Comparison of the specificities of the RGG/RG-domains of FMR1, FXR1 and FXR2 for small RNA

In previous studies, we identified a sequence motif (GDCGG) in almost all microRNAs downregulated in the serum of patients with amyotrophic lateral sclerosis. We found that FMR1, FXR1 and FXR2 directly and specifically interact with microRNAs containing the GDCGG motif via their RGG/RG-domains. Here, we compare the specificities for microRNAs of the RGG/RG-domains of FMR1, FXR1 and FXR2 on a transcriptome-wide scale using the Affymetrix GeneChip™ miRNA 3.0 Arrays. The recombinant RGG/RG-domains of FMR1, FXR1 and FXR2 (2.9 nmole), respectively, were incubated with 50 µg low molecular weight RNA isolated from HEK293 cells in 14 ml of buffer (25 mM Tris, 150 mM potassium chloride, 2.5 mM magnesium chloride, 0.5% NP-40, pH 7.5). After incubation, the recombinant proteins were isolated using Ni-NTA-Agarose (Qiagen #30210), extensively washed and the co-purifying RNA compared to the input-RNA to identify binding microRNAs that get enriched by the different RGG/RG-domains. Three biological replicates were used for each RGG/RG-domain.