Year: 2024
Type: dissertation
Abstract: Advances in in proteomic techniques has uncovered a wide array of putative RNA-binding proteins in eukaryotes. To understand whether these RNA-binding proteins were asymmetrically localized to regulate mRNAs in a compartment specific fashion in mammalian cells, we analyzed the proteomic content of messenger ribonucleoproteins from either cytosolic or ER-enriched cellular fractions. We identified the glycolytic enzyme Pyruvate Kinase M (PKM) as a cytosolic polysome interactor and investigated how it influences mRNA translation. We discovered that the PKM-polysome interaction is directly regulated by ADP levels–providing a link between carbohydrate metabolism and mRNA translation. By performing enhanced crosslinking immunoprecipitation-sequencing (eCLIP-seq), we found that PKM crosslinks to mRNA sequences that are immediately downstream of regions that encode lysine- and glutamate-enriched tracts. Using ribosome footprint protection sequencing, we found that PKM binding to ribosomes causes translational stalling near lysine and glutamate encoding sequences. Lastly, we observed that PKM recruitment to polysomes is dependent on poly-ADP ribosylation activity (PARylation)—and may depend on co-translational PARylation of lysine and glutamate residues of nascent polypeptide chains. Overall, our study uncovers a novel role for PKM in post-transcriptional gene regulation, linking cellular metabolism and mRNA translation.
Source: TSpace (University of Toronto)
Author: Kejiou, Nevraj Singh
Institution: University of Toronto
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W7132872231