A couple of tools would be nice to have for investigating microRNA activity in C. elegans: First, it would be useful to block the activity of a single microRNA or a microRNA family in specific cells or stages without mutations. Second, it would be helpful to have a quantitative assay for microRNA activity that doesn’t use translated reporters like lacZ, GFP or luciferase, so that translation initiation and protein folding and stability don’t muddy the measurements. Several investigators have made artificial RNA circles by placing a splice acceptor before a splice donor in a construct. Such circles are stable because they lack targets for exonucleases. To get splicing to occur efficiently, the sequence is flanked by inverted repeats that bring the splice sites together. We built a vector for expressing RNA circles in C. elegans and have used it to address the two desirables mentioned above.
First, we generated a microRNA “sponge” containing an artificial sequence of 36 repeats of a let-7 seed sequence spaced 18nt apart. (This design is based on the naturally-occurring ciRS-7 sponge that inhibits miR-7 in the mammalian brain (Hansen, Nature, 495, 2013)). The let-7 family microRNAs are required for normal timing of epidermal development, and if they are knocked out, reiteration of L2 and later seam cell fates occurs, which is easily measured. We expressed the circular let-7 sponge in larvae using a col-10 promoter, which is active in the epidermis at all stages. This sponge caused a severe reiterative phenotype, whereas its reverse complement did not.
We next produced an RNA circle that contains a single exact match to a microRNA, in this case lin-4. This match should allow complete base-pairing between the microRNA and its target and subsequent cleavage by “Slicer.” (ALG-1 and -2, the two microRNA-binding Argonautes of C. elegans, have Slicer activity (Bouasker, NAR, 40, 2012)). We expressed this circle in larvae and measured its stability by qRT-PCR in wildtype and lin-4(0). We used the dpy-14 promoter, which is most active in late embryos just prior to hatching, so that the only factor contributing to the circle’s abundance is its stability. Removing lin-4 roughly tripled the amount of the circle, indicating that we can detect microRNA activity independent of a translated reporter. Our next goals are to determine whether flanking sequences affect microRNA activity and whether our Circle Cleavage Assay can be used to measure the contributions of RNA regulatory proteins.
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