Figure 6

Example of a miRNA with false-negative evidence by northern blot (2R:11128979 = miR-137). In this example, four related sequences from four species of insects (Dm, D. melanogaster; Dp, D. pseudoobscura; Ag, A. gambiae; Am, A. mellifera) all adopt a phylogenetically conserved stem-loop structure. One arm has been perfectly preserved among all four species, and we presume that a miRNA is processed from within the conserved sequence (orange). Patterns of nucleotide divergence characteristic of miRNAs are seen, with more related sequences (Dm/Dp and Ag/Am) showing approximately equal amounts of divergence within the loop and along one arm, whereas the Dm/Dp vs Ag/Am comparison shows complete divergence within the loop (blue), with slightly less overall divergence along the putative non-miRNA-encoding arms. We deduce that a mature miRNA may initiate at one of the U residues that are highlighted by asterisks, as the first residue of the conserved region is found in the loop of the drosophilid hairpins and the second G residue is unfavored as the 5' residue of a miRNA. Northern analysis was negative using a probe complementary to the conserved region as well as with a probe identical to the conserved region (in the event that a miRNA is transcribed from the other strand). This sequence was only subsequently discovered to be orthologous to vertebrate miR-137 (which initiates at the second highlighted U). We consider other unverified predicted genes conserved in other insect species with similar characteristics to be potential candidates (see also Table 1).