FMRP RGG box methylation

Identifying the Function of the FMRP RGG box January 2010 Ernest “Tory” Blackwell Ceman Lab

Overview ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]

Fragile X Syndrome (FXS) ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]

The domain structure of Fmrp ,[object Object],[object Object],[object Object],[object Object],RRGDGRRRGGGGRGQGGRGRGGGFKGNDDHSR NLS KH2 KH1 NES RGG

The RGG box affects how well Fmrp associates with polysomes ,[object Object],Mazroui et al., Hum. Mol. Gen. 2003. 10% 50% sedimentation 40S 60S 80S

Recap: ,[object Object],[object Object],[object Object],Are the arginines that are methylated important for polysome association? Fmrp Fmrp methylation Loss of RNA binding Fmrp Loss of RGG domain Abnormal polysome association suggesting loss of RNA binding

WT ∆ RGG 533, 538, 543, 545m The methylated arginines are required for normal polysome association Arginines present/ substituted 533, 538, 543, 545 RGG domain absent 533, 538, 543, 545 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 Arginines present/ substituted 533, 538, 543, 545 533, 538, 543, 545 543,545m 533, 538m 533, 538 / 543, 545 543, 545 / 533, 538 Blackwell et al., Hum. Mol. Genet., Advance Access published on January 11, 2010

Probing RNA association ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]

The RNA capture assay SC1 ggc ugc ggu gug gaa gga gug gcu ggg uug cgc agc u SC1mut ggc ugc ggu gug gaa CCa gug gcu ggg uug cgc agc u Are arginines 533 and 538 required for RNA association? Biotinylated RNA with G-quartet structure Determine amount of FMRP bound Fmrp In vitro synthesized FMRP G G G G G G G G Fmrp Allow FMRP to bind target RNA and capture complex on beads G G G G G G G G

Arginines 533 and 538 are primarily required for sc1 RNA association Captured protein Input SC1 SC1 SC1 SC1 SC1 SC1 mut SC1 mut SC1 mut SC1 mut SC1 mut 533, 538, 543, 545 ∆ RGG 533, 538, 543, 545 ∆ RGG 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 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

Arginines 533 and 538 are NOT required for AATYK RNA association Captured protein Input ∆ RGG 21 67 percent of WT 108 54 ∆ RGG n.s. ** ** ** n.s. ** 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, 545 533, 538, 543, 545 Arginines present/ substituted Blackwell et al., Hum. Mol. Genet., Advance Access published on January 11, 2010

PRMTs are responsible for arginine methylation ,[object Object],[object Object],[object Object],McBride and Silver. 2001. Cell

PRMT1 methylates our FMR proteins ,[object Object],∆ RGG mock BSA BSA PRMT1 3 H FMRP 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

PRMT1 methylation inhibits sc1 RNA binding… Capture Input + - ∆ RGG PRMT1: BSA: 3 H + - + - + - + - + - + - + - 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

… but not AATYK binding? PRMT1: BSA: + - + - + - + - Capture Input n.s. n.s. * n.s. 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

Endogenous PRMT1 activity? VC WT VC Ig PRMT1 lysate Flag IP FMRP WT ∆ RGG mock BSA BSA PRMT1 3 H FMRP 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

PRMT1 methylates Fmrp in cells ,[object Object],34% reduction 3 H-methyl methionine eIF5 PRMT1 Transgene Fmrp Irrel. PRMT1 siRNA 3 H-methyl methionine eIF5 PRMT1 Transgene Fmrp 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 ,[object Object],PRMT3 +/+ PRMT3 -/- PRMT3 endogenous Fmrp 3 H-methyl methionine ~10% reduction eIF5 PRMT3 Transgene Fmrp - siRNA + siRNA 3 H-methyl methionine ~50% reduction ~80% reduction COS-7 MEF eIF5

Recap ,[object Object],[object Object],[object Object],[object Object],[object Object]

Is Fmrp methylated in vivo ? ,[object Object],[object Object],[ 3 H]-methionine C[ 3 H]-Fmrp Block new protein synthesis

Is Fmrp localization affected? ,[object Object],From Mazroui et al., Hum. Mol. Gen. 2002 What about our constructs in neurons? 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 ,[object Object],FMRP FMRP FMRP FMRP

Acknowledgements ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],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 ,[object Object],[object Object],versus Fmrp PRMT1 PRMT3 Fmrp PRMT1 PRMT3 Fmrp Fmrp

FMRP RGG box methylation

FMRP RGG box methylation

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