Figure 5: Roquin binds to miR-146a.
From: Roquin binds microRNA-146a and Argonaute2 to regulate microRNA homeostasis

(a) Normalized counts per million (CPM) of miR-146a and miR-150 in sRNA deep-sequencing from Roquinsan/san versus Roquin+/+ T cells. (b) The ratio of non-templated addition of nucleotides at +1 position in Roquinsan/san and Roquin+/+ T cells. A, adenine; C, cytosine; G, guanine and U, uracil. The two ‘hinges’ are the first and third quartile, the notches extend to ±1.58 × interquartile range/sqrt(no. of observations) and whiskers extend to the data range. (c) The percentage of mono-uridylated to exactly matching mature miR-146a or miR-150 was calculated from Roquin+/+ and Roquinsan/san littermates using the sRNA deep sequencing data set, the percentage change was calculated. sRNA: small RNA. (d) qRT–PCR analysis of endogenous miR-146a and U6 in immunoprecipitates from total human tonsil lymphocytes using anti-IgG, no antibody, anti-Roquin IgG (Roquin) and anti-Roquin IgG pre-incubated with a blocking Roquin peptide (Roquin-pep). 10% of the whole cell lysate (total input) was removed before immunoprecipitation and the results are presented as percentage of total input. **P=0.05 (U-test), NS, not significant. Each dot represents a technical replicate. The results are representative of five independent experiments. (e,f) SPR study of the binding of RoquinM199R1–484 and RoquinWT1–484 to immobilized 5’-biotinylated pre-miR-146a. (e) Blank-subtracted Biacore sensograms for RoquinM199R1–484 (twofold dilutions, 1,000–3.9 nM) and RoquinWT1–484 (2,000–7.8 nM) binding to pre-miR-146a. Protein concentrations increased from the bottom to the top curve. (f) Binding isotherms for equilibrium responses. The solid curves were calculated from the derived KD values for a 1:1 interaction of 110±20 (for M199R) and 370±50 nM (WT). Fit Rmax values were 330±20 (for WT) and 242±13 response units (M199R).