BIOREG DISCUSSION QUESTIONS
February 14, 2003
Tarn, W.-Y. and Steitz, J. A. (1996) Highly diverged U4 and U6 small nulcear RNAs required for splicing rare AT-AC introns. Science 273, 1824-1832.
1. What experiments would you perform to confirm the base pairing predicted in Fig. 1B?
2. The authors identified sites of psoralen crosslinking (from Fig. 2) by primer extension. Why were two stops observed with the U6atac primer and only one with the U4atac primer? What controls were necessary to prove that the stops were due to crosslinks?
3. Why was it expected (p. 1827) that U6atac would not be found in complexes A-1 and A-2 (Fig. 3)?
4. Why were 2'-O-methyl, rather than DNA or RNA, oligonucleotides used to block the splicing reactions (Fig. 4)? Why did the authors use a DNA oligonucleotide to test the importance of U4atac?
5. Why were psoralen crosslinks oberved between U6atac and U12 in the experiments of Fig. 5 and only between U6atac and U4atac in Fig. 2?
6. What is the principle of phosphorothioate interference (mentioned on p. 1831)? What other types of studies commonly use interference experiments?
7. Why might this rare splicing pathway exist? What complications do you envision to having parallel spliceosomes?