Weidner, Julie. Regulation of processing body formation in the budding yeast "Saccharomyces cerevisiae". 2014, PhD Thesis, University of Basel, Faculty of Science.
Official URL: http://edoc.unibas.ch/diss/DissB_11116
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.
|Committee Members:||Stöcklin, Georg Emanuel|
|Faculties and Departments:||05 Faculty of Science > Departement Biozentrum > Growth & Development > Biochemistry (Spang)|
|Bibsysno:||Link to catalogue|
|Number of Pages:||165 p.|
|Last Modified:||30 Jun 2016 10:57|
|Deposited On:||27 Mar 2015 08:59|
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