MicroRNA (miRNA) are endogenous non-coding RNA molecules that exist in all multicellular eukaryotes. MiRNAs are grouped into families based on the identity of nucleotides 2-8 from the 5' end, which are also referred to as the miRNA seed. The seed region is essential for miRNA targeting, while the non-seed region, or 3' supplemental, can also contribute to target specificity. Let-7 family miRNAs have been shown to exist in almost all bilateral animals, and are often present in multiple copies in the genome. Strikingly, almost all species that have
let-7 family miRNAs contain at least one copy of a particular
let-7 isoform (referred to as
let-7a) that shares almost 100% identical sequence across bilaterians. Since the
let-7 family microRNAs have the same seed region sequences, the deep conservation of the non-seed region of
let-7a suggests that
let-7a may have unique target sites and/or other characteristics not shared by other
let-7 family miRNAs. We investigated the specificity of in vivo functions of the
let-7a non-seed sequences by swapping the
let-7a non-seed region at the C. elegans
let-7 locus with the non-seed region of the C. elegans
let-7 family paralog,
mir-84. The
let-7(
mir-84 swap) mutant expresses miR-84 with a developmental profile, and to levels, similar to wild type
let-7. However,
let-7(
mir-84 swap) animals exhibit retarded heterochronic phenotypes and vulva defects characteristic of
let-7(lf). This result suggests the existence
let-7a-specific target sites or other interactions that are determined by the 3' supplemental of
let-7a, and which cannot be effectively engaged by
mir-84. We postulated that
lin-41 could be one
let-7a target that contributes to the phenotypes above, since the
let-7 sites in the
lin-41 3' UTR exhibit extensive non-seed complementarity specific to
let-7. Accordingly, we mutated the
let-7 complementary sites (LCSs) in the endogenous
lin-41 3' UTR to match the 3' region of
mir-84. These
lin-41 LCS mutations should restore full 3' supplemental pairing of
mir-84 to the
lin-41 3' UTR sites, but nevertheless they only partially rescued the
mir-84 swap heterochronic phenotypes. This result suggests that other targets, besides
lin-41, interact with
let-7 by 3' supplemental base pairing, and contribute to the control of developmental timing in C. elegans. Our future direction will be to focus on identifying other
let-7a-specific targets that are regulated by 3' supplemental base pairing to
let-7a, and to characterize the base-pairing patterns and/or other features associated with these interactions.