Institute of Molecular Biology Investigating the function of a member of an organelle-dedicated RNA binding protein family Maritza Duarte Barkan Lab University of Oregon Institute of Molecular Biology

Origin of Mitochondria and Chloroplasts Mito and chloro orginated when … the host pre-eukaryotic cell engulfed prokaryotic cells. After endosymbiosis, the evolution of mitochondria and chloroplast was accompanied by a large scale transfer of their genes to the nucleus. Transfer of some organellar genes to the nucleus

Dual-origin of nuclear genes encoding organellar RNA binding proteins Co-opted host derived During this co-evolution of the 3 separate genomes, both organelles acquired new features unlike their bacterial ancestors. In concert with these newly acquired features new nuclear genes were evolved . these genes encode proteins which are targeted to the organelles and provided functions that confer with these new traits . Resulting in two types of nuclear genes that encoded organelle proteins those that are of bacterial ancestor and those that are host derived. Bacterial ancestors

Many Group II introns in The Chloroplast genome * Group II introns One example of an acquired non-prokaryotic trait is the increased number of introns. These introns are called group 2 intorns which differ from those in the nucleause, which utilize splicesomes. group 2 introns are derived from self splicing RNA. However the cp group 2 introns can no longer self splice and require nuclear encoded proteins. “Self splicing” Group II introns However CP. introns require nuclear encoded proteins for splicing

Nuclear Encoded Proteins required for chloroplast Group II intron splicing My project : Grew out of the discovery of wtf1 . this is an image of the known nuclear encoded proteins which are required for the splicing of cp introns . walk though the pic. One example is wtf1 which is required for splicing several introns ( point out). The barkan lab believes that there are more of these nuc encoded proeteins to be discovered . My project grew out of the discover of this protein WTF1 which was the first characterized member of an unusual family of organelle binding proteins.

Discovery of DUF860/ WTF1 Found: wtf1 Others… transit peptide DUF860 CAF1 ???? van Wijk Lab Mass Spec Found: wtf1 Others… Chloroplast Extract (Stroma) ?? ?? SDS- PAGE Here is how we discovered wtf1 Barkan lab used an antibody to the known splicing factor CAF1. this antibody was used in a co IP to a chloroplast extract revealing a large complex of proteins which were then run an a gel and sent to then van wijik lab for mass spec analysis. One of the proteins that was found associated with CAF1 was WTF1 a member of the DUF 860 family. “ Domain of unknown function” None of these proteins have ever been studied and so we named it WTF1 “ What's this factor” 1 this protein contains a transit peptide which localizes it to the chloroplast and the Duf860 domain. IP with CAF1 antibody WTF1 Protein Architecture DUF860 transit peptide

DUF 860 gene family The DUF860 family in rice and Arabidopsis WTF2 The Duf 860 domain is found in a family of around 15 genes in plant genomes. It is unrelated to any bacterial genes and isn't related to any gene of known function. Genes with in this family are predicted to target either the chloroplast, those in green, or to the mitochondria, those in red. my project will explore another family member Point out wtf2 , in order to expand our understanding of duf 860 and its functions 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. ADD concise project description Investigating the function of Chloroplast targeted DUF860 protein : WTF2. 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 Tiffany Kroeger found the wtf2 mutants by PCR screens of the PML for mu inserts with in the wtf2 gene 1 PML = ~2,000 Transposon (mu) induced non- photosynthetic mutants PML – photosynthetic mutant library consists of around ~2000 transposon induced maize. These plants have had random genes disrupted by mu a transposable element and represent a wide spectrum of cp disruption levels. Tiffany used PCR to screen the entire PML to find plants which had disrupted wtf2 genes. After the screening was complete she found 2 alleles of the mu induced wtf2 gene. Both insertions are with in the open reading frame. 1 2 WTF2

Testing for Phenotype linkage of wtf2 Obtain wtf 2 mutants Plant then collect tissue Run PCR to check for insert Link genotype to phenotype To start my research I began with. Obtaining wtf 2 mutants . I then planted and collected tissue from the plants. After isolating DNA I used PCR genotyping to check for a mu insert within the wtf2 gene. i then linked the mutant genotype to a specific phenotype. The wtf2 mutant was a ivory/ ivoresecent color. 1 WTF2

wtf2 lacks Chloroplast rRNA why1-2 Ladder 28 S The next step was to analyze how wtf2 disruption affects cp RNA. On this RNA gel, we can see that both wtf2 mutants lack cp rRNA, the bottom two bands. ( point out bands) This gel also contained another mutant why1 which also has lower cp rRNA. I included with mutant as another control in other experiments to account for any secondary affects caused by cp. rRna. Lowercase Why, WTF!! Reddish orange 18 S Ribosome subunit Proteins RNA. CP. 16S CP. 23S *

Global analysis of chloroplast RNAs in wtf2 mutants Total RNA wtf 2 why1 Hybridized microarray with wtf2 and why1 Label with fluorescence dyes in order to test how wtf2 affects CP RNA levels I used a microarray chip. This chip contained a tiling of CP genome fragments. The first step of this process is to isolate RNA ---- The data is then converted to statistical data and graphed Tiling Microarray of the CP genome wtf 2 why1

RNA transcriptional Profile why1 vs. wtf2 mutants trnG trnL trnD trnF P24/WTF2 Median Log2 petG Low in wtf2 psaj psac {Label more points} The resulting graph High in wtf2 rpl2 PsaB-1 Fragment #/Chromosomal Position

How are specific CP. RNAs affected?

Psaj-1 probe Pet G probe wtf 2-1 why1-2 wtf2-2 why1-1 WT wtf 2-1 Psaj-1 Mature product is lower in accumulation Not much diff pet G

Rpl2 probe Psa-B probe Psac probe why1-2 wtf 2-1 WT wtf2-2 why1-1 Looks like why1

Possible tRNA loss specific to wtf2 mutants P tRNG 2 probe why1-1 wtf2-2 wtf 2-1 WT why1-2 Heading: possible trna loss specific to wtf2 mutants why1-2 ( pyg) is degraded Why1-1 is IV Mature tRNG

Does wtf2 mutation have different consequences in Dicots vs Monocots? wtf2 disruption no cp translation Maize wtf2 mutant After seeing that wtf2 disruption causes CP Ribosome loss in maize. The next questionis if wtf2 disruption lead to chlorplast ribosome loss in Arabidopsis. The differnce being monocots vs. dicots. The phenotypic result of wtf2 muation in Arabidopsis is shivraled raisin like seeds vs. maize which grow into albino plants. Arabidopsis wtf2 mutant

Can Arabidopsis wtf2 mutants germinate? SAY how I did it first point out seed phenotype I was only able to produce 1 mutant completely albino Wtf2 mutation = No cp= embryo lethality 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 biogenesis of CP Ribosomes Plays a role in CP tRNAs biogenesis? is it involved in metabolism of tRNAs or Ribosomal protein mRNAs or rRNAs … ? Darker softer colors

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 Add tRNA’ s ? Darker

Acknowledgements SPUR Peter O’day And the entire Barkan lab…

Acknowledgements Alice Barkan Susan Belcher Martin Kenneth Watkins Jana Prikryl Nicholas Stiffler Tiffany Kroeger Rosalind Williams Margarita Rojas Sarah Bobby Coalter