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A var gene upstream element controls protein synthesis at the level of translation initiation in Plasmodium falciparum.

Brancucci NM, Witmer K, Schmid C, Voss TS - PLoS ONE (2014)

Bottom Line: Importantly, this 5' UTR element efficiently inhibits translation even in the context of a heterologous upstream region.Further, we found var 5' UTRs to be significantly enriched in uAUGs which are known to impair the efficiency of protein translation in other eukaryotes.Our findings suggest that regulation at the post-transcriptional level is a common feature in the control of PfEMP1 expression in P. falciparum.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Parasitology and Infection Biology, Swiss Tropical and Public Health Institute, Basel, Switzerland; University of Basel, Basel, Switzerland.

ABSTRACT
Clonally variant protein expression in the malaria parasite Plasmodium falciparum generates phenotypic variability and allows isogenic populations to adapt to environmental changes encountered during blood stage infection. The underlying regulatory mechanisms are best studied for the major virulence factor P. falciparum erythrocyte membrane protein 1 (PfEMP1). PfEMP1 is encoded by the multicopy var gene family and only a single variant is expressed in individual parasites, a concept known as mutual exclusion or singular gene choice. var gene activation occurs in situ and is achieved through the escape of one locus from epigenetic silencing. Singular gene choice is controlled at the level of transcription initiation and var 5' upstream (ups) sequences harbour regulatory information essential for mutually exclusive transcription as well as for the trans-generational inheritance of the var activity profile. An additional level of control has recently been identified for the var2csa gene, where an mRNA element in the 5' untranslated region (5' UTR) is involved in the reversible inhibition of translation of var2csa transcripts. Here, we extend the knowledge on post-transcriptional var gene regulation to the common upsC type. We identified a 5' UTR sequence that inhibits translation of upsC-derived mRNAs. Importantly, this 5' UTR element efficiently inhibits translation even in the context of a heterologous upstream region. Further, we found var 5' UTRs to be significantly enriched in uAUGs which are known to impair the efficiency of protein translation in other eukaryotes. Our findings suggest that regulation at the post-transcriptional level is a common feature in the control of PfEMP1 expression in P. falciparum.

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Related in: MedlinePlus

A gene conversion event revokes translational inhibition of hdhfr-gfp transcripts.(A) Southern analysis on digested gDNA from unselected and WR-selected 3D7/pBKminC parasites. Additional hdhfr-containing fragments detected in WR-selected parasites only are highlighted by pink arrows. S, StuI; B, BglII; K, KpnI; i, integration event; p, plasmid fragment. (B) The ends of chromosome 2 and 4 in unselected and 4/2 in WR-selected parasites are schematically depicted. Gene IDs (www.plasmoDB.org) are indicated for a subset of genes as reference. The dashed arrow highlights the site of gene conversion. The blue box represents the duplicated region of chromosome 2. The green box represents the region of chromosome 4 that was deleted. The brown box displays a zoom-in view of the gene conversion event and the resulting recombined locus. Detailed mapping and identification of the recombination site is presented in Figures S1 and S2. (C) hdhfr-gfp transcripts are produced from the var gene intron on chromosome 4 in WR-selected 3D7/pBKminC parasites. Values represent relative var intron-derived hdhfr-gfp (grey bars) and ring stage-specific msp8 (open bars, control) transcript levels at three consecutive time points in WR-selected 3D7/pBKminC parasites (normalised to PF3D7_1331700 transcripts). hpi, hours post invasion. (D) Semi-quantitative analysis of transcript and protein abundance in 3D7/pBKmin (control) and 3D7/pBKminC ring stage parasites (6–14 hpi) cultured in presence of WR99210 (+WR). Top panels: hdhfr-gfp and hsp86 (loading control) transcripts were detected by Northern blot. Ethidium bromide-stained 18S and 28S rRNAs serve as second loading control. Bottom panels: expression of hDHFR-GFP and GAPDH (loading control) in the same parasite samples were analysed by Western blot.
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pone-0100183-g003: A gene conversion event revokes translational inhibition of hdhfr-gfp transcripts.(A) Southern analysis on digested gDNA from unselected and WR-selected 3D7/pBKminC parasites. Additional hdhfr-containing fragments detected in WR-selected parasites only are highlighted by pink arrows. S, StuI; B, BglII; K, KpnI; i, integration event; p, plasmid fragment. (B) The ends of chromosome 2 and 4 in unselected and 4/2 in WR-selected parasites are schematically depicted. Gene IDs (www.plasmoDB.org) are indicated for a subset of genes as reference. The dashed arrow highlights the site of gene conversion. The blue box represents the duplicated region of chromosome 2. The green box represents the region of chromosome 4 that was deleted. The brown box displays a zoom-in view of the gene conversion event and the resulting recombined locus. Detailed mapping and identification of the recombination site is presented in Figures S1 and S2. (C) hdhfr-gfp transcripts are produced from the var gene intron on chromosome 4 in WR-selected 3D7/pBKminC parasites. Values represent relative var intron-derived hdhfr-gfp (grey bars) and ring stage-specific msp8 (open bars, control) transcript levels at three consecutive time points in WR-selected 3D7/pBKminC parasites (normalised to PF3D7_1331700 transcripts). hpi, hours post invasion. (D) Semi-quantitative analysis of transcript and protein abundance in 3D7/pBKmin (control) and 3D7/pBKminC ring stage parasites (6–14 hpi) cultured in presence of WR99210 (+WR). Top panels: hdhfr-gfp and hsp86 (loading control) transcripts were detected by Northern blot. Ethidium bromide-stained 18S and 28S rRNAs serve as second loading control. Bottom panels: expression of hDHFR-GFP and GAPDH (loading control) in the same parasite samples were analysed by Western blot.

Mentions: Noteworthy, after twelve unsuccessful drug challenges we were eventually able to select for a WR-resistant 3D7/pBKminC population. In light of these difficulties in generating a WR-resistant line, we considered a genomic rearrangement the most plausible cause for this altered phenotype. Indeed, Southern blot analysis revealed an additional hdhfr-gfp fragment in WR-selected compared to unselected parasites (Figure 3A). To determine this recombination event in exact detail, we carried out an elaborate mapping strategy based on further Southern blotting, ligation-mediated PCR and DNA sequencing (Figures S1 and S2). These efforts uncovered a major gene conversion event that resulted in the exchange of the end of chromosome 4 with a duplicated version of the end of chromosome 2 (Figure 3B). This occurred through the homologous recombination between a 10 bp sequence directly upstream of the most telomere-proximal hdhfr-gfp gene on chromosome two and an identical 10 bp sequence at the exon 1-intron boundary of var gene PF3D7_0400100 (Figures S1 and S2). Consequently, transcription of a single hdhfr-gfp gene in WR-resistant 3D7/pBKminC parasites was now under control of the reverse strand of a var gene intron. Notably, the var intron possesses bi-directional promoter activity [55], [56]. Indeed, qRT-PCR using primers specific to this recombined locus unambiguously identified active var intron-driven hdhfr-gfp transcription in WR-selected 3D7/pBKminC parasites (Figure 3C), which resulted in successful expression of hDHFR-GFP (Figure 3D). In line with the peak of intron promoter activity late during the IDC [55], synthesis of intron-derived hdhfr-gfp transcripts was higher in trophozoites/early schizonts compared to ring stages (Figure 3C). Attempts to identify intron-derived mRNA by Northern blotting were unsuccessful (data not shown). We explain this by the low abundance of intron-derived transcripts and similar expected size compared to those originating from the kahrpC promoter. Furthermore, intron-mediated antisense transcription initiates at variable sites [55], which additionally hampers detectability in Northern analysis.


A var gene upstream element controls protein synthesis at the level of translation initiation in Plasmodium falciparum.

Brancucci NM, Witmer K, Schmid C, Voss TS - PLoS ONE (2014)

A gene conversion event revokes translational inhibition of hdhfr-gfp transcripts.(A) Southern analysis on digested gDNA from unselected and WR-selected 3D7/pBKminC parasites. Additional hdhfr-containing fragments detected in WR-selected parasites only are highlighted by pink arrows. S, StuI; B, BglII; K, KpnI; i, integration event; p, plasmid fragment. (B) The ends of chromosome 2 and 4 in unselected and 4/2 in WR-selected parasites are schematically depicted. Gene IDs (www.plasmoDB.org) are indicated for a subset of genes as reference. The dashed arrow highlights the site of gene conversion. The blue box represents the duplicated region of chromosome 2. The green box represents the region of chromosome 4 that was deleted. The brown box displays a zoom-in view of the gene conversion event and the resulting recombined locus. Detailed mapping and identification of the recombination site is presented in Figures S1 and S2. (C) hdhfr-gfp transcripts are produced from the var gene intron on chromosome 4 in WR-selected 3D7/pBKminC parasites. Values represent relative var intron-derived hdhfr-gfp (grey bars) and ring stage-specific msp8 (open bars, control) transcript levels at three consecutive time points in WR-selected 3D7/pBKminC parasites (normalised to PF3D7_1331700 transcripts). hpi, hours post invasion. (D) Semi-quantitative analysis of transcript and protein abundance in 3D7/pBKmin (control) and 3D7/pBKminC ring stage parasites (6–14 hpi) cultured in presence of WR99210 (+WR). Top panels: hdhfr-gfp and hsp86 (loading control) transcripts were detected by Northern blot. Ethidium bromide-stained 18S and 28S rRNAs serve as second loading control. Bottom panels: expression of hDHFR-GFP and GAPDH (loading control) in the same parasite samples were analysed by Western blot.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4061111&req=5

pone-0100183-g003: A gene conversion event revokes translational inhibition of hdhfr-gfp transcripts.(A) Southern analysis on digested gDNA from unselected and WR-selected 3D7/pBKminC parasites. Additional hdhfr-containing fragments detected in WR-selected parasites only are highlighted by pink arrows. S, StuI; B, BglII; K, KpnI; i, integration event; p, plasmid fragment. (B) The ends of chromosome 2 and 4 in unselected and 4/2 in WR-selected parasites are schematically depicted. Gene IDs (www.plasmoDB.org) are indicated for a subset of genes as reference. The dashed arrow highlights the site of gene conversion. The blue box represents the duplicated region of chromosome 2. The green box represents the region of chromosome 4 that was deleted. The brown box displays a zoom-in view of the gene conversion event and the resulting recombined locus. Detailed mapping and identification of the recombination site is presented in Figures S1 and S2. (C) hdhfr-gfp transcripts are produced from the var gene intron on chromosome 4 in WR-selected 3D7/pBKminC parasites. Values represent relative var intron-derived hdhfr-gfp (grey bars) and ring stage-specific msp8 (open bars, control) transcript levels at three consecutive time points in WR-selected 3D7/pBKminC parasites (normalised to PF3D7_1331700 transcripts). hpi, hours post invasion. (D) Semi-quantitative analysis of transcript and protein abundance in 3D7/pBKmin (control) and 3D7/pBKminC ring stage parasites (6–14 hpi) cultured in presence of WR99210 (+WR). Top panels: hdhfr-gfp and hsp86 (loading control) transcripts were detected by Northern blot. Ethidium bromide-stained 18S and 28S rRNAs serve as second loading control. Bottom panels: expression of hDHFR-GFP and GAPDH (loading control) in the same parasite samples were analysed by Western blot.
Mentions: Noteworthy, after twelve unsuccessful drug challenges we were eventually able to select for a WR-resistant 3D7/pBKminC population. In light of these difficulties in generating a WR-resistant line, we considered a genomic rearrangement the most plausible cause for this altered phenotype. Indeed, Southern blot analysis revealed an additional hdhfr-gfp fragment in WR-selected compared to unselected parasites (Figure 3A). To determine this recombination event in exact detail, we carried out an elaborate mapping strategy based on further Southern blotting, ligation-mediated PCR and DNA sequencing (Figures S1 and S2). These efforts uncovered a major gene conversion event that resulted in the exchange of the end of chromosome 4 with a duplicated version of the end of chromosome 2 (Figure 3B). This occurred through the homologous recombination between a 10 bp sequence directly upstream of the most telomere-proximal hdhfr-gfp gene on chromosome two and an identical 10 bp sequence at the exon 1-intron boundary of var gene PF3D7_0400100 (Figures S1 and S2). Consequently, transcription of a single hdhfr-gfp gene in WR-resistant 3D7/pBKminC parasites was now under control of the reverse strand of a var gene intron. Notably, the var intron possesses bi-directional promoter activity [55], [56]. Indeed, qRT-PCR using primers specific to this recombined locus unambiguously identified active var intron-driven hdhfr-gfp transcription in WR-selected 3D7/pBKminC parasites (Figure 3C), which resulted in successful expression of hDHFR-GFP (Figure 3D). In line with the peak of intron promoter activity late during the IDC [55], synthesis of intron-derived hdhfr-gfp transcripts was higher in trophozoites/early schizonts compared to ring stages (Figure 3C). Attempts to identify intron-derived mRNA by Northern blotting were unsuccessful (data not shown). We explain this by the low abundance of intron-derived transcripts and similar expected size compared to those originating from the kahrpC promoter. Furthermore, intron-mediated antisense transcription initiates at variable sites [55], which additionally hampers detectability in Northern analysis.

Bottom Line: Importantly, this 5' UTR element efficiently inhibits translation even in the context of a heterologous upstream region.Further, we found var 5' UTRs to be significantly enriched in uAUGs which are known to impair the efficiency of protein translation in other eukaryotes.Our findings suggest that regulation at the post-transcriptional level is a common feature in the control of PfEMP1 expression in P. falciparum.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Parasitology and Infection Biology, Swiss Tropical and Public Health Institute, Basel, Switzerland; University of Basel, Basel, Switzerland.

ABSTRACT
Clonally variant protein expression in the malaria parasite Plasmodium falciparum generates phenotypic variability and allows isogenic populations to adapt to environmental changes encountered during blood stage infection. The underlying regulatory mechanisms are best studied for the major virulence factor P. falciparum erythrocyte membrane protein 1 (PfEMP1). PfEMP1 is encoded by the multicopy var gene family and only a single variant is expressed in individual parasites, a concept known as mutual exclusion or singular gene choice. var gene activation occurs in situ and is achieved through the escape of one locus from epigenetic silencing. Singular gene choice is controlled at the level of transcription initiation and var 5' upstream (ups) sequences harbour regulatory information essential for mutually exclusive transcription as well as for the trans-generational inheritance of the var activity profile. An additional level of control has recently been identified for the var2csa gene, where an mRNA element in the 5' untranslated region (5' UTR) is involved in the reversible inhibition of translation of var2csa transcripts. Here, we extend the knowledge on post-transcriptional var gene regulation to the common upsC type. We identified a 5' UTR sequence that inhibits translation of upsC-derived mRNAs. Importantly, this 5' UTR element efficiently inhibits translation even in the context of a heterologous upstream region. Further, we found var 5' UTRs to be significantly enriched in uAUGs which are known to impair the efficiency of protein translation in other eukaryotes. Our findings suggest that regulation at the post-transcriptional level is a common feature in the control of PfEMP1 expression in P. falciparum.

Show MeSH
Related in: MedlinePlus