Scientific Report 2003-2004 - Cleveland Clinic Lerner Research ...

THE PADGETTLABORATORYPROJECT SCIENTISTSJayendra Prasad, Ph.D.Girish C. Shukla, Ph.D.POSTDOCTORAL FELLOWKrishna P. Kota, Ph.D.TECHNICAL ASSOCIATESRosemary C. Dietrich, M.S.Kelly EmmettJohn D. FullerCOLLABORATORSChristopher Burge, Ph.D. 1Kwaku Dayie, Ph.D. 21Dept. of Biology, Mass. Inst. ofTechnology, Cambridge, MA2Dept. of Molecular Biology, CCFRemoval of intervening sequences (introns)from the primary RNA transcripts of mostvertebrate genes is a required step in theexpression of genetic information. This premRNAsplicing process requires the formation ofa multicomponent complex known as thespliceosome at the sites of splicing. Thespliceosome is composed of over 200 proteinsand five small nuclear RNAs (snRNAs). ThesesnRNAs are involved in splice site recognitionsteps leading to the formationof the spliceosome.They are also believed to beinvolved in the catalyticsteps of RNA cleavage andligation. The research in mylaboratory is directed atunderstanding the processesinvolved in specifying thesites of splicing within a premRNAand the mechanismof the splicing reactions.A few years ago, weidentified a previouslyunsuspected second class ofintrons within eukaryoticgenes. Subsequent work hasshown that this second classis widely distributed innature and must have beenpresent for at least a billionyears. More recent work hasled us to argue that bothclasses of introns werepresent in one of the earliestancestors of eukaryoticorganisms.In modern organisms, these introns arespliced in a spliceosome composed of foursnRNAs unique to this class of introns and onesnRNA that is common to both classes ofintrons. The spliceosomal proteins are also likelyto contain a mixture of unique and sharedproteins. The splicing mechanisms and many ofThe Department of Molecular BiologyIntrons Removed from RNA byRNA-Based MachinesRichard A. Padgett, Ph.D.the RNA-RNA interactions involved in theformation and function of the spliceosome arestrikingly similar in both classes of introns, whichsuggests that they probably share a similar origin.Over the last several years, we have beenmapping the RNA-RNA interactions involved inthe splicing of this new class of introns usinggenetic and biochemical techniques. These studiesare directed toward understanding the structureand function of the spliceosome and gaininginsights into how theseintrons are identified by thesplicing machinery in theprimary RNA transcripts ofgenes.Recently, we havefocused on the function of asmall RNA element in thespliceosome that may beinvolved in the catalysis ofthe splicing reactions. Wehave shown that this elementcan be functionally replacedby a similar RNA elementfrom a class of self-splicingRNA introns. This findingsuggests that both thespliceosome and these selfsplicingintrons use similarcatalytic mechanisms andsupports the idea thatspliceosomal introns evolvedfrom self-splicing introns.We are pursuing structuralstudies of these RNAelements to further definetheir functions in splicing.We are also extending our studies of theminor class spliceosome to the investigation ofthe proteins involved in the splicing of theseunusual introns. We will determine the overlapof protein factors between the two splicingsystems and identify proteins unique to the newlydiscovered spliceosome.Shukla, G.C., and R.A. Padgett (2001) The intramolecular stem-loop structure of U6 snRNA can functionallyreplace the U6atac snRNA stem-loop. RNA 7:94-105.Dietrich, R.C., Peris, M.J., Seyboldt, A.S., and R.A. Padgett (2001) Role of the 3’ splice site in U12-dependentintron splicing. Mol. Cell. Biol. 21:1942-1952 (additional information in Dietrich et al., Mol. Cell.Biol. 2002;21:3563).Shukla, G.C., and R.A. Padgett (2002) A catalytically active group II intron domain 5 can function in theU12-dependent spliceosome. Mol. Cell 9:1145-1150.Padgett, R.A., and G.C. Shukla (2002) A revised model for U4atac/U6atac snRNA base pairing [letter].RNA 8:125-128.Shukla, G.C., Cole, A.J., Dietrich, R.D., and R.A. Padgett (2002) Domains of human U4atac snRNA requiredfor U12-dependent splicing in vivo. Nucl. Acids Res. 30:4650-4657.108