Figure 4.

HuR and miR-26 synergistically repress Rgs4 mRNA. (A) Predicted in silico folding of Rgs4 conserved region (upper panel). The miR-26 binding site (orange) and the ARE6 (blue) are highlighted, with mutated sites marked by black arrows. (B,C) Representative Western blot against HuR and Ago2 (B) and quantification (C) of HuR enrichment from adult rat cortex lysate in in vitro RNA affinity purification using 2xMS2 only, 2xMS2+ Rgs4 3ʹ-UTR WT, 2xMS2+ Rgs4 3ʹ-UTR ARE6 mut and 2xMS2+ Rgs4 3ʹ-UTR miR-26 mut as bait RNA, normalized to input. Paired Student’s t-test. (D) Quantification of eGFP fluorescence intensity in the cell body of hippocampal neurons at 15 DIV co-transfected at 14 + 1 DIV with eGFP-reporter and tagRFP or tagRFP-HuR. Ratio of eGFP-reporter intensity between tagRFP-HuR and tagRFP condition is shown. Paired Student’s t-test. (E) Quantification of tagRFP fluorescence intensity in the cell body of hippocampal neurons at 15 DIV co-transfected at 14 + 1 DIV with tagRFP-reporter and miR-scr or miR-26a. Ratio of tagRFP-reporter intensity between miR-26a and miR-scr condition is shown. Paired Student’s t-test. (F) Quantification of eGFP fluorescence intensity in the cell body of hippocampal neurons at 15 DIV transduced at 10 + 5 DIV with lentiviruses expressing shNTC or shHuR and co-transfected at 14 + 1 DIV with eGFP-reporter and miR-scr or miR-26a. Ratio of eGFP-reporter intensity between Rgs4 3ʹUTR WT and Ctrl reporter is shown. Paired Student’s t-test. All error bars are SEM from ≥ 3 independent biological replicates; asterisks represent p-values (*p < 0.05, **p < 0.01). ARE AU-rich element; WT wild type; KD knock-down; Scr scrambled