The National Institutes of Health this month awarded more than $1 million in funding for the 2010 fiscal year to fund four microRNA-related research projects during 2010, including studies focused on defining the mechanisms of miRNA-target interactions; uncovering the role of a specific miRNA in Caenorhabditis elegans; and determining the impact of the small RNAs in brain cancer and cardiac hypertrophy.
The first grant was awarded to Princeton University's Hilary Coller to continue her efforts to develop a "combined computational and experimental approach to elucidate the mechanisms of miRNA-target interactions for many different miRNA-target pairings," Coller wrote in the grant's abstract.
"In the course of our studies on cellular quiescence in human cells, we discovered that the let-7 miRNA likely regulates not only genes with recognition sequences in their 3' UTRs, but also genes with recognition sequences in their coding regions," Coller noted. "We further discovered evidence for mechanistic differences between the targeting of 3' UTR versus coding region recognition sites. Our results in combination with published data also raise the possibility of target-specificity in miRNA mechanisms in the context of quiescence."
To test the hypothesis that miRNAs have different mechanisms of action depending on the interaction with their targets, Coller and her colleagues will evaluate a combined computational and experimental approach that they expect will be useful for "predicting physiologically relevant targets of miRNAs and for designing maximally effective miRNA therapeutics."
Specifically, they will use comparative genomics and reporter assays to identify distinct mechanisms miRNA use to target different portions of transcripts. They then aim to "analyze cells transfected with one of several miRNAs by performing microarray analysis on total transcripts and polysome-associated transcripts," the abstract states. "These data will allow us to identify characteristics of miRNA-target interactions that result in transcript degradation and those that affect translation initiation."
The team then proposes to examine whether there exists a "target-specific switch in miRNA activity from repression to activation as cells become quiescent," and finally evaluate the impact of miRNA antagonists on total and polysome-associated transcripts.
"Our expectation is that deciphering the mechanisms of miRNA function, including the basis for specificity for miRNA-target interactions, will provide clarity to the field of miRNA research," Coller wrote.
Her grant, which is worth $258,791 during 2010, began in January 2009 and is set to run until the end of 2012.
The second grant was awarded to University of Massachusetts Medical School researcher Molly Hammell to fund her investigations into the highly conserved miR-34, which has been shown to have a role in oncogenesis and tumor suppression in C. elegans.
In collaboration with other UMMS investigators, Hammell has developed a computational target-prediction method that "scores microRNA binding sites based on features enriched in biochemically purified microRNA targets," she wrote in the grant abstract.
This NIH-funded project will "build on the results of that computational study by focusing on … miR-34 and exploring its functional role in C. elegans through identifying functional targets and the phenotypic consequences of misregulating these target genes."
At the same time, because miR-34 has no family members in the worm and no apparent phenotype, Hammell proposes to computationally identify and genetically validate miRNAs that help to regulate miR-34 targets.
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"This hypothesis of cooperating microRNAs is supported by preliminary evidence showing that a miR-34 mutant does have overt phenotypes in a sensitized background where one of the C. elegans Argonaute genes has also been deleted," she wrote.
After validating the top 10 predicted miR-34 targets, Hammell proposes to use genetic epistasis analysis to determine whether the miRNA is capable of regulating its identified targets or whether other miRNAs are working together to do so in the absence of miR-34 function.
The grant, worth $57,194 this year, began in January 2009 and is set to run until the end of 2010.
Brigham and Women's Hospital's Anna Krichevsky received the third grant, which will support her efforts to validate whether miRNAs play a functional role in human glioma formation and to "elucidate intracellular signaling pathways that mediate functions of these miRNAs," according to the grant's abstract.
To do so, specific oncogenic miRNAs will be inhibited in glioma cells, the in vivo and in vitro effects of which will be examined in detail, the abstract notes. "Since miRNA inhibition may potentially produce various off-target effects, [the first aim of the project] will involve validation of highly specific oligonucleotide inhibitors for the miRNAs of interest" to identify key mRNA targets that mediate miRNA functions in glioma cells.
Krichevsky and her colleagues propose to then determine effects of the miRNAs on glioma cell viability, proliferation, and apoptosis in vitro and "further determine effects of the miRNAs and most potent miRNA combinations on growth and invasion of intracranial glioma tumors in animal models," she wrote.
The grant began on Jan. 1, 2010 and is set to run until the end of November, 2014. It is worth $355,592 in its first year.
The last grant was awarded to Maha Abdellatif at the University of Medicine and Dentistry of New Jersey to elucidate the role of Ras GTPase-activating protein in regulating miR-1 during cardiac hypertrophy.
According to the grant's abstract, the miRNA is "acutely down-regulated upon induction of hypertrophy by work overload, growth factors, or RasGAP — an outcome that is necessary for myocyte growth.
"Our preliminary data show that RasGAP SH3-binding protein binds miR-1 in a RasGAP- and Akt-dependent manner," Abdellatif wrote in the abstract. "We hypothesize that hypertrophic stimuli induce Akt-mediated G3BP phosphorylation and its subsequent recruitment by RasGAP-filamin complex," an event that brings it close to miR-1, where it binds and hydrolyzes premature miRNA.
The subsequent down-regulation of miR-1 triggers an up-regulation of its targets including RasGAP, Cdk9, fibronectin, endothelin, and insulin-like growth factors, which Abdellatif notes play a key role in the development of cardiac hypertrophy.
With the NIH funding, Abdellatif and her colleagues propose to examine the mechanism of RasGAP-mediated down-regulation of miR-1 during myocyte hypertrophy by using cultured myocytes in conjunction with recombinant cDNA, adenoviruses, and promoter constructs, and ultimately study the role of RasGAP and miR-1 during cardiac hypertrophy in a mouse model.
The grant begins on Jan. 15 and runs to the end of 2012. It is worth $390,000 in 2010.