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MicroRNAs (miRNAs) are an abundant class of ∼22 nucleotide (nt) long noncoding RNAs that negatively regulate gene expression post-transcriptionally through imperfect base-pairing interactions with sequences in the target messenger RNA (mRNA). We examined the interactions of the  bantam  miRNA with the 3′ untranslated region (UTR) of the  hid  mRNA, and a synthetic derivative, in  Drosophila  S2 cells in order to define the relative contributions of proposed bantam binding sites. The contribution of the  bantam  miRNA to repression of reporter constructs carrying different 3′ UTRs was evaluated by measuring derepression of reporter expression following the transfection of bantam complementary oligoribonucleotides (anti-bantam). Systematic excision of  bantam  miRNA target sequences in the  hid  3′ UTR identified by commonly used miRNA target prediction programs failed to relieve repression to the extent predicted by the anti-bantam experiment. However, removal of additional bantam complementary sequences (with a “seed” match to nucleotide 3–9) derepressed the reporter constructs to the full extent, arguing for a less narrow definition of the seed sequence. Further support for the potential contribution of the 3–9 seed register to microRNA-mediated gene regulation is provided by the experimental validation of several novel bantam targets identified with a more relaxed search algorithm.

An expanded seed sequence definition accounts for full regulation of the hid 3′ UTR by bantam miRNA