The long-term goal of my laboratory is to understand the molecular mechanism(s) by which major developmental transitions are orchestrated. Although precise spatiotemporal regulation of gene expression is essential during these transitions, degradation of key regulators and the mRNAs from which they derive is also critical. Over 60% of the mRNAs associated with early C. elegans development are maternally contributed, of which ~30% will be degraded during the maternal-to-zygotic transition, or MZT, in the first few hours of embryogenesis. We hypothesize that C. elegans utilizes a large and diverse set (or sets) of endo siRNAs to target maternal mRNAs for degradation in early embryos. Any specific maternal transcript fated for degradation will include a target sequence that can be recognized by one or more of the large variety of these early embryo endo siRNAs. These endo siRNAs are derived from a family of highly related genes, some of which are known to be transcribed in early embryos. Central to this model is the Y24F12A.3/.4 locus (previously vet-5), one of the earliest known zygotic genes to be transcribed. We will test two possible mechanisms to explain efficient and robust en masse degradation of maternal mRNAs through endo siRNAs. Our preliminary results show: (1) Degradation of 6 hand-picked maternal mRNAs in C. elegans embryos requires generation of endogenous siRNAi. (2) Endo siRNA derived from the Y24F12A.3/.4 locus plays a central role in endo siRNA-mediated maternal mRNA degradation and embryogenesis. (3) Bioinformatic analyses identified common molecular features in some maternal mRNAs destined for degradation.
The Specific Aims are: (1) To determine whether the endo RNAi pathway functions in degradation of maternal mRNAs. We will take a genome-wide approach to identify mRNAs whose degradation is regulated by endo siRNAs, and Y24F12A.3/.4 siRNAs specifically. (2) To determine the function of Y24F12A.3/.4 in embryos. We will characterize the phenotype(s) of the newly generated Y24F12A.3/.4 null worm strain, with focus on maternal mRNA degradation. (3) To investigate the mechanism by which Y24F12A.3/.4 regulates degradation of maternal mRNAs. We will determine how endo siRNAs from this locus are generated, and how maternal mRNA degradation is affected. We test for direct sequence recognition between siRNA and target transcript. This proposal is highly significant and innovative. Innovations include (1) the concept that the proposed work seeks to establish is novel, (2) the involvement of highly repetitive genes/pseudogenes in the clearance of maternal mRNA during MZT is novel, (3) and the use of state-of-the-art genomic techniques, including RNA-seq and small- RNA-seq of small samples. This model, if true, will shift the paradigm for understanding not only the function of endo siRNAs, but also developmental transitions which require en masse degradation of existing mRNAs. The proposed work may uncover a novel gene regulatory mechanism that has a huge impact in our understanding of development beyond C. elegans.
Very small pieces of RNA are now known to play key regulatory roles - usually by turning specific genes off - in almost every aspect of biology, including embryonic development, tissue homeostasis, aging, and many different disease states, including cancer. This research project uses a small model organism of only 1000 cells to examine the role of a specific subset of small RNAs in early embryonic development when many of the mRNAs that were loaded into the egg to drive early embryogenesis need to be removed to permit normal embryogenesis to continue. Our results may well uncover new small RNA functions which are relevant to their function(s) in other biological processes, including regeneration and disease.
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