Detecting ribosomal association with the 5' leader of mRNAs by Ribosome Density Mapping (RDM).

In eukaryotes, scanning of the 5' leader by the small ribosomal subunit precedes recognition of the start codon. Thus, various sequence elements that are located within this region may affect ribosomes' progression and lead to significant effects on translation. Most notable are short ORFs located upstream of the start codon, which are known to regulate the translation of the main ORF in the transcript. The function of these elements is likely to correlate with altered ribosomal association with the 5' leader of the mRNA. Currently, the only method to determine the ribosomal association of different regions of the mRNA in vivo is the Ribosome Density Mapping (RDM) procedure. This method entails cleavage of the target mRNA by specific oligodeoxynucleotides and RNase H and separation of the cleavage products by velocity sedimentation in a sucrose gradient. In this chapter, we provide a detailed protocol for this procedure and discuss its feasibility.