Mitochondrial Biogenesis Is Positively Regulated by Casein Kinase I Hrr25 Through Phosphorylation of Puf3 in Saccharomyces Cerev
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Genetics: Early Online, published on April 21, 2020 as 10.1534/genetics.120.303191 Mitochondrial biogenesis is positively regulated by Casein Kinase I Hrr25 through phosphorylation of Puf3 in S. cerevisiae Manika Bhondeley and Zhengchang Liu1 Department of Biological Sciences, University of New Orleans, New Orleans, LA 70148 1 Copyright 2020. Running title: Regulation of Puf3 by Hrr25 Key words: Hrr25, Puf3, protein phosphorylation, post-transcriptional regulation, mitochondrial biogenesis 1 Corresponding author: Zhengchang Liu, Department of Biological Sciences, University of New Orleans, 2000 Lakeshore Drive, New Orleans, LA 70148. Telephone: 504-280-6314. Fax: 504-280- 6121. E-mail: [email protected] 2 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’ untranslated region of many 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 Puf3 in 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. Down-regulation of GFP expression due to an hrr25 mutation can be reversed either by puf3 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. INTRODUCTION The maintenance of mitochondrial functions during cell growth and development depends on contributions from the nuclear genome, which encodes the majority of mitochondrial proteins, and the mitochondrial genome, which encodes a small but essential number of mitochondrial proteins that are mostly components of the mitochondrial respiratory complexes (POYTON AND MCEWEN 1996; FOURY et al. 1998; CALVO AND MOOTHA 2010). The prominent function of mitochondria is ATP synthesis. The 3 heteromeric Hap2/3/4/5 transcription factor is an important part of yeast’s metabolic remodeling when cells switch from glycolysis to respiratory metabolism to produce ATP (FORSBURG AND GUARENTE 1989; OLESEN AND GUARENTE 1990; ROSENKRANTZ et al. 1994; MCNABB et al. 1995; BLOM et al. 2000; BUSCHLEN et al. 2003). The Hap2/3/5 trimer binds to CCAAT sequence elements in the promoter of target genes and requires Hap4 to provide the transcriptional activation domain activity (OLESEN et al. 1987; FORSBURG AND GUARENTE 1989; OLESEN AND GUARENTE 1990; MCNABB et al. 1995; MCNABB AND PINTO 2005). A heme-activated protein, Hap1, also contributes to mitochondrial respiratory metabolism by activating the expression of genes under aerobic conditions, including those encoding components of Complexes III and IV of the electron transport chain (HICKMAN AND WINSTON 2007; KUNDAJE et al. 2008). Puf3, a Pumilio RNA-binding domain protein, is a post-transcriptional regulator of mitochondrial biogenesis by binding to the 3’ untranslated region (3’ UTR) of many mitochondrial protein-encoding mRNAs (OLIVAS AND PARKER 2000; GERBER et al. 2004; JACKSON et al. 2004; HOUSHMANDI AND OLIVAS 2005; GARCIA-RODRIGUEZ et al. 2007; SAINT-GEORGES et al. 2008; ZHU et al. 2009; LEE et al. 2010; CHATENAY-LAPOINTE AND SHADEL 2011; FREEBERG et al. 2013; MILLER et al. 2014; KERSHAW et al. 2015; LAPOINTE et al. 2015; WILINSKI et al. 2017). PUF (Pumilio and FBF) proteins typically bind to mRNA targets through a concave face formed typically by eight - helical repeats (EDWARDS et al. 2001; WANG et al. 2002; ZHU et al. 2009). The Puf3 binding motif is the sequence UGUAHAUA (H is A, U or C), with a C often found at the -2 position (GERBER et al. 2004; HOUSHMANDI AND OLIVAS 2005; ZHU et al. 2009). PUF proteins can regulate the stability and/or translation of target mRNAs, often leading to their decay or translational inhibition (MILLER AND OLIVAS 2011; QUENAULT et al. 2011; WANG et al. 2018). Due to its role in promoting target mRNA deadenylation and decay, Puf3 is considered to be a negative regulator of mitochondrial biogenesis 4 under glucose repression conditions (OLIVAS AND PARKER 2000; GERBER et al. 2004; JACKSON et al. 2004; FOAT et al. 2005; LEE et al. 2010; CHATENAY-LAPOINTE AND SHADEL 2011; GUPTA et al. 2014; MILLER et al. 2014). When cells switch from fermentative to respiratory growth, the negative regulatory role of Puf3 is reduced or maybe even converted to a positive regulatory role by promoting the translation of Puf3-bound mRNAs (LEE AND TU 2015; LAPOINTE et al. 2018). Puf3 also promotes mitochondrial biogenesis by localizing mitochondrial protein-encoding transcripts to the mitochondrial outer membrane for co-translational import into mitochondria (GARCIA-RODRIGUEZ et al. 2007; SAINT- GEORGES et al. 2008; ELIYAHU et al. 2010; GADIR et al. 2011; QUENAULT et al. 2011). Consistent with the proposed latter role, puf3 mutant cells show mild growth defects on non-fermentable carbon sources (GERBER et al. 2004; JIANG et al. 2010; LEE AND TU 2015). Puf3 targets are not limited to mRNAs encoding mitochondrial proteins, suggesting that Puf3 may have other cellular roles (GERBER et al. 2004; FREEBERG et al. 2013; KERSHAW et al. 2015; LAPOINTE et al. 2015; WILINSKI et al. 2017). PUF proteins are found in most, if not all, eukaryotes (GERBER et al. 2006; GALGANO et al. 2008; MORRIS et al. 2008; STUMPF et al. 2008; JIANG et al. 2010; TAM et al. 2010; HOGAN et al. 2015; WILINSKI et al. 2017). The number of PUF protein-encoding genes varies in each species (WANG et al. 2018). The Saccharomyces cerevisiae genome encodes six PUF proteins, Puf1-6 (OLIVAS AND PARKER 2000; GU et al. 2004), which associate with different set of functionally related mRNAs (OLIVAS AND PARKER 2000; GERBER et al. 2004; GU et al. 2004; FREEBERG et al. 2013; MILLER et al. 2014; LAPOINTE et al. 2017). Despite the structural conservation of PUF proteins, their functions have diverged. For example, the Puf3 homologs in Saccharomyces cerevisiae, Drosophila melanogaster, and Homo sapiens interact with nearly identical RNA sequence motifs, but their target mRNAs encode proteins with different functions (GERBER et al. 2004; GERBER et al. 2006; GALGANO et al. 2008). Functional diversification of PUF proteins is not limited to major lineages of eukaryotic organisms during evolution 5 (HOGAN et al. 2015). Among 80 fungal species analyzed, only Puf3 homologs from the species in the Saccharomycotina subphylum and Arthrobotrys oligospora, an early-diverging Pezizomycotina species, bind a common set of 176 target mRNAs encoding mitochondrial proteins (GASCH et al. 2004; JIANG et al. 2010; HOGAN et al. 2015; WILINSKI et al. 2017). Although they have been shown or predicted to share a common binding motif with Puf3 of Saccharomyces cerevisiae, Puf3 homologs in Leotiomyceta species (Pezizomycotina species excluding Arthrobotrys oligospora and Tuber melanosporum) have lost association with mRNAs orthologous to most Saccharomycotina Puf3 targets. Instead, they are predicted to bind a set of 409 orthologous genes, 113 of which encode mitochondrial proteins, with remarkable enrichment of subunits of the Complex I of the electron transport chain (HOGAN et al. 2015). More than 150 mRNAs in Leotiomyceta species orthologous to the Saccharomycotina Puf3 targets have been shown to acquire new sets of binding motifs, which are recognized by Leotiomyceta Puf4 (HOGAN et al. 2015). These findings have led to the proposal that rewiring and reprogramming of PUF protein targets is employed to coordinate the expression of genes with related functions during evolution (KEENE 2007). A lot of progress has been made in the identification of mRNA targets of Puf3 orthologs in the budding yeast and other species, but little is known about how Puf3 activity is regulated. Lee and Tu reported that Puf3 phosphorylation is increased when cells switch from fermentation growth to respiratory growth (LEE AND TU 2015). Their data suggest that increased Puf3 phosphorylation leads to a switch from its function as a negative regulator under fermentation conditions to other functions under respiratory growth conditions. Puf3 has an N-terminal domain that contains most of its phosphorylation sites and a C-terminal region that contains the RNA binding domain (OLIVAS AND PARKER 2000; LEE AND TU 2015). Lee and Tu reported that two nutrient-responsive kinases, Sch9 and PKA, and the PP2A- related phosphatase Sit4 might mediate Puf3 phosphorylation. However, it is not clear whether the 6 observed effects are direct or indirect. It is also not clear whether mutations in SCH9, PKA, and SIT4 affect Puf3 activity. Here we show that casein kinase I protein Hrr25 is the primary kinase that phosphorylates Puf3. There are four different casein kinase I isoforms in Saccharomyces cerevisiae, Yck1, Yck2, Yck3, and Hrr25. Yck1 and Yck2 play redundant roles in cell morphogenesis, amino acid sensing, and glucose sensing pathways (ROBINSON et al. 1993; MORIYA AND JOHNSTON 2004; LIU et al. 2008). Yck3 is a vacuolar membrane-localized kinase that mediates vacuolar membrane fusion (LAGRASSA AND UNGERMANN 2005; ZICK AND WICKNER 2012; LAWRENCE et al. 2014). Hrr25 is implicated in multiple cellular processes, including vesicular trafficking, ribosome biogenesis, autophagy, transcriptional regulation, meiosis, endocytosis, microtubule assembly and spindle positioning, and DNA damage response (HOEKSTRA et al. 1991; PETRONCZKI et al. 2006; SCHAFER et al.