Journal: Proceedings of the National Academy of Sciences of the United States of America
Article Title: Phosphoregulated FMRP phase separation models activity-dependent translation through bidirectional control of mRNA granule formation
Figure Lengend Snippet: Arginine perturbations disrupt FMRP LCR -sc1 RNA phase separation but do not notably affect FMRP LCR binding to sc-1 RNA. ( A ) Schematic illustration of the three constructs used. ( B ) Similar binding affinities for interactions between FMRP LCR , Me-FMRP LCR , and RtoK-FMRP LCR with 6-FAM–labeled sc1 RNA (25 nM) are shown using FP assayed in 25 mM Na 2 PO 4 , pH 7.4, 100 mM KCl, 2 mM DTT, 0.02 mg/mL E. coli tRNA, and 0.01% Nonidet P-40. Apparent dissociation constants ( k d,app ) from three experimental replicates. ( C ) Phase diagram of FMRP LCR , Me-FMRP LCR , and RtoK-FMRP LCR at a constant protein concentration of 100 μM with increasing sc1 RNA concentrations in buffer containing 25 mM Na 2 PO 4 , pH 7.4, 30 mM KCl, and 2 mM DTT. Green circles represent an observation of droplet formation, and open circles represent no droplet formation detected. The dotted red line is a visual aid for a qualitative phase boundary of FMRP LCR. ( Right ) Representative DIC microscopy images of droplets for the addition of 2.5 μM of RNA in the conditions described. (Scale bar, 5 μm.) Phase-separation propensity is dramatically decreased for methylated and RtoK mutant proteins, with smaller droplet sizes observed.
Article Snippet: Sc1 RNA, 5′-labeled Cy3-sc1 RNA, 5′-labeled Cy3-miRNA-125b, and 5′-labeled 6-FAM-sc1 RNA were purchased from Sigma as lyophilized samples.
Techniques: Binding Assay, Construct, Labeling, Protein Concentration, Microscopy, Methylation, Mutagenesis