Coccolithophorids are marine phytoplanktons that produce elegantly sculptured calcium carbonate cell coverings known as coccoliths. Emiliania huxleyi (E. huxleyi) is a model coccolithophorid that has potential biomedical applications related to bone formation and diseases. Small silencing RNAs (smRNAs) are known to play a pivotal role in post transcriptional gene regulation in many eukaryotes, from unicellular to multicellular plants and animals. Small RNAs are involved in a variety of cellular processes that are essential to genome stability, development, and adaptive responses to environmental stress. These natural smRNA silencing processes otherwise known as RNA interference, suppress protein expression by triggering the enzymatic cleavage of mRNAs. The specificity of smRNA processes has been exploited for the systematic analysis of gene functions and potential therapeutic purposes. MicroRNAs (miRNAs) and small interfering RNAs (siRNAs) are two major classes of smRNAs known in plants. Very little is currently known about smRNAs and their regulatory roles in E. huxleyi. The specific hypothesis underlying this proposed research is that E. huxleyi contains small RNAs such as miRNAs and siRNAs, which regulate target genes through RNA silencing. The recent release of the E. huxleyi genome by the Joint Genome Institute (JGI) and a large dataset of small RNAs of E. huxleyi generated using high-throughput Solexa sequencing, make it feasible to systematically characterize smRNAs and their regulatory roles in E. huxleyi. Preliminary studies show strong evidence for the existence of miRNAs in E. huxleyi. We propose herein to characterize small RNAs in E. huxleyi using computational methods, combined with experimental validation. Specifically, we propose to 1) identify and characterize miRNA precursors and mature miRNAs in E. huxleyi by computationally analyzing the short RNA sequences;2) computationally predict and characterize targets of E. huxleyi miRNAs and validate the predictions with experimental evidences;and 3) identify and characterize siRNAs and other classes of small RNAs in E. huxleyi. To our knowledge, this proposed research will be the first systemic analysis of smRNAs in E. huxleyi, and as such will provide important insights into post-transcriptional gene regulation in this microalga.
Coccolithophorids are presently being investigated by the biotechnology industry as a potential source for new pharmaceuticals, omega-3 fatty acids, biofuel, and polysaccharides. They also have important implications for human health in terms of understanding the process of calcification as it relates to bone disease, kidney stones, and heart disease. Insights into smRNA controlled gene regulation in E. huxleyi may lead to a better understanding of the fundamental biology of this important microcalcifier, which may offer new strategies to promote healthy biomineralization and mitigate calcification associated with disease states.
