Download Methods Projects Introduction Myotonic dystrophy type 1

Exploring post-transcriptional events in human disease
Damgaard lab
Christian Kroun Damgaard
[email protected]
Room 404, Building 1130 29700599
Molekylærbiologi: Ja/Yes
Molekylær medicin: Ja/Yes
Bioteknologi: Nej/No
Introduction
The eukaryotic cell possesses numerous gene-regulatory mechanisms to
control cell function according to given conditions and environmental
cues. These include rapid changes in gene-expression elicited at almost
every thinkable level inside the cell - events often deregulated in disease.
Historically, much attention has been given to the regulation of transcription and mRNA processing events, which in turn produce a tremendous
diversity from metazoan genes. These important regulated events aside,
there is now increasing evidence that cytoplasmic processes, including
regulation of both global and local protein translation and mRNA stability
are crucial modulatory instruments for the cell during development, cell
growth and as primary responses to environmental changes. These processes are governed by, both RNA-binding proteins (RBPs) and large
classes of ncRNAs, including circular RNAs (circRNAs), long non-coding
RNAs (lncRNAs) and microRNAs (miRNAs). In my laboratory we study the
function of all these types of RNA- and protein regulators in tightly controlled translation and mRNA decay and assess how their deregulated
function impact diseases like myotonic dystrophy (DM1), Neurodegenerative disease and cancer.
DMPK mRNA
Myotonic dystrophy
RBP1
RBP1 RBP1
An
Pre-mRNP
AA
A
UG
Capping
G
AU
mRNA
Processing body (PB)
Decay
AUG
N
RBP2
AUG
UGA
AUG
RISC
AUG
N
3’->5’ decay
A
N
N
N
N
N
N
N
N
UGA
Dcp2
RBP1
5’->3’ decay
N
N
N
N
N
N
Xrn1
N
circular RNA sponge
Stress granule
Stress granules (SGs)
A0
AUG
N
N
Circular RNA
RBP1
RBP1
lncRNA
3’->5’ decay
Exosome
A
N
N
N
N
N
N
N
N
N
N
Exosome
PB
AAAn
An
TIA-1
TIAR
TIA-1
TIAR
TIAR
TIAR
An
TIAR
TIAR
TIAR
PB
TIA-1
TIA-1
TIAR
An
TIA-1
TIA-1 TIAR
TIA-1
TIAR
TIA-1TIAR
TIA-1
TIA-1 TIAR
TIA-1
TIA-1
TIA-1
TIA-1
Deadenylation
A
A
A Pan2/3
A
A0
A A
(PARN)
A
Ccr4/Not
Decapping
miRNA
AAAn
AAAn
UGA
AUG
N
Xrn1
N
UGA
UGA
An
3’->5’ decay
Exosome
Ccr4//Not
TIA AUG
?
AAA
N
N
UGA
Default
Regulated: ARE- or miRNA-mediated decay
5’->3’ decay
N
N
AUG
lncRNA
UGA
An
Stress
?
AAA
Translation
Regulatory
factors
AUG
N
AUG
UGA
eIFs, eEFs, eRFs etc.
RNases
PB
UGA
Nuclear
membrane
(Localization?)
PB
Dcp2
N
AUG
mRNP
mRNP remodeling?
Processing bodies (PBs)
N
5’TOP-mRNA
Splicing (snRNPs)
hnRNPs
Polyadenylation
cell cycle arrest
nutrient starvation
SG
Figure 1: Postranscriptional regulation of gene expression. After a processed mRNA has been exported to the cell cytoplasm it can become regulated at the level of localization, translation and decay. This is mediated by several regulatory factors including
translation- (e.g. green/orange TIA-proteins), decay factors (pacmen) and RNA binding proteins (e.g. “AU-rich binding proteins” (AUBPs)). Current work in the lab is concerned with the regulation of mRNA localization, translation and decay. Many regulated mRNAs accumulate in cytoplasmic foci termed processing bodies (PBs) and stress induce accumulation of certain mRNAs i stress granules (SGs). Both granules contain repessed mRNAs but PB tethered mRNAs are thought to undergo rapid decay.
Stress granules: mRNA-dependent aggregation of
stalled translational complexes (48S) occuring under
stress conditions (oxidative stress, UV-irradiation a.o.).
TIA-1 and TIAR are essential for SG-aggregation, which
occurs via their prion-like domain.
Processing Body: mRNA-dependent aggregation of
decapping enzyme (hDcp2) and its co-factors: hDcp1a, b,
Edc3, Hedls, Lsm4, Rck/p54 among others. ARE-mRNAs,
siRNA targets, miRNA targets, NMD substrates accumulate
in PBs when decay factors are limiting.
Methods
SG/PB
CUG-expanded DMPK mRNA
in DM1
PB
SG
Chase/hrs
0 2 4 8 20
0 2 4
8 20
Control
Human cell culture
Stable inducible cell lines
RNA In Situ Hybridization
Immunofluoresence
Co-localization
Protein Immunoprecipitation
Figure 2: RNA Fluroescent In Situ Hybridization (RNA-FISH) for visualizing “stress granules” (SGs),
RNA Immunoprecipitation
Translation analysis - Polysome fractionation “processing bodies” (PBs) or sequestered nuclear mRNPs from patients with myotonic dystrophy (DM1).
mRNA Decay Assays (Pulse-Chase)
Subcellulær fractionation
Protein-RNA binding
Recombinant protein expression
RNAi
RNA-seq
RIP-seq
Ribosome profiling
Avitag-mediated visualization of
local translation
Control
Unstable
Stable
Figure 5: mRNA pulse-chase decay assay.
Reporter mRNA expression is pulsed/chased.
bead
RBP
Specific
mRN
A
FLAG
IP
EM
P
AU TY
BP
AU 1
BP
AU 2
BP
3
EM
P
AU TY
BP
AU 1
BP
AU 2
BP
3
AAA
Input
Controls
Figure 3: Immunofluorescence revealing co-localization between two proteins in processing bodies (PBs).
Figure 6: “mRNA Immunoprecipitation” (RIP). Northern/RNA-seq.
mRNA
X
10% Sucrose
Y
Z
AAAAAA
P
50% Sucrose
Monosomes
Polysomes
Figure 4: co-immunoprecipitation
Q
Figure 7: Polysome profiling
Poly
Mono
Figure 8: De novo translation assay - BioMIST
Myotonic dystrophy type 1 - DDX6
Projects
1) Studying mechanisms regulating gene expression during cellular stress
Highlights: Translation of so-called 5’-terminal oligopyrimidine tract mRNAs (5’TOP-mRNAs) is selectively inhibited during nutrient
starvation by LARP1 and the integral stress granule proteins TIA-1 and TIAR in a redundant manner.
Project 1:
Project 2:
Project 3:
Identification and functional characterization of posttranslational modifications of AUBPs including LARP1, TIA-1
and TIAR proteins affecting functionality.
Identification of mRNA species that are regulated by LARP1/TIA-1/TIAR during cellular stress by RNA-seq.
Functional analyses of mass-spec identified LARP1/TIA-1/TIAR interaction partners using global and biochemical
assays.
2) Studying mechanisms of miRNA-mediated repression of local translation in polarized cells (epithelial and neuronal cells)
What is the spatiotemporal and functional interplay between mRNAs, miRNAs and RNA sponges in polarized cells?
Highlights: We recently discovered that circular RNAs are able to function as ‘sponges’ for miRNAs and sequesters miRNAs away from
their normal functional locale.
Project 1:
Project 2:
Establishment of a de novo translation assay by in situ visualization
Functional studies of miRNA/sponge interactions in neuronal cells using cellular imaging and biochemical approaches.
3) Studying mechanisms of circRNA-mediated regulation of translation and/or mRNA decay
What is the spatiotemporal and functional interplay between mRNAs and circRNAs in neuronal development?
Highlights: We recently discovered >10000 circular RNAs differentially expressed in developing neurons.
Project 1:
Project 2:
Specific knockdown of candidate circRNA and assess functional impact on cell biology and gene-expression.
Identification of the circRNA interactome by RNA pull-down followed by Mass Spectrometry (MS)
Patient fibroblasts
TF
CUGBP1
CUGBP1
CUG-BP1:
Binding/upregulation
(Splicing)
Transcription factors:
Sequestration?
(Transcription) ds-RNA: Activation hnRNPs/DDX5/17
binding/upregulation
of PKR
(Splicing)
(Global translation)
hnRNP hnRNP
G C
MBNL1:
H
F
DDX17
U
U
Sequestration
C G
G C (Splicing, mRNA decay,
U
U
C G
polyadenylation)
Antisense transcription
DDX5
G C
U
U
(miRNA/RNAi response)
C G
G C
U
U
C G
G C
Non-ATG translation
MBNL1
DDX5 DDX17
U
U
C G
polyglutamine peptides
G C
U
U
DDX5: Unwinding?
cytoplasm (Toxicity)
C G
G C
Stimulation of
MBNL1
U
U
C G
MBNL1 binding
G C
U
U
MBNL1
(Splicing)
C G
G C
U
U
C
G
C
G
UDDX6 U
C
G
C
G
Upf1: Steady-state
U
U
C
G
and foci dynamics
G C
MBNL1
U
U
(mRNA decay - NMD?)
C G
G C
DDX6
U
U
MBNL1
C G
G C
Stau1 Stau1
U
U
C G
G C
U
U
Stau1:Binding/upregulation
DDX6: Binding/unwinding?
C G
(Splicing/nuclear export/translation UG CU
Stripping off MBNL1?
C G
mRNA decay - SMD?)
(Splicing/nuclear export)
G C
MBNL1
+
DDX6 si
Y
X
Cytoplasmic lysate
Contr si
FLAG
Sucrose gradient
Patient muscle
MBNL1
MBNL1
Upf1
MBNL1
+
MBNL1
DDX6 si
bead
IP
+
Figure 9: Molecular mechanisms
in DM1.
Contr si
FLAG-tagged AUBP immunoprecipitation
Input
Figure 10: DDX6 knockdown
increases DMPK mRNA aggregation in patient cells.
Figure 10: DDX6 expression relieves nuclear foci
formation and relieves splicing defect in patient
cells.