Deciphering The Role of RNA Modifications In Maternal Mrna Clearance - Abstract Maternal RNA clearance is an essential process that occurs in the absence of transcription in all sexually reproducing species examined. Stored maternal transcripts sustain early embryonic development prior to activation of the embryonic genome. Perturbations of maternal mRNA degradation can either halt or irreparably alter the maternal-to-zygotic transition, leading to early embryonic demise. Oocytes are unique cells in which translation and RNA clearance are coupled, in the absence of new transcription. Maternal mRNA is eliminated via translationally coupled mRNA degradation, a process that includes recruitment of mRNA to the ribosome, shortening of the poly(A) tail, decapping, and degradation by both 5′ and 3′ exonucleases. Mechanisms that regulate translationally coupled mRNA degradation are poorly understood. Recent studies have established RNA modifications as regulators of maternal mRNA clearance. Our previous work established that inosine RNA modifications within the coding region of mRNA can impact maternal mRNA stability through a translation mechanism. Here, I describe two research directions I will pursue in the next 5 years that address fundamental questions about the role of RNA modifications in maternal mRNA clearance. The first direction will investigate the relationship between inosine mRNA modifications in translation. The second direction will focus on identifying the relationship between inosine RNA modifications and other RNA modifications, and how this combined “RNA modification code” can regulate RNA stability and translation during maternal mRNA clearance. We will use innovative molecular analyses, novel RNA-sequencing approaches, proteomics, in vivo imaging, and combined single molecular/single oocyte assays to test how inosine impacts translation, alters the occurrence of amino acid substitutions, and how this facilitates maternal mRNA clearance. In addition, I will catalog the “RNA modification code” on whole, individual transcripts in oocytes to determine the relationship between multiple RNA modifications and maternal mRNA clearance. While these studies will significantly advance the understanding of RNA processing in oocytes, the discovered principles will also apply to other cells. In fact, inosine RNA modifications were first discovered in oocytes (Xenopus laevis), and this novel discovery led to a change in the entire way we view RNA processing mechanisms. The utility of the oocytes as a model will enable the impact of RNA modifications on the fate of the RNA to be amplified, and will advance our appreciation of the biological roles RNA modifications have within cells, organs, and organisms. These studies will provide a powerful framework of understanding that will build the research focus of the Brachova lab for the future, eventually leading to an understanding of the relationship between RNA modifications and embryo genome activation.
