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Investigating the function of a member of an organellededicated RNA binding protein family Maritza Duarte Barkan Lab University of Oregon Institute of Molecular Biology Origin of Mitochondria and Chloroplasts Transfer of some organellar genes to the nucleus Dual-origin of nuclear genes encoding organellar RNA binding proteins Co-opted host derived Bacterial ancestors Many Group II introns in The Chloroplast genome ** * * * * * * * * * * * “Self splicing” Group II introns * ** However CP. introns require nuclear encoded proteins for splicing * * Group II introns Nuclear Encoded Proteins required for chloroplast Group II intron splicing My project : Grew out of the discovery of wtf1 Discovery of DUF860/ WTF1 van Wijk Lab Mass Spec CAF1 Chloroplast Extract (Stroma) ?? ?? IP with CAF1 antibody transit peptide SDS- PAGE DUF860 WTF1 Protein Architecture Found: wtf1 Others… DUF 860 gene family WTF2 The DUF860 family in rice and Arabidopsis DUF860 family characteristics • the only characterized DUF860 member is a plastid splicing factor, WTF1 • WTF1 binds RNA in vitro and in vivo. • all members predicted to localize to the mitochondria or chloroplast. My Project will investigate the function of another chloroplast targeted DUF860 protein. This protein is named wtf2 and is hypothesized to affect CP. RNA metabolism like wtf1. Outline of approach • Obtaining wtf 2 mutant in maize and Arabidopsis • Analyzing morphological and molecular phenotypes to determine wtf 2 function Reverse Genetics with the Photosynthetic Mutant Library to find wtf mutants 1 Tiffany Kroeger found the wtf2 mutants by PCR screens of the PML for mu inserts with in the wtf2 gene PML = ~2,000 Transposon (mu) induced non- photosynthetic mutants 1 2 WTF2 Testing for Phenotype linkage of wtf2 wtf2 Obtain wtf 2 mutants Plant then collect tissue Run PCR to check for insert Link genotype to phenotype 1 WTF2 wtf2 lacks Chloroplast rRNA 28 S 18 S CP. 16S CP. 23S * Ribosome subunit Proteins RNA. Global analysis of chloroplast RNAs in wtf2 mutants Total RNA wtf 2 why1 Label with fluorescence dyes wtf 2 why1 Tiling Microarray of the CP genome Hybridized microarray with wtf2 and why1 trnD trnL trnF petG psaj rpl2 PsaB-1 Fragment #/Chromosomal Position psac High in wtf2 P24/WTF2 Median Log2 trnG Low in wtf2 RNA transcriptional Profile why1 vs. wtf2 mutants How are specific CP. RNAs affected? Pet G probe Psaj-1 probe Psa-B probe Psac probe Rpl2 probe Possible tRNA loss specific to wtf2 mutants P tRNG 2 probe Mature tRNG Does wtf2 mutation have different consequences in Dicots vs Monocots? wtf2 disruption no cp translation Maize wtf2 mutant Arabidopsis wtf2 mutant Can Arabidopsis wtf2 mutants germinate? Wtf2 mutant WT Conclusion Pleiotrophic effects caused by lack of CP ribosome make it difficult to analyze specific affects of wtf2 disruption However I found wtf2 : Is required for the Plays a role in CP biogenesis of CP tRNAs Ribosomes biogenesis? Future work What RNAs does wtf 2 bind with in vivo? • Raise antibody. Find in vivo target using co-IP & microarray Does wtf 2 bind these target RNAs in vitro? • RNA binding assay Acknowledgements • SPUR • Peter O’day And the entire Barkan lab… Acknowledgements Alice Barkan Susan Belcher Martin Kenneth Watkins Tiffany Kroeger Margarita Rojas Jana Prikryl Rosalind Williams Sarah Nicholas Stiffler Bobby Coalter