Download WTF2 - SPUR - University of Oregon

Investigating the function of a
member of an organellededicated 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 ancestors
Many Group II introns in The
Chloroplast genome
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“Self splicing” Group II introns
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However CP. introns require nuclear
encoded proteins for splicing
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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
van Wijk Lab
Mass Spec
CAF1
Chloroplast
Extract
(Stroma)
??
??
IP with CAF1
antibody
transit
peptide
SDS- PAGE
DUF860
WTF1 Protein Architecture
Found:
wtf1
Others…
DUF 860 gene family
WTF2
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.
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
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Tiffany Kroeger found the
wtf2 mutants by PCR screens
of the PML for mu inserts
with in the wtf2 gene
PML = ~2,000 Transposon (mu) induced
non- photosynthetic mutants
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WTF2
Testing for Phenotype linkage of wtf2
wtf2
Obtain wtf 2 mutants
Plant then collect tissue
Run PCR to check for insert
Link genotype to phenotype
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WTF2
wtf2 lacks Chloroplast rRNA
28 S
18 S
CP. 16S
CP. 23S *
Ribosome subunit
Proteins RNA.
Global analysis of chloroplast
RNAs
in
wtf2
mutants
Total RNA
wtf 2
why1
Label with fluorescence dyes
wtf 2
why1
Tiling Microarray of
the CP genome
Hybridized
microarray with
wtf2 and why1
trnD
trnL
trnF
petG
psaj
rpl2
PsaB-1
Fragment #/Chromosomal Position
psac
High in wtf2
P24/WTF2 Median Log2
trnG
Low in wtf2
RNA transcriptional Profile why1 vs.
wtf2 mutants
How are specific CP. RNAs affected?
Pet G probe
Psaj-1 probe
Psa-B probe
Psac probe
Rpl2 probe
Possible tRNA loss specific to wtf2 mutants
P tRNG 2 probe
Mature tRNG
Does wtf2 mutation have different
consequences in Dicots vs Monocots?
wtf2 disruption
no cp translation
Maize wtf2 mutant
Arabidopsis wtf2 mutant
Can Arabidopsis wtf2 mutants
germinate?
Wtf2
mutant
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 Plays a role in CP
biogenesis of CP tRNAs
Ribosomes
biogenesis?
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
Acknowledgements
• SPUR
• Peter O’day
And the entire Barkan lab…
Acknowledgements
Alice Barkan
Susan Belcher
Martin
Kenneth Watkins
Tiffany Kroeger
Margarita Rojas
Jana Prikryl
Rosalind Williams
Sarah
Nicholas Stiffler
Bobby Coalter