Most metazoan microRNA (miRNA) target sites have perfect pairing to the seed region, located near the miRNA 5' end. Although pairing to the 3' region sometimes supplements seed matches or compensates for mismatches, pairing to the central region has been known to function only at rare sites that impart Argonaute-catalyzed mRNA cleavage. Here, we present "centered sites," a class of miRNA target sites that lack both perfect seed pairing and 3'-compensatory pairing and instead have 11-12 contiguous Watson-Crick pairs to the center of the miRNA. Although centered sites can impart mRNA cleavage in vitro (in elevated Mg(2+)), in cells they repress protein output without consequential Argonaute-catalyzed cleavage. Our study also identified extensively paired sites that are cleavage substrates in cultured cells and human brain. This expanded repertoire of cleavage targets and the identification of the centered site type help explain why central regions of many miRNAs are evolutionarily conserved.
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