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