A research team from Cold Spring Harbor Laboratory this week published details of a new biochemical approach to identify microRNA targets that the investigators claim may shed additional light on the factors that determine miRNA-mediated gene regulation.
The method, which appears online this week in the early edition of The Proceedings of the National Academy of Sciences, combines purification of the RNA-induced silencing complex with microarray analysis of bound messenger RNA.
The results revealed miRNA targets that are down-regulated at the mRNA level, as well as targets that are repressed without changes in mRNA levels, the authors wrote.
Identifying targets not associated with mRNA level changes “will provide an opportunity to glean sequence or structural features of … mRNAs that determine regulatory fate,” they noted.
The new miRNA target-identification method “also provides an important balance to in silico methods of predicting microRNA targets, which, while growing in power, still fail to provide a complete and wholly precise picture of miRNA regulatory networks.”
Researchers from the State University of New York at Stony Brook and from Expression Analysis contributed to the PNAS paper.
“Mature microRNAs function in stable complexes with proteins of the Argonaute family, the core of … RISC,” the researchers wrote in PNAS. “In animals, the 21- to 22-[nucleotide] miRNA targets RISC to mRNA with partial sequence complementarity … [and] results in translational repression that may also be accompanied by mRNA destruction.
“However, the precise factors that determine the extent to which mRNA decay versus translational repression contributes to silencing are not currently well understood,” they noted. Additionally, identifying the targets that mediate miRNA function has proven to be challenging.
Currently, two approaches are used for target identification. The first measures reductions in target mRNA levels triggered by exogenous miRNA, but “targets whose stability is not affected appear as false negatives,” according to the investigators.
The second involves predictive algorithms that use established miRNA-mRNA interaction rules. However, these require “a fully complementary seed sequence in the 3’ UTR and conservation of the site across several species, thus potentially missing targets that do not conform to these rules.”
“Identification of [targets not associated with mRNA level changes] will provide an opportunity to glean sequence or structural features of … mRNAs that determine regulatory fate.”
Seeking to develop a biochemical approach to identify miRNA targets, the researchers devised a method that involves co-immunoprecipitating mRNA with miRNA-programmed Argonaute-2, the member of the Argonaute protein family responsible for cleaving a miRNA target, and showed that “this approach recapitulates the major characteristics of known miRNA-target interactions.”
Examining the “complete spectrum” of targets for miR-124a, which was chosen as a model for the experiments because of the availability of many experimentally determined target candidates, the research team found a set of targets that were down-regulated at the mRNA level as expected, as well as a set whose mRNA levels were unaffected by the miRNA.
“Reporter assays validated both classes, extending the spectrum of mRNA targets that can be experimentally linked to the miRNA pathway,” they wrote.
“Overall, net [immunoprecipitate] enrichment is a highly specific and comprehensive predictor of consequential miRNA-mRNA interactions,” the research team concluded.
The reasons for this include the fact that the down-regulated set of miRNA targets includes both direct targets and mRNA, “whose abundance drops as a secondary consequence of miRNA action. The latter would not be enriched in the IP, thus distinguishing them from primary targets,” the authors noted.
Further, the immunoprecipitate set contains “not only bona fide targets but also those [that] associate non-specifically or bind Ago as a consequence of their interaction with an endogenous microRNA,” they added. “Such mRNAs that are not miR-124a targets, but that increase in abundance as a secondary consequence of miR-124a transfection, would seem to be enriched in the IP” and score as false-positives in the raw IP enrichment set.
“Taking into account both changes in abundance and IP retention focuses attention on the true target set,” they wrote. “Importantly, the Ago IP identifies a large class of potential targets that are not decreased at the mRNA and that would, therefore, be missed using current experimental approaches to target identification.”