Download TARBP2 mediated post-transcriptional regulation of gene

Role of DICER cofactors
during post-transcriptional
regulation of gene expression
in vivo
Central Dogma of Life
Histone modifications
Transcription factors
RNA binding proteins
(TARBP2)
miRNA, siRNA
Ribonucleases
Modifications
Ubiquitination
Double stranded RNA binding
protein TARBP2
TARBP2
 Ubiquitous expression during embryogenesis and postnatally
 Highly expressed in mammalian testis
 Our lab is the first to identify its role in post-transcriptional
regulation
TARBP2 is cytoplasmic and
expressed at high levels in round
spermatids
rs = round spermatids
es = elongated spermatids
Lee et al., 1996
Tarbp2-/- mice have defective spermiogenesis
and translational activation of Prm1
PRM1
Zhong et al., 1999
TARBP2 in canonical miRNA Biogenesis
pre-miRNA
pre-miRNA
pri-miRNA
Xpo5
miRNA duplex
>
>
Ago
Nucleus
Cytoplasm
RISC
Translation
repression
TARBP2/P
RKRA
mRNA
degradation
DICER mutant mouse embryos die by
E7.5 developmental stage
Dicer-/-
Study Questions
• Are TARBP2 and PRKRA cofactors of DICER in
vivo?
• Do TARBP2 and PRKRA exhibit functional
redundancy?
• Is
TARBP2
mediated
post-transcriptional
regulation of gene expression during male
gametogenesis dependent or independent of
miRNA biogenesis?
Genetic mouse models of Tarbp2 and Prkra
Tarbp2tm1Reb
Prkra
Effects of Dicer and its cofactors Tarbp2,
Prkra in embryonic development
Dicer-/Tarbp2 or Prkra single mutant
Tarbp2, Prkra double mutants
Do TARBP2 or PRKRA
influence the mature miRNA
population in vivo?
miR-145a
miR-127
let7e
miR-484
miR-1981
miR-148b
WT vs. Prkra-/-
WT vs. Tarbp2-/-
Differential expression of miRNAs in Tarbp2-/embryos but not in Prkra-/- embryos compared
to wild type embryos
Validation of miRNA sequencing data in
Tarbp2-/- and Tarbp2+/+
Relative Fold Change
1.8
1.6
1.4
1.2
***
***
***
***
***
*
1
0.8
Wild Type
0.6
Mutant
0.4
0.2
0
Conclusions
• TARBP2 and PRKRA are not required
for all functions of DICER
• Tarbp2 but not Prkra is required for
normal expression of a subset of
miRNAs
Role of TARBP2 during
gametogenesis
• Highly expressed in testis and is restricted
to germ cells
• Whole body knock out in B6129S1 mice
are sterile
• The same miRNAs deregulated in E15.5
stage are deregulated in adult testis
Deregulation of mature miRNA in
Tarbp2-/- testis
Relative fold Change
3
*
2.5
2
1.5
***
1
0.5
0
*
**
* *** *** *** *** *
Wild Type
Mutant
Cell specific knock out model
TarbpcKO
Da-Zhi Wang et al., Nature Genetics 47,776–783(2015) doi:10.1038/ng.3324
Tarbp2tm1Reb
Cre recombinase for cell specific
deletion of Tarbp2
PGC’s
Gonocytes
Stra8
Hspa2
Cre-promoter
Spermatogonia
Spermatocytes
(P)3
Leptotene/Zygotene
(pre-meiotic cells)
Spermatid
Tarbp2fl/fl:Stra8icre+ phenotype
Testis cross-section
Wild type
Wild type
Epididymis cross-section
Mutant
Mutant
Mutant
Mutant
Wild type
Aundiff
SSC
Aal
AS
AS
Apr
TARBP2
A1>A2>A3>A4
In>B
Meiosis
Spermiogenesis
Future experiments
 Is TARBP2 essential for miRNA
biogenesis in germ cells?
miRNA sequencing
 Does TARBP2 regulate mRNA levels or
80s
translation?
mRNP
mRNA sequencing
60s
Polysomal sequencing
40s
Polysomes
Chris McCarthy, Braun Lab
Acknowledgements
 Braun Lab
•
•
•
•


Robert E.Braun
Manju Sharma
Chris McCarthy
Elizabeth Snyder
Bill Buaas
Nichelle Gray

Genome technologies Core

Computational Core
• Vivek M. Phillip
• Anuj Srivastav
• Vishal K. Sarsani
Thesis committee
•
•
•
•
•
Dr. Robert E.Braun
Dr. Gregory Cox
Dr. Greg Carter
Dr. Mary Ann Handel
Dr. Voot P. Yin
Funding Source:
 March Of Dimes
foundation
 CCSG grant CA034196 to
The Jackson Laboratory