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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

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FMRP RGG box methylation

  • 1. Identifying the Function of the FMRP RGG box January 2010 Ernest “Tory” Blackwell Ceman Lab
  • 2.
  • 3.
  • 4.
  • 5.
  • 6.
  • 7. 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
  • 8. 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
  • 9.
  • 10. 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
  • 11. 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
  • 12. 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
  • 13.
  • 14.
  • 15. 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
  • 16. … 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
  • 17. 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
  • 18. 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
  • 19.
  • 20.
  • 21.
  • 22.
  • 23.
  • 24.
  • 25.
  • 26.

Editor's Notes

  1. Mention binding of G quartets and some more information on RNA binding Focus here on why methylation would be important
  2. Make sure to explain how polysomes work
  3. Spend more time here emphasizing what we are doing and what we are seein:, both Cos7 and MEF give us a reduction in Fmrp methylation
  4. Remember to add in the possibility of the KH domains playing a more critical role now that the RGG box has been disabled