טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentHaramati Ofir
SubjectRole of Puf Proteins in the Yeast Response to High Calcium
DepartmentDepartment of Biology
Supervisor Professor Yoav Arava
Full Thesis textFull thesis text - English Version


Abstract

RNA binding proteins (RBPs) bind to RNA from its synthesis until its degradation, and are part of a complex machinery that control a wide range of activities on the RNA, such as RNA stability, RNA localization and RNA translation regulation. One of the major goals in current research is to discover and understand the mechanisms and components that are part of the post-transcriptional gene control.

Members of the yeast family of PUF proteins bind unique subsets of mRNA targets that encode proteins with common functions. They therefore became a paradigm for post-transcriptional gene control. To provide new insights into the roles of the seemingly redundant PUF1 and PUF2 members, we used yeast strains with tagged or deleted PUF1 and PUF2. Localization of PMP1, a known target of Puf2p, was similar between wild type strain and a PUF2 knockout strain. Overexpression of PUF1 did not reveal a significant difference in the stability of mRNA targets of PUF1. Additionally, protein levels of tagged PUF1 and PUF2 showed no drastic change under growth in various stress conditions. The lack of strong effects under standard conditions led us to check whether the proteins roles will be more apparent under stressful environments.

We monitored the growth rates of the deletions under many different stress conditions. Under most conditions no effect was observed. However, differential effect was observed at high CaCl2 concentrations, whereby puf1Δ growth was more affected than puf2Δ, and inhibition was exacerbated in puf1Δpuf2Δ double knockout. Importantly, reintroducing PUF1 to knockout strains restored growth. Transcriptome analyses upon CaCl2 application for short and long terms defined the transcriptional response to CaCl2 and showed a major impact to the cell periphery and cell wall genes. In addition, it revealed distinct expression changes for the deletions. puf1Δ showed differential expression associated with the cell wall component, whereas puf2Δ showed differential expression associated with carbohydrate transport. Intriguingly, most of the affected mRNAs were not shown previously to be targets of PUF1 or PUF2. Nevertheless, enrichment for mRNA targets was apparent upon deletion of both genes.

We next focused on the cell-wall regulator ZEO1 and observed that puf1Δpuf2Δ fail to maintain low levels of its mRNA. Complementary, puf1Δpuf2Δ growth defect in CaCl2 was repaired upon further deletion of ZEO1 gene. Thus, these proteins probably regulate the cell wall integrity pathway by maintaining low Zeo1p level.

This work sheds new light on roles of Puf proteins during the cellular response to environmental stress, and further extends our understanding of the impact of RNA-binding proteins in post-transcriptional gene regulation and its physiological roles under stress response. Future studies will define the molecular mechanisms through which Zeo1 is regulated by Puf1 and Puf2 and determine the role of Puf molecules in other stress conditions.