Precise temporal and spatial control of gene expression is essential to ensure normal development and homeostasis and is achieved through multiple regulatory mechanisms acting at every step from gene transcription to protein degradation. MicroRNAs (miRNAs), constitute a large class of highly conserved regulatory short non-coding RNAs that modulate gene expression at the post-transcriptional level. Mechanistically, miRNAs repress their targets as part of a large multicomponent ribonucleoprotein known as the miRNA-Induced Silencing Complex (miRISC), which include Argonaute proteins (AGO) and members of the TNRC6 family of proteins. How miRISC assembly and activity are regulated in vivo remains poorly understood and until recently it was tacitly assumed that miRNAs are constitutively active in all cell types. Recent work from several groups, including ours, has challenged this assumption, revealing that assembly of the miRISC is tightly regulated in vivo, and suggesting the possibility that in many adult tissues and in quiescent cells the bulk of miRNA-bound AGO proteins are not bound to target mRNAs, and are not engaged in their repression. Why many adult tissues and quiescent cells contain high levels of functionally inactive miRNAs and whether, in addition to mitogenic cues, other perturbations?including cancer, aging, infection, and inflammation?regulate miRISC assembly and activity are two major unanswered questions in the field and the main focus of this grant proposal. As a first step to address these two questions we propose a series of experiment that take advantage of a novel genetically engineered mouse strain we have recently generated that allows to control miRISC assembly in vivo in a temporally and spatially controlled fashion. We will use this novel mouse strain to determine the requirement for miRISC activity during normal tissue homeostasis, in development, and during tissue regeneration (Aim 1).
In Aim 2, we will use it to directly test the hypothesis that miRISC activity is required for tumor progression and tumor maintenance in vivo and we will determine the potential of miRISC inhibition as a novel anticancer strategy. Finally, in Aim 3 we will take advantage of a novel mouse strain we have developed to gain mechanistic insights into how miRNAs control these essential processes. Successful completion of this project will greatly advance our understanding of the role of miRNA-mediated gene repression in mammals and could lead to the development of novel anti-cancer strategies.
MicroRNAs are small non-coding RNAs that provide an important layer of gene regulation by negatively controlling the levels of a large fraction of protein coding genes. The research proposed in this grant application has three major goals. First, we want to determine the role of miRNA function during mammalian development, in tissue homeostasis, and during tissue regeneration. Second, we want to test whether that acute inhibition of miRNA function can serve as a novel and effective anticancer strategy. Finally, we want to understand what targets and pathways miRNAs control under these conditions.