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Regulation of processing body formation in the budding yeast "Saccharomyces cerevisiae"

Weidner, Julie. Regulation of processing body formation in the budding yeast "Saccharomyces cerevisiae". 2014, PhD Thesis, University of Basel, Faculty of Science.

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Official URL: http://edoc.unibas.ch/diss/DissB_11116

Abstract

The life cycle of mRNA, from transcription to decay, is a tightly regulated biological process. mRNA is an essential molecule that the cell utilizes to alter gene expression. Degradation is one process through which the cell can regulate mRNA levels. P-bodies constitute the main mRNA decay pathway in yeast cells and are induced in response to various stresses. The presented work aimed to create a better understanding of P-body localization and regulation and to uncover the mRNAs contained within them.
A former graduate student in the lab examined P-body formation in secretory mutants and how this stress differed from other stresses such as starvation. Surprisingly, she found numerous P-bodies were induced in secretory mutants. Similarly, the addition of Ca2+ was able to phenocopy the increased P-body induction as seen in secretory mutants. The calcium-binding protein calmodulin, as well as the core P-body proteins Scd6 and Pat1, were required for the formation of these P-bodies. In addition, different pathways appeared to control P-body number. Lastly, we observed that in yeast, P-bodies occur in close proximity to the ER, implying that the ER may play a role in post-transcriptional regulation.
This finding that P-bodies were associated with the ER led us to examine proteins that may regulate the formation of P-bodies. Through a crosslinking tandem affinity purification approach we were able to identify several P-body components as well as two polysome-associated ER localized proteins: Scp160 and Bfr1. We went on to show that Scp160 is able to associate with P-body components in an mRNA dependent manner at polysomes. Loss of either BFR1 or SCP160 caused the formation of many Dcp2 positive foci under normal growth conditions, suggesting that Bfr1 and Scp160 may be inhibiting the formation of P-bodies. Furthermore, in ?scp160 cells P-bodies failed to properly assemble, indicating the Scp160 is required for P-body assembly. Despite the increase in P-body number under normal growth conditions, general translation was unaffected, thus, uncoupling P-body formation and translation attenuation. Taken together, our results suggest that Bfr1 and Scp160 inhibit P-body formation under normal growth conditions, possibly by limiting the mRNA that is passed from polysomes to P-bodies.
Lastly, we modified a published protocol to uncover the mRNAs that are contained within P-bodies under various stresses. Several rounds of trials and changes were performed and although these modifications were not sufficient to produce a library for deep sequencing, our group has further developed the method and now has a reliably working protocol.
Advisors:Spang, Anne
Committee Members:Stöcklin, Georg Emanuel
Faculties and Departments:05 Faculty of Science > Departement Biozentrum > Growth & Development > Biochemistry (Spang)
Item Type:Thesis
Thesis no:11116
Bibsysno:Link to catalogue
Number of Pages:165 p.
Language:English
Identification Number:
Last Modified:30 Jun 2016 10:57
Deposited On:27 Mar 2015 08:59

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