investigating the function of a member of an organelle- dedicated rna binding protein family maritza...
TRANSCRIPT
![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](https://reader036.vdocuments.us/reader036/viewer/2022062315/5697bfdd1a28abf838cb1d1a/html5/thumbnails/1.jpg)
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](https://reader036.vdocuments.us/reader036/viewer/2022062315/5697bfdd1a28abf838cb1d1a/html5/thumbnails/2.jpg)
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](https://reader036.vdocuments.us/reader036/viewer/2022062315/5697bfdd1a28abf838cb1d1a/html5/thumbnails/3.jpg)
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](https://reader036.vdocuments.us/reader036/viewer/2022062315/5697bfdd1a28abf838cb1d1a/html5/thumbnails/4.jpg)
*
**
*
**
*
*
*
*
*
*
***
*
*
* 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](https://reader036.vdocuments.us/reader036/viewer/2022062315/5697bfdd1a28abf838cb1d1a/html5/thumbnails/5.jpg)
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](https://reader036.vdocuments.us/reader036/viewer/2022062315/5697bfdd1a28abf838cb1d1a/html5/thumbnails/6.jpg)
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](https://reader036.vdocuments.us/reader036/viewer/2022062315/5697bfdd1a28abf838cb1d1a/html5/thumbnails/7.jpg)
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](https://reader036.vdocuments.us/reader036/viewer/2022062315/5697bfdd1a28abf838cb1d1a/html5/thumbnails/8.jpg)
• 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](https://reader036.vdocuments.us/reader036/viewer/2022062315/5697bfdd1a28abf838cb1d1a/html5/thumbnails/9.jpg)
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](https://reader036.vdocuments.us/reader036/viewer/2022062315/5697bfdd1a28abf838cb1d1a/html5/thumbnails/10.jpg)
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](https://reader036.vdocuments.us/reader036/viewer/2022062315/5697bfdd1a28abf838cb1d1a/html5/thumbnails/11.jpg)
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](https://reader036.vdocuments.us/reader036/viewer/2022062315/5697bfdd1a28abf838cb1d1a/html5/thumbnails/12.jpg)
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](https://reader036.vdocuments.us/reader036/viewer/2022062315/5697bfdd1a28abf838cb1d1a/html5/thumbnails/13.jpg)
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](https://reader036.vdocuments.us/reader036/viewer/2022062315/5697bfdd1a28abf838cb1d1a/html5/thumbnails/14.jpg)
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](https://reader036.vdocuments.us/reader036/viewer/2022062315/5697bfdd1a28abf838cb1d1a/html5/thumbnails/15.jpg)
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](https://reader036.vdocuments.us/reader036/viewer/2022062315/5697bfdd1a28abf838cb1d1a/html5/thumbnails/16.jpg)
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](https://reader036.vdocuments.us/reader036/viewer/2022062315/5697bfdd1a28abf838cb1d1a/html5/thumbnails/17.jpg)
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](https://reader036.vdocuments.us/reader036/viewer/2022062315/5697bfdd1a28abf838cb1d1a/html5/thumbnails/18.jpg)
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](https://reader036.vdocuments.us/reader036/viewer/2022062315/5697bfdd1a28abf838cb1d1a/html5/thumbnails/19.jpg)
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](https://reader036.vdocuments.us/reader036/viewer/2022062315/5697bfdd1a28abf838cb1d1a/html5/thumbnails/20.jpg)
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](https://reader036.vdocuments.us/reader036/viewer/2022062315/5697bfdd1a28abf838cb1d1a/html5/thumbnails/21.jpg)
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](https://reader036.vdocuments.us/reader036/viewer/2022062315/5697bfdd1a28abf838cb1d1a/html5/thumbnails/22.jpg)
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](https://reader036.vdocuments.us/reader036/viewer/2022062315/5697bfdd1a28abf838cb1d1a/html5/thumbnails/23.jpg)
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](https://reader036.vdocuments.us/reader036/viewer/2022062315/5697bfdd1a28abf838cb1d1a/html5/thumbnails/24.jpg)
Acknowledgements
Alice Barkan Susan Belcher
Tiffany Kroeger
Jana Prikryl
Margarita Rojas
Nicholas Stiffler
Kenneth Watkins
Rosalind Williams
Bobby Coalter
Martin
Sarah