- Q1. What do you thing brings the Y RNAs to the replication fork? Do that interact with other essential fraction components like PCNA
- Q2. What role are Y RNA playing in replication, is it restart? Why are they highly present in rheumatic diseases? Does that give us a clue to what they normally do? Is it a damage response?
What could be a simple (quick and dirty) experiment to determine what the RNAs are binding to?
- if the fractions from the purification scheme are as pure as they allude to in the paper (they don't show a gel!), they could done something like run the fractions QA and ArFT on a native acrylamide gel plus and minus Y RNAs and look for a band that shifts positions. They should run two gels simultaneously and identical, but one with the RNA radiolabeled to develop in an autoradiogram, the other with unlabeled RNA coomassie or silver stained. This will hint at which position that the RNA is comigrating, and give a clue as to what protein this may bind, if it binds at all. If it does not bind protein, then the radiolabeled RNA will show no shift in the autoradiogram.
Q1: How do the authors proove that human Y RNAs are necessary for semiconserative chromosomal DNA replication.
A1: BrdU triphosphate and density gradient centrifugation (old school!)
Q2: How might you find protien partners for Y RNAs assuming they are ribonuclearproteins.
A1: Fishing with radiolabled Y RNA as bait out from your extracts. gel shift etc.
There appears to be a bulge in the stem structure of Y-RNA’s similar to that present in IRE’s (Iron Responsive Elements); in IRE’s this “stem-bulge” is critical for recognition by the Iron-Responsive Protein (IRP).
Does the bulge on the stem structure serve a specific a function in Y-RNA’s? (First off, is it real, and conserved? The "CG-bulge" is most frequently mutated in psuedogene Y-RNA (Perreault, et al., Nucleic Acids Res. 2005; 33(6): 2032–2041).
Structurally, this could be tested with a variety of Nuclease treatments. e.g., RNase V1 (dsRNA), RNase T1 (ssG), RNaseA (ssU & ssC)). Does mutation of this C-bulge change the binding/association with Ro? Any other ideas for testing experimentally?
Q1. The authors propose that the Y RNAs guide the formation of specific RNP complexes by strand hybridization and that these complexes are necessary for DNA replication. How might you identify nucleic acids which hybridize to a Y RNA?
Q2. How could you test the physiological importance of hybridization between Y RNA and it's target nucleic acid?
Hint: The paper I will be presenting uses some of these techniques to study BC1 RNA.
A1. Zalfa et al. (2003) Cell identify target RNAs which hybridize specifically to BC1 RNA by mixing and pulling down synthetic biotin labeled BC1 with purified total brain mRNA, followed by gene specific RT-PCR (among other experiments). Nucleic acids which hybridize specifically with Y RNAs could be found in a similar manner, by mixing synthetic biotin labeled Y RNA with purified total mRNA from fibroblasts, for example. mRNAs which hybridize to the Y RNA could be identified by gene specific RT-PCR or by microarray analysis.
A2. Zalfa et al. (2003) Cell
design oligos which compete for regions of complementarity between BC1 and target mRNAs and test whether hybridization is necessary for FMRP activity. Once target nucleic acids of Y RNAs are identified, a similar approach could be tested in vivo
by assaying for DNA replication following the introduction of oligos that compete for specific sites of hybridization.