Modified ribosome profiling reveals high abundance of ribosome protected mRNA fragments derived from 3′ untranslated regions

Ribosome profiling identifies ribosome positions on translated mRNAs. A prominent feature of published datasets is the near complete absence of ribosomes in 3' untranslated regions (3'UTR) although substantial ribosome density can be observed on non-coding RNAs. Here we perform ribosome profiling in cultured Drosophila and human cells and show that different features of translation are revealed depending on the nuclease and the digestion conditions used. Most importantly, we observe high abundance of ribosome protected fragments in 3'UTRs of thousands of genes without manipulation of translation termination. Affinity purification of ribosomes indicates that the 3'UTR reads originate from ribosome protected fragments. Association of ribosomes with the 3'UTR may be due to ribosome migration through the stop codon or 3'UTR mRNA binding to ribosomes on the coding sequence. This association depends primarily on the relative length of the 3'UTR and may be related to translational regulation or ribosome recycling, for which the efficiency is known to inversely correlate with 3'UTR length. Together our results indicate that ribosome profiling is highly dependent on digestion conditions and that ribosomes commonly associate with the 3'UTR, which may have a role in translational regulation.