Post-transcriptional Regulation of Gene Expression by Small RNAs and RNA-binding Proteins.

[摘要] Small RNAs and RNA-binding proteins (RBP) are central regulators of gene expression. Yet, the functions of only a handful of them are well understood. The objective of my doctoral research was to better characterize the mechanisms by which small RNAs and RBPs govern post-transcriptional regulation of gene expression.The first half of my thesis examined the role of small RNAs (endogenous siRNAs and microRNAs) in gene regulation and development in C. elegans. First, I uncovered a novel class of endogenous small RNAs (26G RNAs) expressed during germline development. Male 26G endo-siRNAs are expressed during spermatogenesis and are required for sperm production, while female 26G endo-siRNAs are produced during oogenesis and regulate filial gene expression. Second, I discovered that casein kinase 2 phosphorylates a key cofactor of the microRNA-induced Silencing Complex (miRISC) and determined miRISC phosphorylation to be critical for target binding and repression. In addition, I developed a high-throughput approach to sequence the 3’ UTRs of mRNAs, which include the main targets of microRNAs. My efforts produced accurate 3’ UTR annotations for 85% of C. elegans genes and revealed a new mode of gene regulation through alternative 3’ UTR expression across development.The second half of my thesis investigated the global impact of RBP-RNA interactions in budding yeast. First, I isolated RBPs en masse and sequenced the RNA substrates of RBPs. Sequence analysis revealed novel general properties of RBP-RNA recognition as well as highlighting the 3’UTR as the major site of RBP associations. 25% of all RBP-RNA interactions were altered in stress conditions with major changes for genes that encode metabolic factors, reflecting the central role of RBP-mediated post-transcriptional regulation in stress response. Second, protein analysis of in vivo purified RBP-RNA complexes uncovered not only 127 known RBPs, but also 132 novel RBPs that lack recognizable RNA-binding domains. These newly discovered RBPs are highly conserved and fulfill roles in critical biological processes including metabolism and trafficking. Collectively, my findings expand the repertoire of post-transcriptional regulatory mechanisms and provide important insights into analogous mechanisms in higher eukaryotic organisms.

[发布机构] University of Michigan

[效力级别] RNA-binding Proteins

[关键词] Small RNAs;RNA-binding Proteins;Genetics;Molecular;Cellular and Developmental Biology;Science;Cell and Developmental Biology