MicroRNA (miRNA) are endogenous regulatory non-coding RNA molecules. miRNAs are grouped into families based on the identity of nucleotides 2-8 from the 5' end, which are referred as the miRNA seed and are essential for miRNA targeting.
let-7 family miRNAs are present in nearly all bilaterians, and almost all species that have
let-7 family miRNAs contain at least one copy of a particular
let-7 isoform (referred as
let-7a) that is nearly 100% conserved in sequence, including all 3' non-seed nucleotides. This deep conservation suggests that
let-7a may have essential functions that are not redundantly shared by its paralogs. Since all
let-7 family miRNAs share the seed sequence, we hypothesize that
let-7a has special functions that are associated with its non-seed region. We firstly investigated the specificity of
let-7a in C. elegans by swapping
let-7a's non-seed sequence with its family paralog
mir-84, whose functions and expression patterns are different from
let-7a in C. elegans. The swap mutant expresses miR-84 with similar level and developmental profile as wild type
let-7a, but exhibits retarded heterochronic phenotypes and vulva defects, consistent with
let-7a loss-of-function. These results indicate that the specificity of
let-7a function is not determined by its expression pattern alone, but by its non-seed sequence. To further study the functions associated with the
let-7a non-seed region, we individually mutated each of nucleotides 9-22 of
let-7a, and confirmed the normal expression of the mutant
let-7a miRNAs by small RNA sequencing. We observed that each of nucleotides 11-16 are essential for
let-7a function, suggesting a "secondary seed" model, involving pairing to nucleotides 11-16, which is consistent with the structural basis of miRNA (Schirle et al. 2014). Additionally, we found that nucleotides 17-22 functions synergistically with the secondary seed. We also confirmed that derepression of
lin-41 contributes to the phenotypes of
let-7a secondary seed mutants, however restoring
let-7a secondary seed pairing to
lin-41 by compensatory mutations at the
lin-41 3' UTR only partially rescues the phenotypes, suggesting that
let-7a has critical targets other than
lin-41 that also require non-seed pairing. We are employing ribosome profiling and RNA-seq to study the translatome and transcriptome of
let-7a non-seed mutants in order to identify
let-7a target genes that are derepressed in
let-7a non-seed mutants, and to characterize the base pairing patterns of
let-7a to those target mRNAs. To sum up, our study investigated the features of the
let-7a non-seed region and characterized a secondary seed model that essentially associated with
let-7a functions, which may provide insights into miRNA targeting mechanism and conservation.