1. High throughput analysis of a stable protein-associated RNA fraction in different conditions of nutrient starvation in the yeast Saccharomyces cerevisiae
Rodoniki Athanasiadou, David Gresham
Center for Genomics and Systems Biology, Department of Biology, 12 Waverly Place
New York University, New York, NY USA
ABSTRACT
Transcriptome composition changes accompany virtually any alteration in the environmental conditions in which the cells find themselves. These changes lead to specific proteome changes, which in turn lead to specific physiological adaptations that allow the cell to survive in the new conditions and maintain its homeostasis. One interesting physiological adaptation in eukaryotes is quiescence, the reversible exit from cell cycle under adverse environmental conditions or specific signals. The transcriptome composition of these quiescent cells seems to depend, at least partially, on the exact environmental trigger for exiting the cell cycle. When, for example, the transcriptomes of cells starved for carbon, nitrogen or phosphate are compared, a fraction of the changes is shared and reflects the shared physiology of a “quiescent state“, but there are also nutrient-specific starvation responses. Moreover, it has been demonstrated that quiescent yeast cells hold a fraction of their transcriptome in an extraction-resistant protease-labile form. This fraction of mRNAs is believed to be rapidly mobilized upon additional stress. In this study we hypothesize that different nutrient starvation conditions will be accompanied by differences in RNA composition of this extraction-resistant protease-labile fraction in quiescent cells. We test this hypothesis by performing high-throughput directional RNA sequencing on RNA extracted, in the presence or absence of proteases, from quiescent cells starved for carbon, nitrogen or phosphate. The results are discussed in the context of the distinct transcriptional programs of the cells entering quiescence under these conditions, differential UTR utilization, as well as the possible physiological advantages conferred by the mobilization of these protein-bound RNA species
EXPERIMENTAL DESIGN 5. 4. 3. 2. 1.
Directional RNA-seq (Illumina HiSeq)
Yeast cultures were grown in media limited for either N2,P or C.
Equal numbers of cells were harvested at exponential and stationary stages from each culture.
The harvested cells were lysed in the presence or absence of proteinase K.
RNA was extracted.
The samples were subjected to directional RNA-seq (dUTP method).
Growing Stationary
pK- pK+ pK- pK+
12% Acrylamide
Coomasie Stain
1% denaturing agarose
Ethidium Br Stain
RNA extraction (Acid phenol method)
pK pK+
pK pK+
Limiting nutrient
Cell state
Cell density at harvest (cells/ml)
Budding index
N2 (0.4mM Gln)
Growing
3.7x106
0.68 (n=50)
Stationary
18x106
0.07 (n=59)
P (20mg/lt KH2PO4)
5.5x106
0.50 (n=172)
64x106
0.00 (n=127
C (0.08% Glucose)
8.9x106
0.54 (n=99)
25x106
0.25 (n=188)
RNA-seq DATA CONFIRM KNOWN TRANSCRIPTIONAL PROFILES OF STATIONARY vs. GROWNG CELLS
ENRICHMENT OF ANTISENSE TRANSCRIPTS IN THE pK+ SAMPLES OF STATIONARY BUT NOT GROWING CELLS
Growing cells Stationary cells
114 transcripts of which 67 overlap another one at the opposite orientation and are characterized as “dubious ORFS”
The transcriptomes of stationary and growing cells cluster together independently of the growing medium
Genes preferentially expressed in growing cells in all conditions are enriched for GO terms of basal metabolic processes while those of stationary cells are enriched for GO terms reflecting the stress that these cells undergo.
pK pK-
log2(pK+)
log2(pK+)
1 = Plim stationary pK-
2 = Plim stationary pK+
3 = Nlim stationary pK-
4 = Nlim stationary pK+
5 = Plim growing pK-
6 = Plim growing pK+
7 = Nlim growing pK-
8 = Nlim growing pK+
9 = Clim growing pK-
10 = Clim growing pK+
11 = Clim stationary pK-
12 = Clim stationary pK+
log2(pK-) log2(pK-)
FUTURE GOALS
THE IDENTIFIED PROTEIN-BOUND RNAs SHOW LIMITED UPREGULATION AS THE CELLS ENTER THE QUISCENT STATE IN ALL ENVIRONMENTS
P limited media N2 limited media C limited media
Expression (log) at stationary culture
Expression (log) at stationary culture
Expression (log) at stationary culture
Growing (p-value≤0.05)
Stationary (p-value≤0.05)
FUTURE GOALS
Determination of the presence of non-annotated anti-sense transcripts running anti-parallel to annotated genes
Alternative UTR discovery -
REFERENCES
AKNOWLEDGEMENTS
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Kloninska M, Crutchfielad C, Bradley P, Rabinowitz J, Broach J. Yeast cells can access distinct quiescent states. Genes & Development vol.25 (4) pp
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Nishizawa M, Komai T, Katou Y, Shirahige K, Ito T, Toh-E A. Nutrient-regulated antisense and intragenic RNAs modulate a signal transduction pathway in yeast. PLOS Biology 2008 vol. 6(12):
We would like to thank the CGSB core facility and in particular Paul Schied for assistance with RNA-seq. The NYU HPC team and Ashish Agarwal provided invaluable support with analyzing the RNA-seq data
RA would also like to thank the RNA society for providing a travel grant.