of proline isomerization on RNAPII.
The aims in this action were three-fold: (1) to identify new binders RNAPII binders specific to each proline state; (2) to assess transcriptional changes in the absence of RNAPII prolines; and (3), to measure the transcriptional changes of human cells after global dysregulation of proline isomerization.
Within the scope of the first aim, we developed the most comprehensive protein-interactome of RNAPII to date, relying on the synthesis of over 30 different peptides displaying key permutations of phosphorylated aminoacids and proline isomeric states. Importantly, this was only possible due to the collaboration with the mass spectrometry facility within the host institution, which allowed for the use of automated liquid handling robots to perform the necessary key steps in our protocol. This automation allowed for a high-quality dataset, spanning known key RNAPII binders but identifying over 50 new specific binders. We also identified several proteins that uniquely bind to CIS-containing substrates that are missing from the TRANS-dataset, thus achieving the goal of this aim.
For the second and third aim, we took advantage of targeted degradation using dTAG to completely remove endogenous protein targets: RNAPII or PIN1. In this same system, we were then able to complement with mutants of the endogenous targets, in a process we refer to as a “switchover system”. This was critical for the second aim of the project since RNAPII is essential for human cells and cannot be easily replaced. We created mutations where we replaced prolines with other aminoacids for RNAPII, or where the activity of PIN1 is dulled by a single aminoacid change. The production of all these tools was challenging but given we had a well-planned contingency plan, all steps were successful. For both aims we took advantage of mass spectrometry, next-generation sequencing of gene expression, and cell-proliferation assays to measure their impact in gene regulation. All the results in this action have now been presented in at least 2 different international meetings and have led to several cross-institutional collaborations.
