Reflecting the growing interest in microRNAs as regulators of development and biological functioning, the National Institutes of Health last week awarded two miRNA-related grants.
One will study the role of these small, non-coding RNAs in pancreas development, while the other will look at how miRNAs impact the neural processes controlling cognition and behavior.
The grants follow three others awarded last month that support research into miRNAs’ role in aging, the development of a cell-based screen for miRNA inhibitors, and an investigation into how miRNAs inhibit protein synthesis.
miRNAs in Pancreatic Development
The first grant awarded this month went to University of California, San Francisco, researcher Michael German. Issued by the National Institute of Diabetes and Digestive And Kidney Diseases, it funds German’s research into the role that miRNAs play in pancreatic islet development.
The grant runs for five years with $315,444 released in the first year.
“Although miRNAs are known to influence the development of several tissues, and they have been implicated in glucose-stimulated insulin secretion in mature beta cells, the role of miRNAs in pancreatic development has not been explored previously,” according to the grant’s abstract.
The aims of German’s project are to establish the “global role” of miRNAs in pancreas and islet development, then to break out the parts that individual miRNAs play, the abstract notes.
To evaluate the importance of miRNAs in pancreas development, German proposes to remove either Dicer of DGCR8, two components of the miRNA-processing pathway, from early pancreatic precursor cells, early islet precursor cells, and mature beta cells.
MiRNA microarrays developed in-house and transgenic animals will be used to determine the expression pattern of miRNAs during pancreatic development, the abstract states. Meantime, efforts to evaluate the role of specific miRNAs in regulating the expression of pancreatic transcription factors will focus on the regulation of the beta cell differentiation factor Nkx6.1.
German and colleagues will also specifically examine the potential regulation of miRNAs by the pancreatic transcription factors Sox9, Neurogenin3, and Nkx2.2.
“These studies will help us understand how islet cells differentiate and the role of miRNAs in that process,” which in turn is expected to help develop methods to produce islet cells for diabetes patients, the abstract adds.
Cognition, Behavior, and miRNAs
The second grant issued this month was awarded by the National Institute of Mental Health to Massachusetts General Hospital investigator Raymond Kelleher to examine miRNAs in synaptic plasticity and cognitive function.
The project, slated to run five years, has been awarded $396,121 for the first year.
“Disturbances in miRNA-dependent processes have been proposed to contribute to the etiology of mental disorders, but the role of miRNAs in synaptic and cognitive function in the mammalian brain is entirely unknown,” the grant abstract states.
Earlier research by Kelleher and colleagues suggests that “general up-regulation of local protein synthesis through the MARK and mTOR pathways is necessary for persistent modifications of synaptic connectivity and associated cognitive processes,” the abstract notes. “While these results point to the importance of general translational mechanisms, the translational response to synaptic activation is likely to be modulated by mRNA-specific mechanisms mediated by cis-acting elements, such as miRNA target sequences.”
In the grant abstract, Kelleher states that miRNA-dependent translational control may selectively regulate the protein synthesis-dependent consolidation of synaptic plasticity and memory.
To test this hypothesis, he and his colleagues plan to use a “conditional genetic approach to inactivate Dicer and abolish miRNA expression in neurons of the postnatal forebrain,” the abstract states. “Multidisciplinary analysis of the phenotypes caused by conditional ablation of miRNA expression will allow us to evaluate the global contribution of miRNAs to neural processes.”
Specifically, the researchers are focusing on the role of miRNAs in neuronal activity-dependent translational control in primary hippocampal neurons; spatiotemporal patterns of expression of specific proteins implicated in synaptic modifications in the postnatal brain; protein synthesis-dependent and -independent phases of hippocampal synaptic plasticity; and behaviors relevant to mental disorders, particularly protein synthesis-dependent and -independent phases of hippocampus- and amygdala-dependent memory, the abstract notes.
These efforts “should yield important insights into the role of miRNAs in brain function, and the possible involvement of altered miRNA-dependent processes in the pathogenesis of mental disorders,” it adds.
miRNAs and Aging
Last month proved rewarding for miRNA researchers, as well, including Rutgers University’s Monica Driscoll, who was awarded a two-year grant from the National Institute on Aging to conduct a genome-wide analysis of the impact of miRNAs on aging. Driscoll will receive $158,192 in the first year of the grant.
“Next to nothing is known about how miRNAs affect the biology of aging, although very recent work has shown that … lin-4 affects lifespan and age pigment accumulation rates,” the grant’s abstract states.
“We have characterized the expression of all 114 C. elegans miRNAs during adult lifespan and find that the levels of several miRNAs rise or fall in patterns consistent with potential roles in aging and health-span modulation,” the abstract adds.
“Our plan is to exploit the experimental advantages of the C. elegans system to define the roles of age-regulated miRNAs in the biology of aging, with an emphasis on those that impact overall health span and … age-related muscle decline.”
Specifically, Driscoll and her colleagues will test their hypothesis that changes in levels of a specific miRNA, miR-1, influence muscle aging, and that manipulation of this miRNA can extend muscle health span in later life. “This study could provide the first detail on how a miRNA can affect muscle aging and could establish proof-of-principle for novel therapies for extension of muscle health span,” the abstract states.
“Disturbances in miRNA-dependent processes have been proposed to contribute to the etiology of mental disorders, but the role of miRNAs in synaptic and cognitive function in the mammalian brain is entirely unknown.”
The researchers also aim to identify new age-regulated miRNAs and test how their over-expression and deletion impacts lifespan and health.
“Since many miRNAs are conserved in expression pattern and function, we anticipate that our exploratory work in this area will be of high impact and will suggest specific working hypotheses for mammalian aging, perhaps suggesting novel means of clinical intervention,” the abstract adds.
miRNAs and Small-Molecule Inhibitors
Also in March, the National Institute of Neurological Disorders and Stroke awarded Wistar Institute researcher Qihong Huang a one-year grant worth $95,055 to develop a high-throughput cell-based assay for screening small-molecule inhibitors of miRNAs.
To develop this system, Huang proposes to introduce a reporter system consisting of a luciferase gene fused to the binding site of miR-21 into human HeLa cancer cells, according to the grant’s abstract.
“Inhibition of the miRNA pathway by small molecules will lead to an increase of a luciferase reporter signal and thus provide an advantageous positive read-out,” it notes. “This assay will be used to screen small molecule libraries in a high throughput fashion to discover molecules that interfere with the miRNA pathway.”
The grant project also includes the development of three secondary assays to validate and characterize the compound hits from the primary screen. “These assays will exclude non-specific small molecule hits and deliver a more detailed picture of the miRNA pathway steps that are targeted by active compounds,” the abstract states. “Positive hits will be further investigated and improved through structure-activity relationship studies, via synthesis of second-generation compound arrays.”
The abstract states that the small molecules discovered through the project are “expected to have a broad impact on human health, due to the critical roles that miRNAs play in several human diseases such as cancer and viral infection. These molecules will be promising lead compounds for the development of new chemical tools in biomedical research and new therapeutic agents.”
miRNA and Protein Synthesis
The last grant issued last month went to University of California, Berkeley, researcher Keri Silva and funds research into how miRNAs and associated proteins interact with the ribosome to inhibit protein synthesis, according to its abstract.
Silva’s grant will run four years and has received $27,195 for its first year.
“The specific goal of this proposal is to determine the stage at which translation is repressed by miRNAs through [development] of an in vitro assay using rabbit reticulocyte lysate, a robust translational system,” the abstract states. “A biochemical approach will be taken, such that protein fractionation, protein purification, and target mRNA will be assembled in rabbit reticulocyte lysate in order to recapitulate the [miRNA repression] mechanism in vitro under tightly controlled conditions.”
Analysis of in vitro translation and toeprinting assay experiments will be conducted by polyacrylamide gel electrophoresis, autoradiography, and polysome profiling.
“In humans, the process of protein biosynthesis is regulated by microRNAs during many stages of development,” the abstract concludes. “Defects in this regulation may contribute significantly to the onset of many diseases, including cancer. Understanding the molecular mechanism of microRNA function will greatly aid in future development of therapies for diseases affecting development and leading to cancer.”