Investigating how mRNA decapping factors regulate gene silencing programs

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Post-transcriptional control (PTC) programs regulate protein production after a messenger (m) RNA is transcribed from its DNA template. Many PTC programs control protein production by repressing mRNA translation and/or initiating mRNA destabilization, events collectively referred to as `silencing'. A number of PTC mediators, including micro (mi)RNAs and RNA binding proteins bring about gene silencing by recruiting the CCR4-NOT deadenylase complex to target mRNAs. Others, such as the RNA binding protein MARF1 endoribonuclease, internally cleave targeted mRNAs to engender their decay. Both the miRNA machinery and MARF1 interface with mRNA decapping factors, which in turn can enhance or inhibit their activities, respectively. Many decapping factors associate with each other through a network of multivalent interactions. These protein-protein interaction networks can in turn can lead to formation of dynamic ribonucleoprotein granules in cells called processing bodies, or `P'-bodies. How these P-bodies and their protein-protein interactions help regulate mRNA biology is not well known and is a topic of significant research. Here, I propose a research program that aims to shed light upon how the mRNA decapping machinery regulates different classes of mRNA silencing programs in the context of RNA granule integrity. We hypothesize that (i) mRNA decapping factors inhibit MARF1 endonuclease activity and (ii) modulating RNA granule integrity in turn regulates MARF1- and microRNA-mediated gene silencing programs, as well those mediated by RNA binding proteins that recruit the CCR4-NOT deadenylase complex. To test these hypotheses, we propose the following aims: 1.To elucidate the molecular underpinnings of how MARF1 interacts with the decapping machinery and how it targets select mRNAs for decay. 2.To determine how P-body granule integrity regulates MARF1-mediated mRNA decay. 3.To determine how P-body integrity modulates the mode by which microRNAs repress targeted mRNAs. The proposed research program is highly significant and builds upon key published and unpublished observations our group has made over the course of our previous NSERC Discovery grant. Ultimately, we are confident that this research proposal will generate novel insights into the modes and mechanisms by which interactions between mRNA decapping and decay factors help regulate PTC silencing programs in mammalian cells.