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. Author manuscript; available in PMC: 2011 Aug 1.
Published in final edited form as: Nat Struct Mol Biol. 2011 Jan 23;18(2):237–244. doi: 10.1038/nsmb.1991

Figure 3. Ago2-CLIP identified GCACUU-motif containing cluster is sufficient to confer miR-295-mediated repression.

Figure 3

Box plots of relative activity of Renilla luciferase transgenes bearing CLIP clusters plus 25nt flanking regions in the 3′UTR. All raw Renilla luciferase values are normalized first to values from Firefly luciferase transfection control. Error bars are S.D. (a) Exemplary plot of a luciferase reporter assay testing an Ago2-CLIP 3′UTR cluster, Slc31a1 plus 25nt flanking regions at both ends. Shown are box plots for the ratio of the expression level of a luciferase reporter containing a wild-type Slc31a1 CLIP cluster in its 3′UTR to an identical luciferase reporter where the GCACUU seed was mutated to CCUCAU. This ratio is defined as “WT/Seed Mutant Activity” and calculated in three different cellular states: Dicer−/− mESCs (black), wild-type mESCs (white), and Dicer−/− mESCs transfected with miR-295 mimic (grey). For panels b-c, “Relative Luciferase Activity” is defined as “WT/Seed mutant activity” in specific cellular state normalized to the corresponding value in Dicer−/− mESCs. Genes that passed both the Normalization and Multi-library filters (i.e. “Overlap” set) are marked with an asterisk. All remaining genes passed the Normalization filter in one WT library. All box plots are from at least 3 independent experiments. (b) Relative luciferase activity of CLIP-identified 3′UTR clusters plus 25nt flanking regions in wild-type mESCs (grey) or Dicer−/− mESCs transfected with miR-295 mimic (white). Relative luciferase activity is significantly less than one in all genes examined (p<0.05, two-tailed paired t-test). (c) Relative luciferase activity of CLIP-identified CDS clusters plus 25nt flanking regions in wild-type mESCs (grey) or Dicer−/− mESCs transfected with miR-295 mimic (white). Herc1 and Eif2c2 each have two GCACUU motifs. Relative luciferase activity is significantly less than one for all genes examined except Tnrc6a (p<0.05, two-tailed paired t-test).