A molecular switch in the efficiency of translation reinitiation controls expression of var2csa, a gene implicated in pregnancy-associated malaria

Abstract

Plasmodium falciparum malaria parasites export the protein PfEMP1 to the surface of infected erythrocytes, enabling them to adhere to receptors in the microvasculature and thereby avoid clearance by the spleen. The gene var2csa encodes the form of PfEMP1 that binds specifically within the placenta, causing pregnancy-associated malaria, and appears to not be expressed in the absence of a placenta. We previously described an upstream open reading frame (uORF) that is responsible for repression of translation of the downstream ORF (dORF) that encodes VAR2CSA, thus keeping the gene silent when parasites infect non-pregnant individuals. To elucidate the molecular mechanism by which this repression is overcome during pregnancy, we stably transformed parasites with reporter gene constructs designed to detect switches in the efficiency of dORF translation. We found that proper regulation of switching relies on two separate components, (i) active translation of the uORF and (ii) sequence-specific characteristics of the surrounding transcript, which together control the ability of the ribosome complex to reinitiate a second round of translation and thus express VAR2CSA. These results provide the first details of a molecular switch that allows parasites take advantage of the unique niche provided by the placenta.

Figure 1. Typical structure of a bicistronic transcript and mechanisms that allow downstream ORF translation

(A) The basic structure of a bicistronic mRNA containing a CAP, 5′ untranslated region (5′UTR), an upstream ORF (uORF), an intercistronic region (ICR) and a downstream ORF (dORF) is shown. The start codons (AUG) and stop codon (STOP) are also identified. (B) The ribosome will typically recognize and translate the uORF. Once it reaches the stop codon, it will dissociate, thus preventing the dORF from being translated into protein. The usual repression of initiation at the start codon of the dORF can be circumvented by different mechanisms: (C) uORF “skipping” and recognition of a downstream start codon in a more optimal context, (D) translation of dORF by initiation at an internal ribosomal entry site (IRES) and (E) reinitiation of translation at dORF after first translating the uORF.

Figure 2. Selection of parasites translating the dORF of the var2csa bicistronic transcript

(A) The structure of the var2csa gene is shown. The transcription start site is marked with an arrow, the upstream ORF (uORF) is shown as a solid box, and the VAR2CSA coding region is shown as an empty box. (B) The construct V2B includes the transcription start site and regulatory region, the intact uORF and the intercistronic region from the var2csa gene, but the VAR2CSA coding region has been replaced by the coding region of the blasticidin S deaminase gene (bsd). The middle panel shows growth curves of parasites cultured with and without blasticidin (Ø/+ blast). The additional time (in days) required for the parasites to reach 5% parasitemia under blasticidin selection compared to the parasites cultured without the drug is shown. The right panel shows quantitative RT-PCR measurements of steady state mRNA levels for both the uORF and bsd (dORF) regions of the transcript from parasite grown with or without blasticidin (5 μg/ml). (C) V2mB is identical to V2B, except a single base pair mutation in the uORF start codon has abolished it as a start site for translation. Growth curves and quantitative RT-PCR measurements are also shown. (D) V2BØHpaI is identical to V2B except the HpaI site near the initiation codon of the bsd open reading frame has been removed by site-directed mutagenesis to restore the wildtype sequence found at this position in the var2csa gene. Growth curves with and without blasticidin are very similar to those observed for V2B transfected parasites. Restriction sites used for construction of the plasmids are shown.

Figure 3. uORF and ICR requirements for dORF translational repression

Parasites were stably transfected with plasmid constructs and selected for their ability to translate the dORF encoding blasticidin S deaminase (bsd). Schematic diagrams of the constructs are shown on the left along with corresponding growth curves of parasites cultured with and without blasticidin (5 μg/ml). The additional time (in days) required for the parasites to reach 5% parasitemia under blasticidin selection compared to parasites cultured without the drug is shown. V2cB (A) was made by replacing the 10 bp upstream of the start codon of the uORF with the analogous sequence found immediately upstream of the dORF start codon. In V2GB and V2RB (B, C) the uORF element was replaced with the coding regions of the reporter genes Gaussia and Renilla luciferase. V2cGB (D) has the 10 bp upstream of the start codon of the uORF replaced by the sequence found immediately upstream of the dORF start codon and the Gaussia luciferase coding region in the place of uORF. For V2GB, V2RB and V2cGB (B-D), luciferase expression levels and steady state RNA levels are also shown and demonstrate that translation of the uORF remains steady after selection of blasticidin resistant parasites. Parasites were cultured with or without blasticidin pressure and synchronized to ring stages. Luciferase activity was normalized to levels of mRNA expression. In V2dsB (E) the uORF sequence was replaced with a divergent sequence that encodes the same amino acids, but that uses alternative codons, and the ICR sequence was “scrambled”. V2B0.4×5′ICR, V2B1.5×ICR and V2B2×ICR (F, G, and H, respectively) are constructs with altered ICR length to 0.4, 1.5 and 2 times the length of the region found in the endogenous var2csa transcript. V2B0.4×5′ICR (F) contains the 5′ 0.4 portion of the ICR. In V2B1.5×ICR and V2B2×ICR (G, H) the ICR length was increased adding the scrambled sequence used in V2dsB (E). In V2BsICR (I) the ICR was replaced by a sequence of identical size and AT content, but with a scrambled sequence. Restriction sites used for construction of the plasmids are shown.

Figure 4. Sequence requirements for repression of dORF translation

Parasites were stably transfected with plasmid constructs and selected for their ability to translate the dORF encoding blasticidin S deaminase (bsd). Schematic diagrams of the constructs are shown on the left and corresponding growth curves of parasites cultured with and without blasticidin (5 μg/ml) are shown on the right. Assays were performed as described for Figure 3. The additional time (in days) required for the parasites to reach 5% parasitemia under blasticidin selection compared to parasites cultured without the drug is shown. V2B0.6×3′ICR (A) contains only the 3′ 0.6 of the ICR. In V2BinvICR (B) the ICR was inverted relative to its orientation in the var2csa transcript. V2BunstICR (C) is identical to V2B (Figure 2B), except 9 point mutations were introduced at critical sites of a predicted ICR hairpin with the goal of disrupting it. V2BstICR (D) is similar to V2BunstICR (C) but compensatory mutations were introduced that were predicted to re-establish the putative hairpin. In V2BØICR (E) the ICR element was completely removed. In V2GBØICR and V2RBØICR (F and G) the uORF was replaced with the coding regions of the reporter genes Gaussia and Renilla luciferases and the ICR element was removed. For these constructs, luciferase expression levels and steady state RNA levels are also shown (right). In each of these constructs (E-G) the two ORFs remain separate and the ORF in the upstream position contains an in frame stop codon. In V2BduORF (H) the uORF sequence was replaced with a divergent sequence that encoded the same amino acids, but that used alternative codons. V12uB (I) was made by replacing the entire sequence upstream of the AUG of the uORF with the promoter, upstream regulatory region and 5′UTR from a var gene on chromosome 12 (PF3D7_1200100). Restriction sites used for construction of the plasmids are shown.

Figure 5. Trans acting factors are implicated in the var2csa reinitiation mechanism

Parasites stably co-transfected with two plasmids were used to study trans effects on translation of the dORF. Schematic diagrams of the plasmid constructs are shown on the left, growth curves with and without blasticidin pressure are shown in the center, Gaussia luciferase expression is shown in the central bar graph, and bsd mRNA levels are shown at the right. Selection of blasticidin resistant parasites co-transfected with V2B and V2uGU is delayed when compared to parasites transfected with V2B alone (see Figure 2B), and luciferase expression is reduced (A). In contrast, parasites co-transfected with V2B and V2mGU behave similarly to parasites transfected only with V2B (B). In addition, luciferase expression increased in parasite populations after blasticidin resistance was established. Selection with blasticidin results in a dramatic increase in transcript levels of V2B in parasites co-transfected with V2uGU, while a much small increase is detected in parasites co-transfected with V2mGU. This suggests that the V2B transcript competes with the V2uGU transcript for the ability to translate the dORF and consequently significantly more transcript is required, while it does not compete (or competes much less) with the V2mGU transcript. Restriction sites used for construction of the plasmids are shown.

Figure 6. A model for control of VAR2CSA translation

Under standard conditions (A), the 40S subunit and initiation factors begins scanning at the CAP and initiates translation when it reaches the start codon of the uORF. Sequence dependent secondary structure slows transit time of the translating ribosome, allowing nearly complete release of initiation factors from the ribosome complex. In the absence of initiation factors, the ribosome dissociates from the transcript upon reaching the stop codon of the uORF, thus preventing translation of the dORF encoding VAR2CSA. If initiation factors are more stably associated with the ribosome while it is translating the uORF (B), the ribosome can resume scanning after it passes the uORF stop codon and reinitiate a second round of translation when it encounters the start codon of the dORF. If the secondary structure of the transcript is disrupted (C), transit time of the translating ribosome through the uORF is reduced and initiation factors are less likely to be shed. This enables the ribosome to resume scanning when it reaches the ICR and increases the efficiency of a second round of translation.