Context-dependent regulation of P-body composition and mRNA turnover in Saccharomyces cerevisiae

Control of gene expression is crucial for cells surviving in a changing environment. In
eukaryotes, gene expression can be regulated at different levels, among which posttranscriptional
control is of special importance, as it can rapidly modulate the level of gene
products. It typically includes mRNA processing, export, translation, decay as well as protein
degradation. One type of cytoplasmic granules, which is involved in repression of translation,
mRNA decay, mRNA surveillance/quality control and mRNA storage are P-bodies. They
consist of mRNP aggregates, constitute the 5’ to 3’ mRNA decay machinery in yeast cells and
can be induced in response to various stress conditions. This thesis contains three studies
carried out in S. cerevisiae aimed to uncover the mRNA contents of P-bodies, their role in
mRNA turnover, as well as to better understand how P-body formation and function are
regulated.
Unlike protein components, the mRNA species sequestered by P-bodies are poorly
characterized. In the first part of this thesis, an approach to isolate P-body localized transcripts
was established. This method was used to identify, subsequently, common and stress-specific
mRNA subsets associated with P-bodies. We further examined the fates of these transcripts,
and discovered two major types of transcripts. One type was decayed within P-bodies, yet the
decay occurred with different kinetics. The second type remained stable for at least 1 h after
stress induction. Moreover, we identified transcript-specific cis- and trans-elements that affect
P-body targeting and/or degradation including the 3’UTR and RNA binding protein Puf5p.
In yeast, P-bodies were observed in close proximity to the ER, implying that the ER
may play a role in mRNA regulation (Kilchert et al., 2010). In the second study, we identified
Scp160p and Bfr1p as polysome-associated ER localized proteins. Loss of either Bfr1p or
Scp160p led to numerous Dcp2p positive foci under normal growth condition. Dcp2p is a core
constituent of P-bodies, and served as a marker in this study. Therefore, this observation
suggests that they might serve as inhibitors preventing P-body formation under unstressed
condition. However, general translation was unaffected, indicating that P-body formation and
translation attenuation were uncoupled in the absence of Bfr1p and Scp160p.
In the third part of this thesis, we identified Pby1p as a facultative P-body component,
Pby1p P-body localization was observed under glucose starvation but not when hyper osmotic
shock was applied. Interestingly, loss of Pby1p caused a great reduction of cellular Dcp2p
concentration without preventing P-body formation. Furthermore, the decay of particular
P-body associated transcripts was slightly delayed in a Δpby1 strain, suggesting its possible
role in regulating mRNA stability and P-body function.