Reversible HuR-microRNA Binding Controls Extracellular Export of miR-122 & Augments Stress Response (2016)
Figure 5

Figure 5. HuR binds and replaces miRNA bound to Ago2

(A) Time course experiment of HuR binding of miR-122 replaced from Ago2 miRNPs. A schematic representation of in vitro miRNA binding assay of HuR (left panel). Equal amounts of recombinant HuR (rHuR), after indicated time of the miRNP interaction on FLAG-beads, were immunoprecipitated and HuR associated miR-122 levels were detected by qRT-PCR (right top panel). Ago2 western blot detects its presence in the FLAG beads used in the assay and its absence in HuR immunoprecipitated materials. HuR western blot was used to confirm its presence and quantification of its levels in the immunoprecipitated materials, at different time points (right bottom panel).

(B) A schematic diagram of in vitro miRNA binding assay of HuR, using wild type full length and delta RRMIII deletion mutant of HuR. Relative amount of miR-122 bound to full length and delta RRMIII after 60 minutes of their incubation with miR-122 containing miRNPs.

(C) Coomassie stained gel picture denotes the purity of the recombinant HuR used in the assay. The same proteins were western blotted for HuR to show the specificity of the antibody used for immunoprecipitation. The recombinant P and N proteins of Chandipura virus was used as negative control to confirm the specificity of 3A2 anti-HuR antibody.

(D) HuR western blot in middle right panel confirmed the presence of HuR and the truncated version of it in the immunoprecipitated materials. A representative domain picture of HuR and HuRΔIII are shown in the upper panel. Relative levels of HuR bound miR-122 were determined by qRT-PCR (mean+/- s.e.m., n=3) (middle left panel). Amount of miR-122 recovered from the immunoprecipitated materials were normalized against the amount of HuR or HuRΔIII present in the Immunoprecipitated materials. Ago2 western blot confirmed its presence in the FLAG beads used in the assay while Ago2 was not detected in HuR immunoprecipitated materials (bottom panel).

(E) RNA electrophoretic mobility shift assay done with 32P-labeled synthetic miR-122 and recombinant HuR proteins. Radiolabelled miR-122 (100 fmol) was incubated with increasing concentration of FL-HuR (5-100nM) or HuRΔIII (100 nM) and gel shifting of miR-122 were marked by arrowhead. Position of the free probes is marked by an arrow. In the right panel, 1pmol of unlabelled TNF-alpha AU-rich element encoding synthetic RNA was used to compete with miR-122 binding of full length HuR. Amount of HuR or HuRΔIII used for gel shift assay were premeasured by densitometric estimation of coomassie stained gel bands from SDS page.

(F) Association of miRNAs with recombinant HuR in vitro. Small RNA pool of Huh7 cell isolated by miRVana miRNA isolation kit were incubated with recombinant HuR in an in vitro binding reaction and HuR associated RNA, recovered from immunoprecipitated HuR after the binding reaction, were quantified along with the input RNA used for binding. The mean Ct values of RNA from samples from three independent binding reactions along with input RNA were measured and plotted (mean+/- s.e.m., n=3). The Input recombinant HuR used in the binding assay and in the immunoprecipitated materials were detected by western blot.

* P<0.05.P values were determined by paired t test. Position of size markers in protein gels used for respective western blot analysis are shown against each panels. HC; Heavy Chain and LC; Light Chain of IgG used for immunoprecipitation reaction. For estimations of relative level of either miRNAs or mRNAs Ct values only within the range of 20-32 were considered for analysis.