Phosphorylation of mRNA-Binding Proteins Puf1 and Puf2 by TORC2-Activated Protein Kinase Ypk1 Alleviates Their Repressive Effects.

Members of the Puf family of RNA-binding proteins typically associate via their Pumilio homology domain with specific short motifs in the 3'-UTR of an mRNA and thereby influence the stability, localization and/or efficiency of translation of the bound transcript. In our prior unbiased proteome-wide screen for targets of the TORC2-stimulated protein kinase Ypk1, we identified the paralogs Puf1/Jsn1 and Puf2 as high-confidence substrates. Earlier work by others had demonstrated that Puf1 and Puf2 exhibit a marked preference for interaction with mRNAs encoding plasma membrane-associated proteins, consistent with our previous studies documenting that a primary physiological role of TORC2-Ypk1 signaling is maintenance of plasma membrane homeostasis. Here, we show, first, that both Puf1 and Puf2 are authentic Ypk1 substrates both in vitro and in vivo. Fluorescently tagged Puf1 localizes constitutively in cortical puncta closely apposed to the plasma membrane, whereas Puf2 does so in the absence of its Ypk1 phosphorylation, but is dispersed in the cytosol when phosphorylated. We further demonstrate that Ypk1-mediated phosphorylation of Puf1 and Puf2 upregulates production of the protein products of the transcripts to which they bind, with a concomitant increase in the level of the cognate mRNAs. Thus, Ypk1 phosphorylation relieves Puf1- and Puf2-mediated post-transcriptional repression mainly by counteracting their negative effect on transcript stability. Using a heterologous protein-RNA tethering and fluorescent protein reporter assay, the consequence of Ypk1 phosphorylation in vivo was recapitulated for full-length Puf1 and even for N-terminal fragments (residues 1-340 and 143-295) corresponding to the region upstream of its dimerization domain (an RNA-recognition motif fold) encompassing its two Ypk1 phosphorylation sites (both also conserved in Puf2). This latter result suggests that alleviation of Puf1-imposed transcript destabilization does not obligatorily require dissociation of Ypk1-phosphorylated Puf1 from a transcript. Our findings add new insight about how the TORC2-Ypk1 signaling axis regulates the content of plasma membrane-associated proteins to promote maintenance of the integrity of the cell envelope.

A genome-scale CRISPR interference guide library enables comprehensive phenotypic profiling in yeast.

BACKGROUND: CRISPR/Cas9-mediated transcriptional interference (CRISPRi) enables programmable gene knock-down, yielding loss-of-function phenotypes for nearly any gene. Effective, inducible CRISPRi has been demonstrated in budding yeast, and genome-scale guide libraries enable systematic, genome-wide genetic analysis. RESULTS: We present a comprehensive yeast CRISPRi library, based on empirical design rules, containing 10 distinct guides for most genes. Competitive growth after pooled transformation revealed strong fitness defects for most essential genes, verifying that the library provides comprehensive genome coverage. We used the relative growth defects caused by different guides targeting essential genes to further refine yeast CRISPRi design rules. In order to obtain more accurate and robust guide abundance measurements in pooled screens, we link guides with random nucleotide barcodes and carry out linear amplification by in vitro transcription. CONCLUSIONS: Taken together, we demonstrate a broadly useful platform for comprehensive, high-precision CRISPRi screening in yeast.