Analysis of Ribosomal Shunting During Translation Initiation in Eukaryotic MRNAs

In eukaryotes, translation initiation involves recruitment of ribosomal subunits at either the 5' m7G cap structure or at an internal ribosome entry site (IRES). For most mRNAs, the initiation codon is located some distance downstream, necessitating ribosomal movement to this site. Although the mechanistic details of this movement remain to be fully resolved, it appears to be nonlinear for some mRNAs (i.e., ribosomal subunits appear to bypass [shunt] segments of the 5' leader as they move to the initiation codon). This chapter describes various experimental approaches to assess ribosomal shunting and to identify mRNA elements (shunt sites) that facilitate shunting. In addition, we provide an overview of approaches that can be used to investigate the mechanism used by individual shunt sites, along with a detailed protocol for investigating putative base pairing interactions between shunt sites and 18S rRNA.