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. Author manuscript; available in PMC: 2017 Jun 1.
Published in final edited form as: Trends Parasitol. 2016 Apr 6;32(6):446–457. doi: 10.1016/j.pt.2016.03.005

Figure 1. Key Figure. Overview of ribosome profiling protocol.

Figure 1

Ribosome profiling provides an unbiased means of identifying those regions of mRNAs associated with assembled ribosomes and thus engaged in protein production. Additionally, it allows quantitation of the extent of ribosome association with each mRNA (or a portion thereof) of interest. (A) Procotol summary. Two mRNA libraries are made from each sample: one from ribosome footprints (RF) produced by nuclease digestion of a cell lysate and the other from fragmented purified polyadenylated (polyA+) mRNA, as detailed [7,68]. Following sequencing and alignment with the relevant genome, read counts for each coding sequence (CDS) are quantified. The green structures in the cartoon represent ribosomes that are associated with the blue and red mRNAs, generating four ribosome footprints for each CDS in this example. However, since the mRNA abundances differ, the corresponding translation efficiencies (TEs) of the mRNAs differ, with the TE for the red mRNA being four-fold higher. (B) Two adjacent Trypanosoma brucei genes with similar mRNA levels, but different protein production levels. More ribosome footprints correspond to the red CDS, reflecting more protein production and hence a higher TE than for the blue gene. Data for an in vivo derived slender bloodstream form (BF) sample are visualized here and elsewhere using Artemis [28]. Each small horizontal bar represents an aligned sequence read, and the arrow shows the direction of transcription. Note that the extended 3′ UTR of the red gene, as defined by mRNA reads (top), lacks ribosome footprints (middle), as expected for untranslated region of mRNA. A map of the region of chromosome 7 where these genes are located is shown at the bottom. (C) Trypanosomatid mRNA structure. Trypanosomatids genes are organized into clusters with adjacent protein-coding sequences (CDSs, shown as yellow boxes) that are transcribed as a polycistronic precursor RNA, which is processed by trans-splicing and polyadenylation to produce mature mRNAs. Each mature mRNA contains a 5′- and 3′- untranslated region (UTR), flanked by the CDS and spliced leader (SL) and polyadenylation (polyA) sites, respectively. The SL is donated by a distinct RNA.