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. Author manuscript; available in PMC: 2011 Feb 9.
Published in final edited form as: Curr Biol. 2010 Jan 28;20(3):265–270. doi: 10.1016/j.cub.2009.12.042

Figure 4. Repressed bulged luciferase transcripts are not degraded and do not localize to P-bodies.

Figure 4

(A) Schematic of the miR-30 reporters is shown. (B) Oocytes were microinjected with miR-30 reporter mRNAs shown in panel A, and after one day of culture the relative reporter mRNA abundance was measured by qRT-PCR, and reporter mRNA translation efficiency was monitored by the dual luciferase assay. Renilla luciferase reporter activities were normalized to co-injected firefly luciferase control and are shown relative to RL-C control, which was set to one. The experiment was performed three times and similar results were obtained in each case. Shown are data (mean ± SEM) from one experiment. (C) mRNA harboring a let-7-binding sequence fails to localize to P-bodies in oocytes. Schematic depiction of reporters bound and not bound by endogenous let-7 is shown on the top of the figure. Below are confocal images showing cytoplasmic localization of corresponding reporter mRNAs. NIH 3T3 cells (upper images) were transfected with the corresponding reporter plasmids and fully-grown GV oocytes (lower images) were microinjected with in vitro transcribed mRNAs as described in Experimental Procedures. Co-transfected (co-injected) YFP-MS2 fusion protein containing NLS is retained in the cytoplasm upon binding to reporter transcripts, thus visualizing their localization [18]. White arrowheads depict P-bodies visualized by let-7 targeted reporter mRNA in NIH 3T3 cells. Scale bars represent 20 μm.