The long term goal of this work is to understand the fundamental problem of how tissues and organs are specified at the correct time and synchronized with respect to one another during development. We and others are systematically exploiting the power of C. elegans genetics to identify the important players as mutants that express cell fates (and hence, tissues) earlier or later than wild-type animals do. These mutants define genes, termed heterochronic genes, that control the timing of cell fate determination. Two heterochronic genes, the small RNA gene, let-7 and its target, lin-41 control the timing of a proliferation versus differentiation decision by epidermal cells through an unknown mechanism that we would like to understand. We would also like to understand how a proposed duplex between the let-7 and the 3'UTR lin-41 RNAs controls gene expression of lin-41. Since LIN-41 has orthologues in mammals, the principles for control of developmental timing discovered in our work may be more general, and may shed light on the mechanism of oncogenesis by the poorly-understood RBCC family of regulatory proteins, of which LIN-41 is a member. In this proposal we will explore the mode of action of the let-7 and lin-41 genes. While there is currently much interest in how small RNAs (like those involved in RNA interference) control gene expression, little is known about their mechanism of action. We propose to test the hypothesis that an RNA/RNA duplex forms between the let- 7 and lin-41 mRNAs and examine how this duplex controls gene expression. We would like to identify new factors that act with the small RNA let-7 to control gene expression, in the hope that the molecular nature of the new genes will shed light on the mechanism of let-7. The first major aim of the proposal is to identify new genes that modify or cause Let-7 phenotypes through saturating genetic screens. We will also generate mutations in the 3'UTR of lin-41 that are compensatory to the let-7(n2853) allele and test for restoration of normal regulation in a let- 7(n2853) mutant. Can the two RNAs bind each other in vitro? If so, then we propose to illuminate the structure using chemical and enzymatic means. If we can show evidence for a duplex, then we would like to identify potential proteins that bind the duplex, using biochemical means or a yeast three hybrid screen. The second major aim of the proposal is to elucidate the mechanism of action of lin-41, a member of the RBCC family, many of which are human cancer genes. We will seek genetic suppressors of lin-41 phenotypes and search for lin-41 interacting proteins using the yeast two hybrid screen in an effort to identify partners and targets of lin-41. We will also study the role of lin-41 in the control of one of its target genes, the transcription factor, lin-29. Control of developmental timing and gene regulation by 3'UTR elements are fundamental problems faced by all animals. Our analysis should shed light on both a novel mechanism of gene regulation by small RNA genes and temporal inputs to cell-cycle control, which may direct work in analogous systems in human biology and disease.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Genetics Study Section (GEN)
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Greenberg, Judith H
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Yale University
Schools of Arts and Sciences
New Haven
United States
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Slack, Frank J (2013) MicroRNAs regulate expression of oncogenes. Clin Chem 59:325-6
Van Wynsberghe, Priscilla M; Chan, Shih-Peng; Slack, Frank J et al. (2011) Analysis of microRNA expression and function. Methods Cell Biol 106:219-252
Corbin, Rachel; Olsson-Carter, Katherine; Slack, Frank (2009) The role of microRNAs in synaptic development and function. BMB Rep 42:131-5
Chan, Shih-Peng; Slack, Frank J (2009) Ribosomal protein RPS-14 modulates let-7 microRNA function in Caenorhabditis elegans. Dev Biol 334:152-60
Nimmo, Rachael A; Slack, Frank J (2009) An elegant miRror: microRNAs in stem cells, developmental timing and cancer. Chromosoma 118:405-18
Chan, Shih-Peng; Ramaswamy, Gopalakrishna; Choi, Eun-Young et al. (2008) Identification of specific let-7 microRNA binding complexes in Caenorhabditis elegans. RNA 14:2104-14
Maller Schulman, Betsy R; Liang, Xianping; Stahlhut, Carlos et al. (2008) The let-7 microRNA target gene, Mlin41/Trim71 is required for mouse embryonic survival and neural tube closure. Cell Cycle 7:3935-42
Ding, Xavier C; Slack, Frank J; Grosshans, Helge (2008) The let-7 microRNA interfaces extensively with the translation machinery to regulate cell differentiation. Cell Cycle 7:3083-90
Nolde, Mona J; Saka, Nazli; Reinert, Kristy L et al. (2007) The Caenorhabditis elegans pumilio homolog, puf-9, is required for the 3'UTR-mediated repression of the let-7 microRNA target gene, hbl-1. Dev Biol 305:551-63
Johnson, Charles D; Esquela-Kerscher, Aurora; Stefani, Giovanni et al. (2007) The let-7 microRNA represses cell proliferation pathways in human cells. Cancer Res 67:7713-22

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