UC Berkeley

Rapid modulation of gene expression is critical for cells to respond to environmental challenges and initiate developmental programs. Cells employ a variety of mechanisms to achieve tight regulation of gene expression, including post-transcriptional control of active messenger RNA (mRNA) levels by inhibition of translation or by mRNA degradation. While mRNA production via transcription has been extensively characterized, our understanding of how mRNAs are partitioned between an actively translating state and an inactive state is limited. In this thesis I examine the role of a highly evolutionarily conserved protein, the DEAD-box ATPase Dhh1, in mRNA inactivation and turnover in S. cerevisiae.

Previous work from our lab puts Dhh1 at the crossroads of mRNA fate. For example, artificially tethering Dhh1 to an mRNA is sufficient to trigger its degradation. In contrast, in cells compromised in the 5'-3' decay pathway, tethered Dhh1 can no longer direct degradation of the message, yet still possesses the ability to repress its translation. Moreover, ATPase activity of Dhh1 is critical for mRNA localization in the cell, as ATPase-deficient mutants of Dhh1 induce the constitutive formation of mRNA-protein (mRNP) foci known as Processing Bodies (PBs) - enigmatic cellular structures that can direct storage or degradation of mRNAs. However, mechanistically how Dhh1 functions in translation repression and mRNA decay, as well as its role in PB assembly has remained elusive.

In the following dissertation, I further characterize Dhh1 activities in translation repression, mRNA degradation, and PB formation. Using the previously established tethering assay, I identified protein factors that are distinctly required for translational repression or mRNA decay by Dhh1. Furthermore, I discovered that a mutant of Dhh1 that cannot bind to ATP is unable to interact with the Ccr4-NOT deadenylase complex - a major intracellular machine involved in transcriptional regulation and mRNA turnover. Finally, I show that Not1, the major scaffold of the Ccr4-NOT complex, controls Dhh1 localization to PB foci. In summary, my work suggests that the ATPase activity of Dhh1 is regulated in vivo, and this regulation may ultimately determine the fate of an mRNA - whether it is actively translated in the cytoplasm, or delivered to Processing Bodies for degradation or storage.