investigating the function of a member of an organelle- dedicated rna binding protein family maritza...

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

Transfer of some organellar genes to the nucleus

Dual-origin of nuclear genes encoding organellar RNA binding

proteins

Co-opted host derived

Bacterial ancestorsBacterial ancestors

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* Group IIintrons

However CP. introns require nuclear encoded proteins for splicing

Many Group II introns in The Chloroplast genome

“Self 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

CAF1??

??

Mass Spec

Chloroplast Extract (Stroma) IP with CAF1

antibody

SDS- PAGE

WTF1 Protein Architecture

DUF860transit peptide

????

van Wijk Lab

Found:wtf1Others…

DUF 860 gene family

WTF2

The DUF860 family in rice and Arabidopsis

• 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.

DUF860 family characteristics

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

PML = ~2,000 Transposon (mu) induced non- photosynthetic mutants

WTF2

1

21

Tiffany Kroeger found the wtf2 mutants by PCR screens of the PML for mu inserts with in the wtf2 gene

Testing for Phenotype linkage of wtf2

wtf2

Plant then collect tissue

Run PCR to check for insert

Link genotype to phenotype

Obtain wtf 2 mutants

WTF2

1

wtf2 lacks Chloroplast rRNAw

tf2-2

wtf2

-1

WT

WT

why

1-2

Ladd

er

28 S

18 SCP. 16SCP. 23S *

Ribosome subunitProteins RNA.

Global analysis of chloroplast RNAs in wtf2 mutants

Total RNA

Tiling Microarray of the CP genome

Hybridized microarray with wtf2 and why1

Label with fluorescence dyes

why1

why1wtf 2

wtf 2

RNA transcriptional Profile why1 vs. wtf2 mutants

PsaB-1

petG

psaj psac

Fragment #/Chromosomal Position

L

ow

in w

tf2

H

igh

in w

tf2

trnG trnD

trnL

rpl2

trnF

How are specific CP. RNAs affected?

Psaj-1 probe Pet G probe

Rpl2 probe Psac probe Psa-B probe

P tRNG 2 probe

Possible tRNA loss specific to wtf2 mutants

Mature tRNG

Does wtf2 mutation have different consequences in Dicots vs Monocots?

no cp translation

Maize wtf2 mutant

Arabidopsis wtf2 mutant

wtf2 disruption

Can Arabidopsis wtf2 mutants germinate?

Wtf2 mutant

WT

Conclusion

Future work

Acknowledgements

• SPUR • Peter O’day

And the entire Barkan lab…

Acknowledgements

Alice Barkan Susan Belcher

Tiffany Kroeger

Jana Prikryl

Margarita Rojas

Nicholas Stiffler

Kenneth Watkins

Rosalind Williams

Bobby Coalter

Martin

Sarah