Date of Award

Spring 1-1-2011

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

First Advisor

Kenneth Krauter

Second Advisor

Jens Lykke-Andersen

Third Advisor

Tom Blumenthal

Abstract

Messenger RNA degradation is important for the control of gene expression. The major mRNA decay pathway requires the coordination of proteins involved in deadenylation, decapping, and exonucleolysis to function properly. Interestingly, many of those proteins, as well as translationally repressed mRNAs, localize to discreet cytoplasmic foci called processing bodies (PBs). It remains unclear how PBs form and their functional significance is, as yet, unknown. To better understand how PB assembly may be regulated, I tested whether the cytoskeleton is required for PB dynamics in human cells. I found that the cytoskeleton is likely not required for overall PB assembly, integrity, or disassociation; moreover, disruption of the cytoskeleton does not inhibit mRNA decay efficiency. However, the localization of AU-rich element (ARE)-containing mRNAs in PBs was inhibited upon cytoskeleton disruption, which suggests a possible role for the cytoskeleton in transcript-specific delivery to PBs.

In an assay designed to identify novel PB factors, I found two proteins (PRMT5 and MEP50) that are known to be involved in splicing as a part of the methylosome complex, to co-purify with PB proteins. PRMT5 is a methyltransferase that has an affinity for methylating arginine residues within GRG-tripeptide repeats. Interestingly, the PB protein Lsm4 contains a large GRG-repeat domain in its C-terminus. I confirmed that the Lsm4 C-terminus is methylated and wished to determine if this methylation was important for Lsm4 function in PBs and mRNA decay. Unlike yeast Lsm4, I found that the C-terminus of Lsm4 in humans is neither necessary, nor sufficient, to form PBs. Knockdown of Lsm4 revealed it is important for efficient mRNA decay; however, I found that this is not dependent on the GRG-rich c-terminal domain. Taken together, these studies add to our understanding of PBs assembly and mRNA decay in human cells.

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