|Ph.D Student||Melamed Daniel|
|Subject||Regulation of mRNA Translation by Signaling Pathways in|
|Department||Department of Biology||Supervisor||Professor Yoav Arava|
|Full Thesis text|
Eukaryotic cells respond to various changes in their surrounding environment by changing their protein repertoire. These responses are often elicited by signaling pathways and involve both transcriptional and post-transcriptional regulatory mechanisms. One of the most important regulatory mechanisms at the post-transcriptional level involves the control of protein synthesis. However, the contribution of signaling cascades to the translation of specific mRNAs remains largely unknown. To uncover principles that govern the translational response of individual genes to environmental stimuli, we analyzed the effects of changes in external salinity on mRNA translation in the yeast Saccharomyces cerevisiae, and the contributions of the HOG and the PKC cascades to these responses.
We analyzed the translational response to high salinity and its coordination with transcriptome changes, by subjecting nonpolysomal, polysomal and total mRNA pools to DNA microarray analysis. Results indicated a strong repression in polysomal association for most mRNAs, with an accumulation of many mRNAs as a nontranslating pool. Yet, some mRNAs displayed a significant increase or decrease in association with polysomes, with a few of them demonstrating also a correlated change in their transcript levels (i.e., potentiation). In particular, genes showing a positive effect for both parameters (i.e., positive potentiation) were enriched with targets of the transcription factors Msn2/Msn4. Genes showing a negative effect for the two parameters (i.e., negative potentiation) were found to encode mainly for ribosomal proteins and for other highly abundant transcripts. Genome-wide analysis of a strain lacking the Hog1p kinase revealed that the group of translationally affected genes is significantly enriched with sequence motifs that were shown to be associated with the ARE-binding protein Pub1. This suggests that Hog1p and Pub1p cooperate to regulate the translation of specific mRNAs in response to high salinity.
A rapid decrease in external salinity activates the PKC signaling pathway. We studied the connection between this cascade and the ribosomal protein Asc1. asc1∆ cells exhibited several cell-wall sensitivity phenotypes that are consistent with defects in the PKC pathway. Co-immunoprecipitation and polysomal profiling experiments suggest that Pkc1p associates with Asc1p on translation ribosomes. Furthermore, DNA microarray analysis of asc1∆ cells revealed reduction in translation levels for genes that are involved in cell-wall integrity. Finally, following Asc1p and Pkc1p localization within cells indicate these two proteins may interact at the cortical ER near the budding site. These results suggest that Asc1p and Pkc1p act together to affect localized translation that is important for cell-wall integrity.