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 10003 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. 1 2 3 4 5 6 7 8 9 10 11 12 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 Gresham D, Boer D, Caudy A, Ziv N, Brandt NJ, Storey JD, Botstein D. 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PLOS Biology 2008 vol. 6(12):2817-30 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.