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Hidden in plain sight: Masking RNA Pol II phosphorylation via proline isomerization during gene expression

Project description

Proline isomers in regulation of RNA polymerase II phosphorylation

Proline isomerase PIN1 catalyses the transition between the naturally existing CIS and TRANS isomeric states of prolines. Recent studies suggest PIN1 involvement in the phosphorylation of the C-terminal domain (CTD) of RNA polymerase II (Pol II), while mutations in PIN1 are associated with cancer progression. Insufficient knowledge of the proline isomerisation role is linked to technological difficulties in differentiating CIS and TRANS isomers using mass spectrometry. The EU-funded Iso-Proline CTD project aims to elucidate the role of proline isomerisation in Pol II function, which underlies the regulation of gene expression. The study will involve new technological advances in peptide synthesis, allowing locking of prolines in CIS or TRANS state to identify isomer-specific interacting factors.

Objective

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.

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MSCA-IF - Marie Skłodowska-Curie Individual Fellowships (IF)

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Call for proposal

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(opens in new window) H2020-MSCA-IF-2020

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Coordinator

KOBENHAVNS UNIVERSITET
Net EU contribution

Net EU financial contribution. The sum of money that the participant receives, deducted by the EU contribution to its linked third party. It considers the distribution of the EU financial contribution between direct beneficiaries of the project and other types of participants, like third-party participants.

€ 219 312,00
Address
NORREGADE 10
1165 KOBENHAVN
Denmark

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Region
Danmark Hovedstaden Byen København
Activity type
Higher or Secondary Education Establishments
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Total cost

The total costs incurred by this organisation to participate in the project, including direct and indirect costs. This amount is a subset of the overall project budget.

€ 219 312,00
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