Temporally Regulated MicroRNAs in C. elegans

Yale University, New Haven CT

Investigators

Abstract

b. Summary: Intellectual merits: MicroRNAs (miRNAs) are a novel class of regulatory RNAs found in plants and animals that regulate gene expression by binding to complementary sequences in the 3'untranslated region (UTR) of their target gene mRNAs. lin-4 and let-7, founding members of the miRNA family, are critical for timing of stage-specific developmental events in C. elegans. Both are temporally regulated during development and are conserved in higher animals, including humans. Despite being the best understood miRNAs, little is known about their mechanism of action, their targets, or how they are temporally regulated. lin-4 and let-7 RNAs first accumulate during the first and fourth larval stages, respectively. They act as timing switches to initiate a cascade of developmental changes in the somatic cells between the L1 and L2 stages (lin-4), and the L4 and adult stages (let-7). Since the appearance of these miRNAs correlates with temporal transitions in cell fates, we can distill the timing of temporal transitions down to the timing of appearance of these miRNAs. In this work, the Slack laboratory will systematically identify the sequences and factors important for the temporal up-regulation of lin-4 and let-7. The lin-4 and let-7 promoters contain elements conserved in other nematode species. In the case of let-7, a conserved element called the temporal regulatory enhancer (TRE), which binds an unknown factor called TRE binding factor (TREB), is required for temporal up-regulation of let-7. This work will identity TREB. In a similar manner, the regulatory factors that bind to important sequences in the lin-4 promoter will be identified. This should help define the pathways upstream of these important miRNAs. Since both are conserved and temporally regulated in other animals, this work will provide valuable insights into the regulatory mechanisms used by the miRNA family. Since lin-4 RNA accumulates during the L1 stage and is responsible for the L1/L2 transition, and let-7 RNA accumulates during the L4 stage and is responsible for the L4/Adult transition, it seems plausible that additional temporally regulated miRNAs control other developmental transitions in C. elegans. Over 100 miRNAs, encoded by mir genes, have been identified in C. elegans, about one third of which are temporally regulated. This work will focus on the three let-7 homologues, e.g. mir-84, and the lin-4 homologue, mir-237, since they are temporally regulated and complementary to the 3'UTR of known heterochronic genes. The Slack laboratory will determine the expression pattern of these miRNAs by developmental northern blot and mir::gfp fusion technology. In parallel, this work will also genetically test the developmental role of these miRNAs by examining the phenotype caused by over-expressing each selected mir gene in a wild-type background. Knowledge of the expression patterns may additionally lead the Slack laboratory to identify additional potential miRNA target genes. Broader impacts: Not only will this work aid in our understanding of the heterochronic pathway in C. elegans, but it will also provide important information on the regulation and complexity of miRNA control of developmental timing. In addition, since many of the temporally regulated miRNAs are conserved in other animals, including humans, this work should provide important information about their regulation and even their potential targets. One postdoc, 2 graduate students and a number of undergraduate students will be trained during the course of this proposal. The experiments outlined here are mostly accessible to undergrads, and a small project can be completed within a summer or semester. The Slack lab has historically provided research opportunities to 3 undergrads a year, and it is anticipated that this involvement will increase over time, e.g., there are 4 undergrads here this year. Most of these students are from the Yale SURF and STAR programs, which cater to minorities underrepresented in science. If interested, they attend the current C. elegans meeting, where they present a poster and interact with other scientists. Two are second authors on manuscripts.