Illuminating Parasite Protein Production by Ribosome Profiling

Abstract

While technologies for global enumeration of transcript abundance are well-developed, those that assess protein abundance require tailoring to penetrate to low-abundance proteins. Ribosome profiling circumvents this challenge by measuring global protein production via sequencing small mRNA fragments protected by the assembled ribosome. This powerful approach is now being applied to protozoan parasites including trypanosomes and Plasmodium. It has been used to identify new protein-coding sequences (CDSs) and clarify the boundaries of previously annotated CDSs in Trypanosoma brucei. Ribosome profiling has demonstrated that translation efficiencies vary widely between genes and, for trypanosomes at least, for the same gene across stages. The ribosomal proteins are themselves subjected to translational control, suggesting a means of reinforcing global translational regulation.

Keywords: Plasmodium; Trypanosoma; genome curation; ribosomal proteins; stage-regulation; translation.

Figure 1. Key Figure. Overview of ribosome profiling protocol

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.

Figure 2. A putative coding sequence encoding a hypothetical protein that was eliminated by analysis of ribosome profiling data

The top three panels depict the RNA-seq reads (using a linear scale) derived from procyclic culture forms (PCF) and bloodstream forms (BF) for the spliced leader (SL), mRNA, and ribosome footprints (RF), with each trace color-coded according to the developmental stage as indicated. Note that traces represent raw read counts and the number at right of each panel indicates the read count of the highest peak. The bottom panel shows the coding sequences (CDS) aligned across the corresponding region of chromosome 7 of the Trypanosoma brucei TREU 927 genome. The CDS for Tb927.7.7370 (enclosed by the dashed cyan line) is truncated at the blue arrow by a major SL site (shown in the top panel) with most of the CDS lying in the 3′ UTR of gene to the right. A region in the middle of the CDS lacks mRNA reads (pink box), while all ribosome footprints that map to Tb927.7.7370 (purple box) lie in the 5′ UTR of the adjacent gene (Tb927.7.7360), which encodes the protein kinase CRK2. Thus Tb927.7.7370 does not lie on a contiguous transcript and the ribosome footprints do not span from start to stop codon, so a full-length protein cannot be produced. The arrow shows the direction of transcription.

Figure 3. Parasite life cycles

Trypanosoma brucei and Plasmodium falciparum undergo development in both insect and mammalian hosts as shown. The stages that have been examined by ribosome profiling are identified by text in green font. (A) Trypanosoma brucei life cycle. Metacyclic forms are transmitted from the tsetse fly to the mammalian host, where they develop into long, slender bloodstream forms that actively divide (as indicated by the curved arrow). Occasionally, parasites will exit the cell cycle, and develop into short stumpy bloodstream forms, which can be transmitted to the tsetse fly. There, they become dividing procyclic forms in the fly midgut. These develop into epimastigotes which divide in the salivary gland, ultimately yielding the transmissible metacyclic forms. (B) Plasmodium falciparum life cycle. The bite of an infected mosquito injects sporozoites into the human host, where they migrate to the liver. There, a single sporozoite will yield thousands of progeny that subsequently enter red blood cells. The asexual erythrocytic cycle allows for continuous parasite replication via an ordered sequence of development from ring stages to merozoites. Occasional parasites exit the asexual erythrocytic cycle to become gametocytes that are then transmissible to the mosquito. In the mosquito, mating, cell division, and development occur to yield numerous sporozoites. Figures were adapted from the Public Health Image Library of the CDC (www.CDC.gov).

Figure 4. Differential abundance of mRNA and ribosome footprints for adjacent genes in polycistronic units

Panels are as described in Figure 2. Shown are the first genes in two divergent polycistronic transcription units, which are separated by a region with very low levels of stable transcripts. The direction of transcription is marked by arrows. The asterisk marks a coding sequence (CDS) encoding a conserved hypothetical protein that shows different translation efficiency (TE) in procyclic culture forms (PCF) and bloodstream forms (BF).

Figure 5. Genes encoding translation machinery in Trypanosoma brucei show stage-regulated protein production

Clustering analysis groups genes with similar expression patterns, in this case for ribosome footprints (protein production) and mRNA abundance. The 1557 genes showing at least a four-fold difference in ribosome footprint normalized read counts were clustered by K-Means and HCL using MEV (for details see [12]). Biological samples included three replicates of PCF, animal-derived slender BF (slBF), and cultured BF (cBF). Each gene is represented by a separate line in the cluster diagram with the magnitude of the fold-change (relative to PCF median) indicated by color intensity (magenta for up and green for down). Bars at right indicate genes within each cluster that can be classified into the listed functional groups. Cluster A3 is highly enriched for proteins involved in translation, particularly structural components of the cytosolic ribosome. Reproduced from [12].

Figure 6. A transcript with ribosome footprints mapping to the 5′ end while larger downstream open reading frames are not translated

This transcript (Tb7.NT.1) has two small open reading frames (ORFs) (9 and 27 nt) immediately following the spliced leader (SL) site that are spanned by ribosome footprints (RF) (see enlargement, turquoise boxes). However, downstream ORFs (including one of 468 nt, indicated by the dotted box), do not bear ribosome footprints. Black bars represent stop codons and pink bars, start codons. Traces are color-coded as indicated. PCF, procyclic culture forms; BF, bloodstream forms.