Validating microRNA Targets
Although microRNAs control many aspects of an organism’s development, the specifics of microRNA-mRNA regulatory interactions is largely unknown. Liang Zhang, Ph.D., who is currently working at The Rockefeller University, also developed an approach using a C. elegans model while in the laboratory of Min Han, Ph.D., at Howard Hughes Medical Institute and the University of Colorado at Boulder. Dr. Zhang creates and expresses a fusion protein in which green fluorescent protein (GFP) is tagged to AIN-2, an essential member of miRISC (microRNA associated RISC).
“This model can be used to generate a more global look at the dynamics of microRNA-mediated regulation of gene expression during the worm’s development,” Dr. Zhang says. “We prepare lysates during five developmental stages and immunoprecipitate the AIN-2:GFP containing miRISC with an antibody to GFP. Then, we analyze the microRNAs and mRNAs in the immunoprecipitated complexes using deep sequencing and microarrays, which allows us to access the profiles of microRNAs, microRNA targets, and interactions. We found that more than 2,000 mRNAs associate with the AIN-2 family during worm development.”
Using that data, Dr. Zhang then identified thousands of microRNA targets related to each developmental stage. “We used this data to predict more than 1,500 microRNA family-mRNA interactions. Our data indicates that microRNAs have a high degree of specificity and preferentially target genes involved in signaling and do not regulate genes involved in housekeeping functions.
“Further, although perfect complementarity between bases 2–8 of the microRNA and its targets is a highly enriched feature of these microRNA family-mRNA interactions, additional matching between bases 9–10 of the miRNA and its targets are depleted, suggesting that perfect target matchings in the center region of the microRNA are avoided by functional interaction in vivo.”
Dr. Zhang reports that his studies indicate that microRNAs target and have preferences for different stages of development. “MicroRNAs are trying to temporally coordinate processes in different stages of development. Our future studies will begin to analyze spatial patterns of microRNA regulations in specific tissues of the worm, since our current studies used lysates of the whole worm.”