Analysis of the Condition-Specific Regulation of Puf3p Activity and Puf3p-Mediated Translational Repression of mRNA in \u3ci\u3eSaccharomyces cerevisiae\u3c/i\u3e

The Puf family of proteins regulates aspects of eukaryotic development such as embryonic development, and memory formation by promoting translational repression and/or degradation of targeted mRNAs in the cytoplasm. Yeast Puf3p regulates mitochondria biogenesis and function by modulating the stabilities of nuclear-transcribed mitochondrial mRNAs in response to different carbon sources. Dextrose simulates rapid Puf3p-mediated degradation of its mRNA targets via decay complex recruitment. Ethanol, galactose, or raffinose promotes stabilization of mRNA targets, as Puf3p-mediated decay activity is severely inhibited or abolished. In this work, I have established that carbon source-induced inhibition of Puf3p activity is not due to decreased transcription or translation, but is likely regulated by post-translational phosphorylation. In the absence of Puf3p activity, binding interactions between the Puf3 repeat domain and the deadenylation factors Ccr4p and Pop2p are disrupted, while interactions with COX17mRNA are maintained. Analysis of Puf3p localization demonstrated that Puf3p aggregates in multiple cytoplasmic foci in all carbon source conditions, but these foci increase in size in Puf3p inactivating conditions. Puf3p aggregate size is increased in all P-body inducing conditions, with concomitant co-localization of Puf3p with P-bodies. However, the co-localization of Puf3p aggregates with mitochondria is only observed in Puf3p inactivating conditions. These observations present multiple schemes to regulate Puf protein activity, such that post-translational phosphorylation may regulate Puf3p activity and allow rapid changes in mRNA target repression. Inhibition of Puf3p activity may be due to Puf3p’s inability to recruit decay factors to a target mRNA. In Puf3p activating conditions, Puf3p is expressed in the cell cytoplasm to bind target mRNAs and recruit deadenylases, presumably to repress translation. Puf3p-bound transcripts targeted for decay localize within multiple P-bodies where they are decapped and degraded. When Puf3p is inactive, altered Puf3p localization to P-bodies might serve to temporarily store inactive Puf3p pools. Additionally, Puf3p may shuttle its mRNA targets to mitochondria for translation and subsequent mitochondrial import of nascent proteins. Together, this work provides a greater understanding of the role of Puf3p in mRNA decay regulation, and provides insight into the conditional control of Puf3p activity and how Puf3p accomplishes fine tuning of mitochondrial protein production