Whi3, a Developmental Regulator of Budding Yeast, Binds a Large Set of mRNAs Functionally Related to the Endoplasmic Reticulum

Abstract

Whi3 is an RNA-binding protein associated with the endoplasmic reticulum (ER) that binds the CLN3 mRNA and plays a key role in the efficient retention of cyclin Cln3 at the ER. In the present work, we have identified new Whi3-associated mRNAs by a genomic approach. A large and significant number of these Whi3 targets encode for membrane and exocytic proteins involved in processes such as transport and cell wall biogenesis. Consistent with the genomic data, we have observed that cell wall integrity is compromised in Whi3-deficient cells and found strong genetic interactions between WHI3 and the cell integrity pathway. Whi3-associated mRNAs are enriched in clusters of the tetranucleotide GCAU, and mutation of the GCAU clusters in the CLN3 mRNA caused a reduction in its association to Whi3, suggesting that these sequences may act as cis-determinants for binding. Our data suggest that Whi3 is involved in the regulation and/or localization of a large subset of mRNAs functionally related to the ER and, since it is important for different molecular processes such as cytoplasmic retention or exocytic traffic of proteins, we propose that Whi3 is a general modulator of protein fate in budding yeast.

Ras/cAMP-dependent Protein Kinase (PKA) Regulates Multiple Aspects of Cellular Events by Phosphorylating the Whi3 Cell Cycle Regulator in Budding Yeast

“…The RRM of Whi3 would bind the CLN3 mRNA and restrict Cln3 synthesis in the endoplasmic reticulum (3,4,6,7). Here, we showed that PKA phosphorylated Ser-568 of this RRM, thus inhibiting Whi3 function.…”

“…Whi3 function is important not only for control of the G 1 /S transition but also for the switch to developmental options such as invasive growth and meiosis (3,4,6,7). None of the above studies addressed the role of the phosphorylation of Whi3 in such cellular functions.…”

“…Thus, cells harboring mutated clusters (CLN3 mGCAU ) in their CLN3 mRNA due to mutations in the 14 GCAT sites in the CLN3 gene show a decrease in cell size similar to ⌬whi3 cells (7). If Whi3-S568A binds through GCAU sites in the CLN3 mRNA, Whi3-S568A would not be able to bind to the CLN3 mGCAU mRNA, and so Whi3-S568A cells harboring CLN3 mGCAU should show a decrease in cell size.…”

The RNA-Binding Protein Whi3 Is a Key Regulator of Developmental Signaling and Ploidy inSaccharomyces cerevisiae

“…For this purpose, we determined the influence of Whi3 on the protein/mRNA ratio of selected biofilm regulators including components of the cAMP-PKA pathway (Tpk1, Tpk2, and Flo8), the PKA-regulated Yak1 branch (Yak1 and Phd1), and the Fus3/Kss1 MAPK cascade (Tec1) as well as the RNA-binding protein Mpt5. We also included Mig2, which has not been associated with biofilm formation, but whose mRNAs are bound by Whi3 in vivo (Colomina et al 2008).…”

“…For this purpose, we determined the influence of Whi3 on the protein/mRNA ratio of selected biofilm regulators including components of the cAMP-PKA pathway (Tpk1, Tpk2, and Flo8), the PKA-regulated Yak1 branch (Yak1 and Phd1), and the Fus3/Kss1 MAPK cascade (Tec1) as well as the RNA-binding protein Mpt5. We also included Mig2, which has not been associated with biofilm formation, but whose mRNAs are bound by Whi3 in vivo (Colomina et al 2008).To determine the effect of Whi3 on production of these regulators, we used the same experimental approach as for the analysis of G 1 cyclins and created a series of WHI3 and whi3Δ yeast strains that carry endogenous fusions of corresponding genes to GFP. Resulting strains were assayed for biofilm formation, showing that all fusion genes are fully functional except FLO8-GFP that conferred partially reduced functionality ( Figure S1).…”