A B1 SINE element within the mouse Malat1 long noncoding RNA acts as an internal ribosome entry site mediating micropeptide translation

is exported to the cytoplasm where it is translated to generate the M1 micropeptide. Here, we characterize an internal ribosome entry site (IRES) required for M1 translation that is sufficient to induce cap-independent expression of a downstream open reading frame. In vivo chemical probing and structural modeling defined a three-stem-loop RNA secondary structure that encompasses the active IRES segment and coincides with a mouse B1 SINE (short interspersed element) repetitive element. In affinity purification experiments, a minimal IRES/B1 RNA selectively bound ribosomal subunits, translation factors, and the known IRES cofactors Rack1 and hnRNP A2/B1. Depletion of Rack1 and hnRNP A2/B1 inhibited IRES-dependent translation of the reporter and expression of the endogenous M1 peptide. Our study identifies an unexpected functional unit hidden within a widely studied long noncoding RNA.