Investigations of mRNP regulation through post translational modifications to the RNA binding proteins PABP and Lsm4
Chapter I is an introduction to the unifying theme of my thesis, competition between translation initiation and mRNA decay with a focus on my proteins of interest.
Chapter II is a reprint of a review co-authored by myself and Donghui Wu. The review covers structural insights into the mechanisms of decapping, catalytic decapping enhancers, and enhancers that function primarily by repressing translation.
Chapter III focuses on the role of the decay factor Lsm4 in regulating Processing Bodies (PBs). PBs are cytoplasmic foci that contain mRNA decay factors decay intermediates. PBs are lost when human cells are depleted of Lsm4, which contains an arginine/glycine rich RGG C-terminal domain that has been shown to undergo arginine dimethylation. Using immunofluorescence and Lsm4 mutants, I show that arginine methylation, likely by the methyltransferase PRMT5, in the RGG domain of Lsm4 is critical for PB accumulation. Assays for mRNA decay and translational activity, and mass spectrometry analysis all suggest that the RGG domain is not required for efficient decay, translation inhibition or decay factor recruitment.
Chapter IV shows the results of a collaboration investigating the decapping enhancer PNRC2. PNRC2 can interact with and stimulate the activity of the Dcp1/Dcp2 decapping complex. Disrupting this interaction leads to a loss in decapping stimulation. Chapter V focuses on PABP, a highly conserved protein that binds to the 3’ poly(A) tail of nearly all mRNA and stimulates translation. We wondered if modifications to PABP could regulate mRNP function similar to how histone modifications regulate transcription. I used PABP mutants and assays designed to identify protein modifications to investigate this question.
Chapter VI is a look at the most pressing questions arising from my research.