Abstract
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.
Highlights
Translation can be subdivided into initiation, elongation and termination
We stimulated translation with additional 10% fetal bovine serum (FBS) for 1 h before lysis and the samples for RNase I and micrococcal nuclease (MN) digestions were derived from the same cell lysates and same digestion conditions were used for both nucleases
We did not attempt ribosomal RNA removal during sample preparation and used high salt digestion conditions to minimize weak ionic protein–mRNA interactions, which could lead to false positive footprints derived from other RNA binding proteins (RBPs)
Summary
Translation can be subdivided into initiation, elongation and termination. In addition to the core ribosome, a number of auxiliary proteins interact with ribosomes and mRNA to assist through each phase. When ribosome encounters a stop codon, the newly synthesized polypeptide is released and the ribosome subunits dissociate from mRNA. The mRNAs engaged in active translation are thought to form a loop due to interactions of polyA binding protein with initiation factors [2,3,4], suggesting that incomplete dissociation and recycling at the stop codon may potentially facilitate transfer of the ribosome back to the 5 untranslated regions (5 UTRs) for additional rounds of translation. It was recently described that yeast mutant, where translation termination was modified, displayed high levels of ribosomes in the 3 UTRs [5]. One further situation where ribosomes have been observed in 3 UTR is stop codon read-through [8]
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