Ribosome Profiling: Genome-Wide Analysis of Translation at High Resolution

The above described classical methods have been informative, but they are labor intensive and limited in resolution, and none of them is suited to genome-wide and/or high-throughput analyses. These deficits were addressed by ribosome profiling, an approach that enables the quantitative genome-wide analysis of translation in unprecedented depth and resolution (Ingolia et al., 2009). Ribosome profiling takes advantage of the remarkable stability of translating ribosomes, which protect the mRNA sequence they physically cover from attack by nucleases, thereby producing protected fragments, so-called ribosome footprints (Wolin and Walter, 1988; Figure 1C). Next-generation sequencing analysis of these footprints determines the in vivo positions and abundances of translating ribosomes. Considering that each elongating ribosome produces one protein, ribosome footprint abundances reflect the protein synthesis rate for each reading frame (Ingolia et al., 2009). Footprint abundance is typically normalized to mRNA abundance (assayed by RNA sequencing), so that relative translation efficiencies can be inferred (Ingolia et al., 2009). Consequently, the approach measures the two determinants of gene expression that define the final protein output: transcript amount and translational activity. In recent years, ribosome profiling has been extensively used to study translation in prokaryotes and eukaryotes (Ingolia, 2016).

In chloroplasts, ribosome profiling was first applied in a modified approach, exchanging the next-generation sequencing analysis of footprints by microarray hybridization (Zoschke et al., 2013a; Figure 1C). More recently, deep sequencing was used to study chloroplast translational dynamics in maize and Arabidopsis (Chotewutmontri and Barkan, 2016; Lukoszek et al., 2016; Gawroński et al., 2018).

Despite the compelling attractions of ribosome profiling, it should be noted that the method cannot distinguish actively translating from paused ribosomes. This may be problematic if translation is regulated at the level of elongation, as described for some chloroplast genes (see below). Application of inhibitors of initiation or early elongation (e.g., lincomycin) and examination of the run-off kinetics of ribosomes over time should allow distinguishing pausing from elongating ribosomes.