Structure and function of a small RNA that selectively inhibits internal ribosome entry site-mediated translation

A 60 nt long RNA termed IRNA, isolated from the yeast Saccharomyces cereves iae, was previously shown to selectively block internal ribosome entry site (IRES)-mediated translation without interfering with cap-dependent transla tion of cellular mRNAs both in vivo and in vitro. IRNA specifically bound c ellular proteins believed to be important for IRES-mediated translation, We demonstrate here that a complementary copy of IRNA (cIRNA) is also active in blocking IRES-mediated translation and that it binds many of the same ce llular proteins that IRNA does. We have probed the secondary structure of b oth IRNA and cIRNA using single-strand- and double-strand-specific nuclease s as well as using oligonucleotide hybridization followed by RNase H digest ion. Both IRNA and cIRNA share secondary structural homology, although dist inct differences do exist between the two structures. Mutational analysis o f IRNA shows that sequences that form both the main stem and one loop are c ritical for its translation inhibitory activity. Maintenance of the establi shed secondary structure appears to be required for both IRNA's ability to bind cellular trans-acting proteins believed to be required for IRES-mediat ed translation and its ability to block IRES-mediated translation.