microRNAs (miRNAs) are small, noncoding RNAs that may regulate more than half of human genes, yet the molecular mechanism of miRNA-mediated repression remains obscure. Using a cell-free assay of miRNA activity, we show that miRNA-targeted mRNAs are enriched for components of the 40S, but not 60S ribosomal subunit. Additionally, a molecular toeprint of 18 nucleotides 3' relative to the start codon, consistent with nucleotide protection by 40S ribosomal subunits, is enriched on miRNA-targeted mRNAs. Our results suggest that miRNAs repress translation initiation in a cell-free system by preventing 60S ribosomal subunit joining to 40S subunits positioned at the start codon.
