University of Massachusetts Medical School investigators from the lab of Victor Ambros have begun a two-year project to analyze the function of miR-34 in C. elegans using a combination of computational, biochemical, and molecular techniques.
In addition to providing new insights into the function of miR-34, which has been associated with oncogenesis and tumor suppression, the effort is expected to help refine a recently published tool for miRNA target identification.
According to UMMS postdoc Molly Hammell, who is leading the National Institutes of Health-funded project, the decision to focus on miR-34 in part stems from the fact that it is one of the most highly conserved miRNAs in a variety of model organisms.
At the same time, “we know that in human and mouse tissues, it seems to … [play] roles in regulating the cell cycle and apoptosis, and can actually act as a tumor suppressor,” she told RNAi News this week. In addition, miR-34 appears to be critical to an organism’s early development.
In nematodes, the miRNA is up-regulated as the animal passes through the larval stages and peaks in adulthood, she explained. “So it must have some sort of role for the developing animal, and that’s what I’m interested in finding out.”
Hammell said she expects the project, which is being funded by a $100,000 grant from the National Institute of General Medical Sciences, will also help her improve a computational miRNA target-identification tool she developed to address certain shortcomings of a biochemical method published last year.
In late 2007, Min Han and colleagues at the University of Colorado at Boulder published a study that identified nearly 3,500 potential miRNA targets in C. elegans through an approach combining immunoprecipitation, pyrosequencing, and microarray analysis of RNAs associated with the RISC components AIN-1 and AIN-2.
Despite the robustness of the approach, Han’s team was “not able to actually match up individual microRNAs to all of the individual mRNAs — they just got a big pool of everything that came down,” Hammell said. “That’s where I started: computationally trying to look at the features of these transcripts that came down with [miRNAs] and their protein components … to see if I could find identifying features that are particular to microRNA targets.”
The result was an miRNA-prediction method termed mirWIP, short for miRNA Targets by Weighting RISC-IP Enriched Parameters, which was published in Nature Methods in August.
“One of the explanations for [our previous findings] would be that the microRNAs actually buffer each other — they’re cooperating and can compensate for loss of one or more of the other microRNAs.”
“Our analysis of [Han’s] AIN-IP dataset revealed enrichment for defining characteristics of functional miRNA-target interactions, including structural accessibility of target sequences, total free energy of miRNA-target hybridization, and topology of base-pairing to the 5' seed region of the miRNA,” Hammell and her colleagues wrote in that paper.
These features formed the basis for mirWIP, “which optimizes sensitivity to verified miRNA-target interactions and specificity to the AIN-IP dataset … [and] can be used to capture all known conserved miRNA-mRNA target relationships in Caenorhabditis elegans at a lower false-positive rate than can the current standard methods,” the authors added. The mirWIP tool can be accessed at www.mirtargets.org.
In her grant project, Hammell aims to identify putative miR-34 targets using mirWIP and construct “translational reporters for the top predicted [target] genes,” according to the grant’s abstract. Then, applying Han’s approach, she and her colleagues plan to “biochemically purify targets of miR-34 by immunoprecipitating proteins in the RNA-induced silencing complex in a miR-34 mutant background.”
Re-applying the RISC-immunoprecipitation method in C. elegans mutants “should result in the loss of miR-34 targets from the pool of RISC-associated mRNA,” the abstract notes.
In the end, though, “what you’re really interested in is the biology of that interaction,” Hammell said. “So the last step [of the NIH project] is to make … a GFP gene that reports on where [a target] gene is expressed. And if you include the 3’ UTR, hopefully you can know where that gene is being regulated by microRNAs or other UTR elements.”
Hammell noted that her project could also help confirm a hypothesis that miR-34 works in concert with other miRNAs. In 2007, Ambros and the Massachusetts Institute of Technology’s Robert Horvitz published the results of an effort to knock out all C. elegans miRNAs individually in order to identify their phenotypes. Surprisingly, most of the miRNAs, including miR-34, were found to be individually non-essential for development or viability.
“One of the explanations for that [result] would be that the microRNAs actually buffer each other — they’re cooperating and can compensate for loss of one or more of the other microRNAs,” Hammell said.
Supporting this theory are data from experiments in which Hammell crossed nematodes lacking miR-34 with mutants lacking Argonaute-1. “It turns out that when you have a double-mutant for miR-34 and this Argonaute [protein], you get some problems in adult cell-fate specification,” as well as improper gonad formation, she said.
Following up on these findings, Hammell also plans to computationally identify and genetically validate miRNAs that work together to regulate miR-34 target genes.