Generating and validating tissue-specific miRNAs in body muscle and hypodermis of C. elegans
Post-transcriptional gene regulation mechanisms exert control over cell and tissue identity. Many of the regulatory elements on which these mechanisms rely target the 3’ untranslated region (3’ UTR) of mRNAs. MicroRNAs (miRNAs) are small non-coding RNAs (ncRNAs) which target 3’UTRs, allowing translational repression and mRNA degradation. They are essential for post-transcriptional gene regulation and directly influence cell identity. In the past years, our lab used the RNA immunoprecipitation approach to identify miRNA targets in the intestine and the body muscle tissues of the soil nematode C. elegans. However, this approach doesn’t detect the tissue-specific miRNAs. To rectify this issue, our lab has now developed a novel approach using fluorescence-activated cell sorting (FACS), followed by RNA extraction and sequencing. Here, we have profiled tissue specific miRNAs from two worm strains in the body muscle (myo-3p) and hypodermis (dpy-7p) tissues. To express fluorescence in the nuclei, paralyzed worms were injected with constructs containing tissue-specific promoters driving the expression of his-58 fused to an mCherry fluorochrome. Worms expressing this construct were homogenized, filtered, and centrifuged for nuclei isolation, and then sorted. We have identified several tissue specific regulatory networks controlled by miRNAs. To validate our results, we injected a miRNA-specific promoter driving expression of GFP fluorochrome in the C. elegans gonads and screened for GFP fluorescence in the tissue from which the nuclei are isolated. While we await the sequencing results for the hypodermis-specific miRNA, we were able to validate the let-7 miRNA expressed in the body muscle. This work will help to better understand the role of miRNAs in controlling tissue specific cell identity and in turn provide tools to probe post-transcriptional gene regulation in vivo.
