Insights into the mechanisms of eukaryotic translation gained with ribosome profiling

Abstract

The development of Ribosome Profiling (RiboSeq) has revolutionized functional genomics. RiboSeq is based on capturing and sequencing of the mRNA fragments enclosed within the translating ribosome and it thereby provides a 'snapshot' of ribosome positions at the transcriptome wide level. Although the method is predominantly used for analysis of differential gene expression and discovery of novel translated ORFs, the RiboSeq data can also be a rich source of information about molecular mechanisms of polypeptide synthesis and translational control. This review will focus on how recent findings made with RiboSeq have revealed important details of the molecular mechanisms of translation in eukaryotes. These include mRNA translation sensitivity to drugs affecting translation initiation and elongation, the roles of upstream ORFs in response to stress, the dynamics of elongation and termination as well as details of intrinsic ribosome behavior on the mRNA after translation termination. As the RiboSeq method is still at a relatively early stage we will also discuss the implications of RiboSeq artifacts on data interpretation.

Figure 1.

Types of alterations in ribosome density observed with ribosome profiling. (A) Changes in translation efficiency (TE), represented here by decrease of TE (compare with left part); Left panel represents control conditions and the right panel corresponds to changed conditions. (B) Translation in 5′ leader. (C) Presence of ribosomes in 3′ trailer. (D) Site specific pause originating from ribosomes stalled within acORF at a specific location. (E) Ribosomes paused at the stop codon and queued upstream ribosomes; green and blue shapes represent 40S complexes and 60S subunits respectively, only 80S ribosomes (40S+60S complexes) produce footprints using the conventional RiboSeq protocol. Red bars show the number of RPFs aligned to a particular location, due to biases of library preparation and sequencing the bar heights do not fully correspond to actual ribosome occupancies. Orange ovals highlight specific locations of ribosome footprints.

Figure 2.

Examples of some known and potential artifacts associated with ribosome profiling. (A) Antibiotic pretreatments may cause increased initiation on upstream translation initiation sites (TISs) because arrested elongating ribosomes prevent scanning. (B) Antibiotics may not block elongation completely (on NNN codon in the A-site) but allow slow elongation (until preferred XYZ codon is in the A-site), which affects the estimation of local decoding rates. (C) Ribosome protected fragments shorter than 26 and longer than 34 nt may be excluded upon size selection. In contrast, mRNA protected by RNA binding proteins may be selected as RPFs.