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. Author manuscript; available in PMC: 2014 Jan 17.
Published in final edited form as: Cell. 2013 Jan 17;152(1-2):262–275. doi: 10.1016/j.cell.2012.11.052

Figure 3. miR-185 directly targets and represses 2310044H10Rik (Mirta22).

Figure 3

(A) Structure of the 3′UTR of 2310044H10Rik (Mirta22) showing miRNA binding sites predicted by TargetScan or mirDB. Blocks in mouse 2310044H10Rik (Mirta22) 3′UTR that are highly conserved in rat and human orthologues are shown below the mouse 3′UTR. Evolutionary conservation is also assessed by the “30-way multiz alignment and conservation analysis” in the USCS browser, with conserved blocks indicated by green peaks. miR-185 and miR-485 binding sites located within the conserved blocks are shown in red.

(B, C) qRT-PCR quantification of endogenous 2310044H10Rik (Mirta22) in DIV7 hippocampal neurons. Expression levels in anti-miR-185-treated and pre-mir-185-treated neurons were normalized to expression levels under respective controls. (B) Increased expression levels of Mirta22 in neurons transfected with anti-miR-185 at DIV5 (n = 5, each treatment). (C) Reduced expression levels of Mirta22 in DIV9 hippocampal neurons transfected with pre-mir-185 mimic at DIV7 (n = 3, each treatment).

(D, E) qRT-PCR quantification of endogenous 2310044H10Rik (Mirta22) in N18 cells. Expression levels in pre-mir-185-treated and anti-miR-185-treated cells were normalized to expression levels under respective controls. (D) Reduced expression levels of Mirta22 in cells transfected with pre-mir-185 mimic (n = 3, each treatment). (E) Up-regulation of Mirta22 in cells transfected with an anti-miR-185 LNA oligo (n = 3, each treatment).

(F–H) Repression effects of pre-mir-185, pre-mir-485 and pre-mir-491 on Mirta22 3′UTR were examined by a dual-luciferase reporter assay (see Methods). Values are Renilla luciferase levels relative to firefly luciferase levels and normalized to the relative expression levels under pre-scramble treatment (F, H) or to the relative expression levels from plasmid with no 3′UTR (G) (n = 3 for each condition). Pre-mir-185 significantly decreases the 2310044H10Rik (Mirta22) 3′UTR reporter expression over a concentration range of 10nM to 0.01nM (F). Pre-mir-185 mediated repression on 2310044H10Rik (Mirta22) 3′UTR reporter expression depends on conserved miRNA binding sites (G). Pre-mir-485 and pre-mir-491 significantly decreases the 2310044H10Rik (Mirta22) 3′UTR reporter expression (H).

Results are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 (Student’s t- test). See also Figure S3 and Table S3.