Eukaryotes from yeast to metazoans respond to cellular stressors through a precisely coordinated interplay between regulatory and signal transduction pathways controlling gene expression. The regulation of mRNA translation is an important aspect of the eukaryotic gene expression program, and the assembly of cytoplasmic mRNA-protein (mRNP) complexes is presumably a significant mechanism by which post-transcriptional control is achieved. Among eukaryotes, mRNPs are well conserved and form in response to numerous stressors, such as heat shock, osmotic or oxidative stress, and starvation. In yeast, processing bodies and stress granules are among the most well-studied mRNPs, but their exact functions and regulation remain to be fully resolved.
Previous studies have identified that Snf1p (AMPK) phosphorylation activates the RNA processing Xrn1p exonuclease and Ste20p (PAK) phosphorylation of Dcp2p affects both its decay of certain mRNAs and its localization to p-bodies. However, the extent to which kinase signaling pathways regulate mRNP activity is likely much broader. Under the hypothesis that kinase phosphorylation events regulate mRNP dynamics, we performed a genetic screen on homozygous yeast mutants deleted for kinase genes. For this screen, mRNPs were initially visualized by live-cell imaging using a PGK1 reporter mRNA containing binding sites in its 3’-UTR for the U1A-GFP fusion protein. Plasmids encoding the modified PGK1 gene and U1A-GFP were transformed into 114 yeast strains, each deleted for a single kinase gene. This kinomic screen identified eight deletion mutants exhibiting mRNP localization phenotypes upon acute glucose deprivation. These mRNP phenotypes were validated using an integrated fusion of mCherry to sequence encoding the mRNP marker protein Dcp2p.
In particular, a homozygous strain deleted for GIN4 exhibited a striking loss of mRNPs. Under conditions of heat shock, osmotic or oxidative stress, and starvation, mRNPs form in wild type but not gin4 ∆/∆ cells. Interestingly, Gin4p is involved in bud growth and septin ring assembly with several kinase-dependent and independent functions. When a plasmid bearing GIN4 and its native promoter is re-introduced, wild type levels of mRNPs are restored in gin4 ∆/∆. The kinase dead Gin4K48A mutant displays wild type numbers of mRNPs, which suggests that a Gin4p kinase-independent function is modulating mRNP activity. Notably, inhibition of bud growth and septin ring assembly does not perturb wild type levels of mRNPs. Therefore, our data imply a novel Gin4p kinase-independent function affecting mRNP formation, which we are actively investigating further.