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Similar to FMRP RGG box methylation
Similar to FMRP RGG box methylation (20)
FMRP RGG box methylation
- 1. Identifying the Function of the FMRP RGG box January 2010 Ernest “Tory” Blackwell Ceman Lab
- 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
- 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
- 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
Editor's Notes
- Mention binding of G quartets and some more information on RNA binding Focus here on why methylation would be important
- Make sure to explain how polysomes work
- 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
- Remember to add in the possibility of the KH domains playing a more critical role now that the RGG box has been disabled