5615 assignment 1 DUE Feb 16

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Biochem 5615 Literature Assignment #1 Name: _________________________________ 25 points Assignment: Table 1 from "Proteome-wide search reveals unexpected RNA-binding proteins in Saccharomyces cerevisiae ". PLoS ONE, September 2010. (Paper is on Carmen) The following is one possible statement of hypothesis that could be made about the work in this paper. Many biological processes are controlled by proteins that interact with RNA, but the identities of these RNA- binding proteins are not all known and many important RNA-binding proteins remain to be discovered. 1) From the four choices below, pick two that best provide the basis for this hypothesis (2 points). a) Currently over 600 proteins in S. cerevisiae are thought to bind RNA. b) Measurements of individual RNA and protein amounts for the same gene do not correlate well with each other, suggesting that extensive post-transcriptional control must exist to explain these differences. c) Of "known" RNA binding proteins, many don't have obvious RNA-binding domains, so RNA-binding proteins are difficult to predict based on sequence characteristics alone. d) Glyceraldehyde-3-phosphate dehydrogenase is known to bind to AU-rich sequences in specific RNAs in humans and in S. cerevisiae . 2) Write the approach that was used by the authors to obtain the data in Table 1 addressing this hypothesis. (2 points) 3) In one sentence, give the significance of the work in the paper (2 points)

4) Why was ASH1 mRNA chosen as the specific RNA target for this experiment? (2 points) 5) From the list of statements below, indicate (Yes or No on each line) whether this detail should be used for the most concise but complete description of the experiment that led to the data shown in Table 1. (5 points) ____ a) ~4700 S. cerevisiae proteins were purified and were attached to a microarray through their GST-tags. ____ b) ASH1 mRNA was labeled with either Cy3 or Cy5 dye. ____ c) Dye incorporation was quantified using a Nanodrop. ____ d) Prior to hybridization, the microarray slides were pre-blocked in Blocking Buffer at 4°C ____ e) Fluorescent-labeled RNA was incubated with the microarray, then unbound RNA was washed off. ____ f) The microarray was dried in a centrifuge. ____ g) The amount of fluorescence on each spot was measured. ____ h) Microarray data were loaded onto the Stanford Microarray Database. ____ i) The hybridization experiment was performed four times with different dyes used in each hybridization. ____ j) The average fluorescence signal for each dye-labeled RNA was calculated and compared to the average of fluorescence from all spots on the array. 6) The following questions are about the data for ASH1 -interacting mRNAs shown in Table 1 a) What were the specific statistical criteria used to select the 9 proteins shown in Table 1? (2 points) b) Name the highest rank protein that met the authors criteria in the experiment with Cy3 label. (1 point) c) Why are proteins that ranked higher than the protein you identified in (b) (in terms of their signal with Cy3 labeled ASH1 ) not included in Table 1? (1 point) d) How many of the 9 proteins were not previously known to bind to ASH1 ? (1 point) e) What structural feature did the authors identify in one of the proteins that had not been previously known to bind to any RNA, giving them further confidence in their results? (1 point)

7) Co-purification of GST-tagged proteins with interacting proteins is a potential pitfall identified by the authors. a) Give one example of a false negative result that could arise from the presence of co-purifying proteins on the microarray. (2 points) b) Give one example of a false positive result that could arise from the presence of co-purifying proteins on the microarray. (2 points) 8) The authors noted that the pI values for the RNA-interacting proteins they identified in their protein microarray did not follow any specific trend. a) What would be the overall predicted charge for Mdh3 at neutral pH? (positive, negative, neutral) (1 point) b) If the authors had identified a trend of proteins that all had the same electrostatic properties as Mdh3 in their RNA-interacting proteins, this could have indicated a potential problem with their approach to identifying RNA- binding proteins using the protein microarray. What would be the concern about the proteins they identified if they had observed this result? (1 point)

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