Post on 17-Aug-2015
Identifying the Function of the FMRP RGG box
January 2010
Ernest “Tory” BlackwellCeman Lab
Overview
• Background• Arginine residues and polysome
association• Arginine residues and RNA association • In vivo methylation• Model*
*may contain modest conjecture and pure speculation
Fragile X Syndrome (FXS)
• Mental retardation• Impaired language skills• Echolalia
• Autistic-like behavior• Epilepsy
• Caused by loss of the fragile X mental retardation protein (Fmrp)
• Fmrp is an RNA binding protein
• Fmrp is thought to be a translational regulator
The domain structure of Fmrp
• Methylated on arginines 533, 538, 543, and 545• Methylated by PRMT1 and PRMT3 in vitro• Methylation inhibits RNA binding in vitro
Stetler A. et al., Hum. Mol. Genet. 2006. Denman, Cell. Mol. Biol. Lett. 2002.
RRGDGRRRGGGGRGQGGRGRGGGFKGNDDHSR
NLS KH2KH1 NES RGG
The RGG box affects how well Fmrp associates with polysomes
Fmrp lacking the RGG box accumulates in early polysomal fractions
Mazroui et al., Hum. Mol. Gen. 2003.
10% 50%sedimentation
40S60S
80S
Recap:• Four arginines within the RGG domain are methylated
• Methylation inhibits RNA association in vitro
• The RGG domain is required for proper polysome association
Fmrp
Fmrp
methylation
Loss of RNA binding
Fmrp
Loss of RGG domain
Abnormal polysome association suggesting loss of RNA binding
Are the arginines that are methylated important for polysome association?
WT
∆RGG
533, 538, 543, 545m
The methylated arginines are required for normal polysome association
The methylated arginines are required for normal polysome association
Arginines
present/ substituted
533, 538, 543, 545
RGG domain absent
533, 538, 543, 545
0
0.1
0.2
0.3
0.4
1 2 3 4 5 6 7 8
0
0.1
0.2
0.3
0.4
1 2 3 4 5 6 7 8
0
0.1
0.2
0.3
0.4
1 2 3 4 5 6 7 8
Blackwell et al., Hum. Mol. Genet., Advance Access published on January 11, 2010
WT
533, 538, 543, 545m
Arginine 533 and 538 are required for normal polysome association
Arginine 533 and 538 are required for normal polysome association
Arginines
present/ substituted
533, 538, 543, 545
533, 538, 543, 545
543,545m
533, 538m
533, 538 /
543, 545
543, 545 /
533, 538
0
0.1
0.2
0.3
0.4
1 2 3 4 5 6 7 8
0
0.1
0.2
0.3
0.4
1 2 3 4 5 6 7 8
0
0.1
0.2
0.3
0.4
1 2 3 4 5 6 7 8
0
0.1
0.2
0.3
0.4
1 2 3 4 5 6 7 8
Blackwell et al., Hum. Mol. Genet., Advance Access published on January 11, 2010
Probing RNA associationAssumption: Polysome association of Fmrp reflects RNA binding
Hypothesis: Arginines 533 and 538 are required for Fmrp RNA association
Consideration:Fmrp has multiple RNA binding domains and many interacting
RNAs.
Need:An assay that is specific for binding of G-quartet-containing RNAs
by the RGG box
The RNA capture assay
SC1ggc ugc ggu gug gaa gga gug gcu ggg uug cgc agc u
SC1mutggc ugc ggu gug gaa CCa gug gcu ggg uug cgc agc u
Determine amount of FMRP bound
Are arginines 533 and 538 required for RNA association?
Biotinylated RNAwith G-quartet structure
Fmrp
In vitro synthesized FMRPGG
GG
GGGG
Fmrp
Allow FMRP to bind target RNA and capture complex on beads
GGGG
GGGG
Arginines 533 and 538 are primarily required for sc1 RNA association
Captured protein
Input
SC1 SC1 SC1 SC1 SC1SC1 mut
SC1 mut
SC1 mut
SC1 mut
SC1 mut
533, 538, 543, 545 ∆RGG
533,
538
, 543
, 545
∆RG
G
Arginines
present/ substituted
533, 538, 543, 545
533, 538, 543, 545
533, 538, 543, 545
533,
538
, 543
, 545
533,
538
, 543
, 545
15% 23% 44%percent of WT
0
0.2
0.4
0.6
0.8
1
533, 538, 543, 545
∆RGG
*
*
*
533, 538, 543, 545
533, 538, 543, 545
533, 538, 543, 545
533,
538
, 543
, 545
Blackwell et al., Hum. Mol. Genet., Advance Access published on January 11, 2010
Captured protein
Input
∆RG
G
21 67percent of WT 108 54
0
0.2
0.4
0.6
0.8
1
1.2
1.4
∆RGG
n.s.
**
****
n.s.
**53
3, 5
38, 5
43, 5
45
533,
538
, 543
, 545
533,
538
, 543
, 545
533,
538
, 543
, 545
533, 538, 543, 545
533, 538, 543, 545
533, 538, 543, 545
533, 538, 543, 545
Arginines 533 and 538 are NOT required for AATYK RNA association
Arginines
present/ substituted
Blackwell et al., Hum. Mol. Genet., Advance Access published on January 11, 2010
PRMTs are responsible for arginine methylation
• Fmrp is mono and asymmetrically dimethylated• PRMT1 and PRMT3 have been shown in vitro to methylate
FmrpStetler A. et al., Hum. Mol. Genet. 2006. Denman, Cell. Mol. Biol. Lett. 2002.
McBride and Silver. 2001. Cell
∆RGG mock
BSA BSAPRMT1
3H
FMRP
PRMT1 methylates our FMR proteins
Hypothesis: If all four arginines are required for RNA binding and methylation inhibits binding, then the combination of substituting arginines and methylating the remaining arginines should result in loss of RNA binding
533, 538, 543, 545
Arginines
present/ substituted
533, 538, 543, 545
533, 538, 543, 545
533, 538, 543, 545
533, 538, 543, 545
Blackwell et al., Hum. Mol. Genet., Advance Access published on January 11, 2010
Capture
Input
+ -
∆RGG
PRMT1: BSA:
3H
+ - + -+- +- +-
+ -+-
PRMT1 methylation inhibits sc1 RNA binding…
Arginines
present/ substituted
533, 538, 543, 545
533, 538, 543, 545
533, 538, 543, 545
Blackwell et al., Hum. Mol. Genet., Advance Access published on January 11, 2010
PRMT1: BSA: + -
+-+ -
+-Capture
Input
0
0.5
1
1.5
2
533,538m +PRMT1
533,538m +BSA
543,545m +PRMT1
543,545m +BSA
n.s.n.s.
*n.s.
…but not AATYK binding?
Arginines
present/ substituted
533, 538, 543, 545
533, 538, 543, 545
533, 538, 543, 545
533, 538, 543, 545
Blackwell et al., Hum. Mol. Genet., Advance Access published on January 11, 2010
Different RNAs have different molecular requirements for FMRP binding
sc1 RNA AATYK RNA
Requires RGG box:
Requires arginines 533, 538, 543, and 545:
Requires arginines 533 and 538:
Requires arginines 543 and 545:
Association inhibited by methylation:
*
*
* = required, but not to the same degree as sc1
VC WT VC
IgPRMT1
lysate Flag IP
FMRP
WT
∆RGG mock
BSA BSAPRMT1
3H
FMRP
533, 538, 543, 545
Arginines
present/ substituted
533, 538, 543, 545
533, 538, 543, 545
533, 538, 543, 545
533, 538, 543, 545
Endogenous PRMT1 activity?
Blackwell et al., Hum. Mol. Genet., Advance Access published on January 11, 2010
PRMT1 methylates Fmrp in cells
• siRNA treatment against PRMT1 results in reduced Fmrp methylation in COS-7 and HeLa cells
34% reduction
3H-methyl methionine
eIF5
PRMT1
TransgeneFmrp
Irrel. PRMT1
siRNA
3H-methyl methionine
eIF5
PRMT1
TransgeneFmrp
Irrel. PRMT1
siRNA
48% reduction
COS-7 HeLa
Blackwell et al., Hum. Mol. Genet., Advance Access published on January 11, 2010
PRMT3 methylates Fmrp in cells
• Reduction in methylation seen in “knockout” cell line and using siRNA treatment in COS-7 cells
PRMT3+/+ PRMT3-/-
PRMT3
endogenous Fmrp
3H-methyl methionine ~10%
reduction
eIF5
PRMT3
Transgene Fmrp
- siRNA + siRNA
3H-methyl methionine
~50% reduction
~80% reduction
COS-7 MEF
eIF5
Recap
• Arginines 533 and 538 are required for normal polysome association
• Arginines 533 and 538 are required for sc1 RNA association; 543 and 545 play a smaller role
• Arginines 543 and 545 are important for AATYK RNA association but 533 and 538 are not
• Methylation of either arginines 533 and 538 or arginines 543 and 545 inhibits sc1 RNA association but not AATYK association
• PRMT1 and 3 are responsible for methylating Fmrp in cells
Is Fmrp methylated in vivo?
• Mass spectrometry to determine whether Fmrp from brain is methylated within the RGG box
• Metabolic labeling of cultured neurons to determine whether Fmrp is methylated in neurons
[3H]-methionine
C[3H]-Fmrp
Block new protein synthesis
Is Fmrp localization affected?
Loss of the RGG box leads to a diffuse cytoplasmic localization pattern and the failure of Fmrp to accumulate in granules
From Mazroui et al., Hum. Mol. Gen. 2002
What about our constructs in neurons?
QuickTime™ and aTIFF (LZW) decompressor
are needed to see this picture.
QuickTime™ and aTIFF (LZW) decompressor
are needed to see this picture.
An Fmrp isoform lacking the C-terminus (NES and RGG box) does not form dendritic granules in hippocampal neurons.
From Levenga et al., Neuro. of Disease 2009
A speculative model
FMRP
FMRP
FMRP
FMRP
Methylation of the RGG box affects the identity of bound mRNAs and how those bound mRNAs are transported
AcknowledgementsThe Ceman Lab(current)• Dr. Stephanie Ceman• Miri Kim• Claudia Winograd• Adriana Jang
(former)Dr. Anne CheeverKevin McNerney
Dr. Edward KhandjianDr. Xing ZhangDr. Andre HoogeveenDr. Mark Bedford
NIH HD41591-01
Spastic Paralysis Research Foundation of the Illinois-Eastern Iowa District of Kiwanis International
Developmental Psychobiology and Neurobiology Training Grant
Two models for how PRMT1 and 3 function on Fmrp
Are they redundant or do they have specific roles?
Currently purifying PRMT3 to test its effects on Fmrp compared to PRMT1
Fmrp
PRMT1 PRMT3
Fmrp
PRMT1 PRMT3
Fmrp Fmrpversus