Mitochondrial Biogenesis Is Positively Regulated by Casein Kinase I Hrr25 Through Phosphorylation of Puf3 in Saccharomyces cerevisiae

Abstract

Mitochondrial biogenesis requires coordinated expression of genes encoding mitochondrial proteins, which in Saccharomyces cerevisiae is achieved in part via post-transcriptional control by the Pumilio RNA-binding domain protein Puf3. Puf3 binds to the 3′-UTR of many messenger RNAs (mRNAs) that encode mitochondrial proteins, regulating their turnover, translation, and/or mitochondrial targeting. Puf3 hyperphosphorylation correlates with increased mitochondrial biogenesis; however, the kinase responsible for Puf3 phosphorylation is unclear. Here, we show that the casein kinase I protein Hrr25 negatively regulates Puf3 by mediating its phosphorylation. An hrr25 mutation results in reduced phosphorylation of Puf3in vivo and a puf3 deletion mutation reverses growth defects of hrr25 mutant cells grown on medium with a nonfermentable carbon source. We show that Hrr25 directly phosphorylates Puf3, and that the interaction between Puf3 and Hrr25 is mediated through the N-terminal domain of Puf3 and the kinase domain of Hrr25. We further found that an hrr25 mutation reduces GFP expression from GFP reporter constructs carrying the 3′-UTR of Puf3 targets. Downregulation of GFP expression due to an hrr25 mutation can be reversed either by puf3Δ or by mutations to the Puf3-binding sites in the 3′-UTR of the GFP reporter constructs. Together, our data indicate that Hrr25 is a positive regulator of mitochondrial biogenesis by phosphorylating Puf3 and inhibiting its function in downregulating target mRNAs encoding mitochondrial proteins.

Yeast Puf3p‐mediated mRNA decay is regulated by carbon source‐specific differential interaction of Puf3p with Pop2p and Yak1p

“…A number of potential kinases have been predicted to facilitate these modifications. Recently, Bhondeley and Liu [49] identified Hrr25 as the likely dominant kinase involved in phosphorylation of Puf3p. A loss of function hrr25 mutation resulted in severely abrogated cell growth on mitochondria‐reliant media, a defect that was rescued upon PUF3 deletion.…”

“…A loss of function hrr25 mutation resulted in severely abrogated cell growth on mitochondria‐reliant media, a defect that was rescued upon PUF3 deletion. Moreover, this hrr25 mutation led to downregulation of various Puf3p target proteins under respiratory growth conditions [49].…”

“…A phosphonull Puf3p mutant resulted in decreased Puf3p association with polysomes and a decrease in mitochondria‐associated protein accumulation compared to WT Puf3p upon glucose depletion [48]. The dominant kinase responsible for negatively regulating Puf3p during mitochondria‐reliant conditions is casein kinase I, Hrr25p [49].…”

Multiple layers of gene-expression regulatory mechanisms during fermentation and respiration

“…However, Puf3p associates with actively translating polysomes upon glucose depletion and promotes mitochondrial biogenesis [198,204], indicating its bidirectional functions. These dual functions of Puf3p are regulated by phosphorylation via casein kinase Hrr25p [198,205]. Mutations in the PUF3 gene, such as PUF3(24A), act dominantly negative and even more strongly inhibit cell growth upon the diauxic shift than the complete deletion of PUF3 [198].…”

Pbp1 associates with Puf3 and promotes translation of its target mRNAs involved in mitochondrial biogenesis

“…Depending on glucose availability, Puf3 can switch the fate of its target mRNAs from decay to translation [9]. In the presence of non-fermentable sugars that require respiratory metabolism, Puf3 becomes heavily phosphorylated in its N-terminal low complexity region [9,10], leading to the stabilization and translation of its bound transcripts. Furthermore, Puf3 has been found in ribonucleoprotein (RNP) granules [9].…”