Two microRNA-regulated transcription factors,
cog-1 and
die-1, are required for the establishment and maintenance of left/right asymmetry in the ASE cell type. The microRNA
lsy-6 has been shown to regulate the translation of
cog-1 in ASEL through deletion analysis and 3 UTR sensor constructs (1). We are systematically mutating the
lsy-6 target site in the
cog-1 3UTR in the context of sensor constructs in order to determine the specific requirements for functional microRNA/target interaction. This analysis will provide valuable insight into microRNA/target interaction, which will provide prediction specialists with a verified data set. The status of this analysis will be presented. Furthermore, we hypothesize that a battery of microRNAs, including
mir-273,
mir-55,
mir-56 and
mir-265 are acting in a combinatorial manner to regulate
die-1 via several evolutionarily conserved target sites in its 3 UTR.
die-1 encodes a Zn finger transcription factor essential for the generation of ASE asymmetry (2). GFP-reporter analysis shows a strong bias for
die-1 expression in ASEL vs. ASER.
die-1 is primarily responsible for inhibiting the expression of
cog-1 in ASEL via activation of the microRNA
lsy-6. Through the use of a
ceh-36::gfp-
die-1 3 UTR sensor construct it was determined that
die-1 is down regulated in ASER through its 3 UTR (2). This sensor construct analysis indicates that two phylogenetically conserved sites in the
die-1 3 UTR are required for ASER down-regulation; both sites display complementarity to
mir-273,
mir-55 and
mir-56 and an as yet untested site displays similarity to
mir-265. All of these microRNAs are expressed predominantly in the ASE cell type, with some showing a bias toward ASER expression. Additionally, ectopic expression of
mir-55,
mir-56, and
mir-273 is each sufficient to disrupt ASE asymmetry; while
mir-265 has yet to be tested. We are determining which miRNAs act on which site/s in the 3 UTR of
die-1 to regulate its expression. This analysis will involve a systematic scanning deletion analysis of the
die-1 3 UTR, in conjunction with an analysis of microRNA deletion alleles that we retrieved from a deletion library. This analysis will provide a paradigm for combinatorial regulation of target genes by microRNAs. References: (1) Johnston RJ, Hobert O. Nature. 2003 Dec 18;426(6968):845-9. (2) Chang S, Johnston RJ Jr, Frokjaer-Jensen C, Lockery S, Hobert O. Nature. 2004 Aug 12;430(7001):785-9.