1. 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

2. 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

3. 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

4. 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

5. 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.

6. 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

7. 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

8. 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.

9. Outline of approach Obtaining wtf 2 mutant in maize and Arabidopsis
Analyzing morphological and molecular phenotypes to determine wtf 2 function

10. 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

11. 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

12. 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 *

13. 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

14. 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

15. How are specific CP. RNAs affected?

16. 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

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

18. 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

19. 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

20. 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

21. 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

22. 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

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

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