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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

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Page 1: Investigating the function of a member of an organelle- dedicated RNA binding protein family Maritza Duarte Barkan Lab University of Oregon Institute of

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

Page 2: Investigating the function of a member of an organelle- dedicated RNA binding protein family Maritza Duarte Barkan Lab University of Oregon Institute of

Origin of Mitochondria and Chloroplasts

Transfer of some organellar genes to the nucleus

Page 3: Investigating the function of a member of an organelle- dedicated RNA binding protein family Maritza Duarte Barkan Lab University of Oregon Institute of

Dual-origin of nuclear genes encoding organellar RNA binding

proteins

Co-opted host derived

Bacterial ancestorsBacterial ancestors

Page 4: Investigating the function of a member of an organelle- dedicated RNA binding protein family Maritza Duarte Barkan Lab University of Oregon Institute of

*

**

*

**

*

*

*

*

*

*

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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

Page 5: Investigating the function of a member of an organelle- dedicated RNA binding protein family Maritza Duarte Barkan Lab University of Oregon Institute of

Nuclear Encoded Proteins required for chloroplast Group II intron splicing

My project :Grew out of the discovery of wtf1

Page 6: Investigating the function of a member of an organelle- dedicated RNA binding protein family Maritza Duarte Barkan Lab University of Oregon Institute of

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…

Page 7: Investigating the function of a member of an organelle- dedicated RNA binding protein family Maritza Duarte Barkan Lab University of Oregon Institute of

DUF 860 gene family

WTF2

The DUF860 family in rice and Arabidopsis

Page 8: Investigating the function of a member of an organelle- dedicated RNA binding protein family Maritza Duarte Barkan Lab University of Oregon Institute of

• 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.

Page 9: Investigating the function of a member of an organelle- dedicated RNA binding protein family Maritza Duarte Barkan Lab University of Oregon Institute of

Outline of approach

• Obtaining wtf 2 mutant in maize and Arabidopsis

• Analyzing morphological and molecular phenotypes to determine wtf 2 function

Page 10: Investigating the function of a member of an organelle- dedicated RNA binding protein family Maritza Duarte Barkan Lab University of Oregon Institute of

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

Page 11: Investigating the function of a member of an organelle- dedicated RNA binding protein family Maritza Duarte Barkan Lab University of Oregon Institute of

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

Page 12: Investigating the function of a member of an organelle- dedicated RNA binding protein family Maritza Duarte Barkan Lab University of Oregon Institute of

wtf2 lacks Chloroplast rRNAw

tf2-2

wtf2

-1

WT

WT

why

1-2

Ladd

er

28 S

18 SCP. 16SCP. 23S *

Ribosome subunitProteins RNA.

Page 13: Investigating the function of a member of an organelle- dedicated RNA binding protein family Maritza Duarte Barkan Lab University of Oregon Institute of

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

Page 14: Investigating the function of a member of an organelle- dedicated RNA binding protein family Maritza Duarte Barkan Lab University of Oregon Institute of

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

Page 15: Investigating the function of a member of an organelle- dedicated RNA binding protein family Maritza Duarte Barkan Lab University of Oregon Institute of

How are specific CP. RNAs affected?

Page 16: Investigating the function of a member of an organelle- dedicated RNA binding protein family Maritza Duarte Barkan Lab University of Oregon Institute of

Psaj-1 probe Pet G probe

Page 17: Investigating the function of a member of an organelle- dedicated RNA binding protein family Maritza Duarte Barkan Lab University of Oregon Institute of

Rpl2 probe Psac probe Psa-B probe

Page 18: Investigating the function of a member of an organelle- dedicated RNA binding protein family Maritza Duarte Barkan Lab University of Oregon Institute of

P tRNG 2 probe

Possible tRNA loss specific to wtf2 mutants

Mature tRNG

Page 19: Investigating the function of a member of an organelle- dedicated RNA binding protein family Maritza Duarte Barkan Lab University of Oregon Institute of

Does wtf2 mutation have different consequences in Dicots vs Monocots?

no cp translation

Maize wtf2 mutant

Arabidopsis wtf2 mutant

wtf2 disruption

Page 20: Investigating the function of a member of an organelle- dedicated RNA binding protein family Maritza Duarte Barkan Lab University of Oregon Institute of

Can Arabidopsis wtf2 mutants germinate?

Wtf2 mutant

WT

Page 21: Investigating the function of a member of an organelle- dedicated RNA binding protein family Maritza Duarte Barkan Lab University of Oregon Institute of

Conclusion

Page 22: Investigating the function of a member of an organelle- dedicated RNA binding protein family Maritza Duarte Barkan Lab University of Oregon Institute of

Future work

Page 23: Investigating the function of a member of an organelle- dedicated RNA binding protein family Maritza Duarte Barkan Lab University of Oregon Institute of

Acknowledgements

• SPUR • Peter O’day

And the entire Barkan lab…

Page 24: Investigating the function of a member of an organelle- dedicated RNA binding protein family Maritza Duarte Barkan Lab University of Oregon Institute of

Acknowledgements

Alice Barkan Susan Belcher

Tiffany Kroeger

Jana Prikryl

Margarita Rojas

Nicholas Stiffler

Kenneth Watkins

Rosalind Williams

Bobby Coalter

Martin

Sarah