Ribosome Footprint Profiling of Translation throughout the Genome

Abstract

Ribosome profiling has emerged as a technique for measuring translation comprehensively and quantitatively by deep sequencing of ribosome-protected mRNA fragments. By identifying the precise positions of ribosomes, footprinting experiments have unveiled key insights into the composition and regulation of the expressed proteome, including delineating potentially functional micropeptides, revealing pervasive translation on cytosolic RNAs, and identifying differences in elongation rates driven by codon usage or other factors. This Primer looks at important experimental and analytical concerns for executing ribosome profiling experiments and surveys recent examples where the approach was developed to explore protein biogenesis and homeostasis.

Figure 1. Schematic of Ribosome Footprint Profiling of Translation

The workflow for ribosome profiling in different cell types follows the same basic steps: isolation of mRNAs on polysomes, nuclease digestion of the mRNA sequences unprotected by bound ribosomes, and purification of the remaining mRNA fragments followed by library generation, deep sequencing, and computational analysis.

Figure 2. Annotating Translated Sequences with Ribosome Profiling Data

(A) Detecting translated sequences from elongating ribosome footprint profiling on model transcripts. Differences in footprint density and triplet periodicity indicate translated regions. Truncated protein products cause subtle changes in ribosome density. (B) Initiation profiling highlights alternative initiation sites clearly. (C) Alternate translation products can be identified relative to the annotated ORF on a transcript.

Figure 3. Quantifying Expression from Ribosome Profiling and mRNA-Seq

(A) Ribosome density indicates protein production. Ribosomes initiate and elongate at the same speed, yielding a correspondence between the number of protein molecules produced and the ribosome density. (B) Deep sequencing quantifies the fraction of all ribosome footprints derived from a transcript because absolute read count does not reflect input RNA quantity and inactive ribosomes produce no footprints. (C) Ribosome footprint density encompasses mRNA abundance and translation. Higher mRNA abundance or increased translation will yield more ribosome footprints. (D) Regulatory effects illustrated on a plot of ribosome footprint and mRNA abundance changes.

Figure 4. Monitoring the Speed of Translation Elongation with Ribosome Profiling

(A–C) (A) Individual ribosomes spend more time where elongation is slowest and so in (B) a snapshot of the full ensemble ribosomes in the cell, (C) footprint density is higher where translation elongation is slower.

Figure 5. Ribosome Footprint Profiling of Co-translational Protein Maturation

(A) Factors such as chaperones associate with nascent proteins on the ribosome. (B) Selective co-purification of ribosome nascent chain complexes with a co-translational chaperone. (C) Ribosome footprints enriched by the purification indicate the regions of the protein where the chaperone binds.