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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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Integration of the upsC 5′ upstream sequence into a heterologous context at the kahrp locus.(A) Schematic map of the transfection construct pBKminC. Single-crossover integration was guided by kahrp 5′ homology. The position of the kahrp TSS is indicated [81]. Numbers refer to the nucleotide positions relative to the ATG start codon. The bsd resistance cassette selects for stably transfected parasites. The var intron is indicated by a bold dashed line. hsp86 5′, hsp86 promoter; Pb DT 3′, P. berghei dhfr-thymidylate synthase terminator; rep20, 0.5 kb TARE6 repeat element; hrp2 3′; histidine-rich protein 2 terminator. MEE, location of the 101 bp mutual exclusion element MEE [54]. (B) Genomic situation after integration of the pBKminC concatamer into the endogenous kahrp locus. Restriction sites used in Southern analysis and fragment lengths are indicated and colour-coded. S, StuI; B, BglII. The Southern blot on BglII/StuI-digested gDNA shows integration of pBKminC into the endogenous locus of kahrp. The membrane was hybridised with hdhfr (top) and kahrp (bottom). Fragments are colour-coded according to the integration map. wt, size of the kahrp fragment in 3D7 wild-type parasites. i, integration event; p, plasmid fragment. (C) The upsC 5′ UTR sequence represses kahrp promoter activity. The bars represent the ratio of relative hdhfr-gfp and msp8 transcript levels in 3D7/pBKminC parasites (open bars) compared to the 3D7/pBKmin control (black bars) cultured in absence of WR. Results are the mean +/− s.d. of three independent experiments. Values are normalised for PF3D7_1331700 transcripts.
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pone-0100183-g001: Integration of the upsC 5′ upstream sequence into a heterologous context at the kahrp locus.(A) Schematic map of the transfection construct pBKminC. Single-crossover integration was guided by kahrp 5′ homology. The position of the kahrp TSS is indicated [81]. Numbers refer to the nucleotide positions relative to the ATG start codon. The bsd resistance cassette selects for stably transfected parasites. The var intron is indicated by a bold dashed line. hsp86 5′, hsp86 promoter; Pb DT 3′, P. berghei dhfr-thymidylate synthase terminator; rep20, 0.5 kb TARE6 repeat element; hrp2 3′; histidine-rich protein 2 terminator. MEE, location of the 101 bp mutual exclusion element MEE [54]. (B) Genomic situation after integration of the pBKminC concatamer into the endogenous kahrp locus. Restriction sites used in Southern analysis and fragment lengths are indicated and colour-coded. S, StuI; B, BglII. The Southern blot on BglII/StuI-digested gDNA shows integration of pBKminC into the endogenous locus of kahrp. The membrane was hybridised with hdhfr (top) and kahrp (bottom). Fragments are colour-coded according to the integration map. wt, size of the kahrp fragment in 3D7 wild-type parasites. i, integration event; p, plasmid fragment. (C) The upsC 5′ UTR sequence represses kahrp promoter activity. The bars represent the ratio of relative hdhfr-gfp and msp8 transcript levels in 3D7/pBKminC parasites (open bars) compared to the 3D7/pBKmin control (black bars) cultured in absence of WR. Results are the mean +/− s.d. of three independent experiments. Values are normalised for PF3D7_1331700 transcripts.

Mentions: The 101 bp MEE element is located downstream of the transcriptional start site (TSS) in the upsC upstream region and controls inclusion of the locus into the programme of mutually exclusive var activity [54]. Here, we aimed at a more detailed functional characterisation of this regulatory sequence. First, we asked whether an upsC upstream sequence including the MEE is able to modulate gene expression autonomously when placed in a conserved position downstream of the TSS of a heterologous promoter. To achieve this, we used our previously published transfection vector pBKmin as a vehicle to target the endogenous kahrp (knob-associated histidine rich protein) locus [54]. pBKmin contains the blasticidin deaminase (bsd) resistance gene followed by a reporter cassette in which a minimal kahrp promoter (Kmin) controls expression of the hdhfr-gfp (human dihydrofolate reductase fused to green fluorescent protein) reporter gene that confers resistance to the antifolate WR99210 (WR). Here, we replaced the region spanning bps −445 to −1 downstream of the TSS of the minimal kahrp promoter with the upsC sequence (bps −519 to −1) containing the MEE (Figure 1A). Transfected 3D7 parasites were selected on blasticidin-S-HCl (BSD) and the plasmid was integrated into the endogenous kahrp locus by single-crossover homologous recombination (3D7/pBKminC). This event created the kahrp-upsC hybrid upstream sequence kahrpC that drives expression of the hdhfr-gfp gene (Figure 1B). In this context, the wild-type kahrp promoter drives transcription of hdhfr-gfp and produces transcripts in which the 5′ UTR of kahrp had been swapped with that of var upsC. Each of the downstream reporter cassettes on the integrated concatamer is flanked by the minimal KminC 5′ upstream region, whereas the endogenous kahrp gene is controlled by the minimal Kmin sequence. Note that these units are essentially inactive because Kmin has negligible promoter activity [54].


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)

Integration of the upsC 5′ upstream sequence into a heterologous context at the kahrp locus.(A) Schematic map of the transfection construct pBKminC. Single-crossover integration was guided by kahrp 5′ homology. The position of the kahrp TSS is indicated [81]. Numbers refer to the nucleotide positions relative to the ATG start codon. The bsd resistance cassette selects for stably transfected parasites. The var intron is indicated by a bold dashed line. hsp86 5′, hsp86 promoter; Pb DT 3′, P. berghei dhfr-thymidylate synthase terminator; rep20, 0.5 kb TARE6 repeat element; hrp2 3′; histidine-rich protein 2 terminator. MEE, location of the 101 bp mutual exclusion element MEE [54]. (B) Genomic situation after integration of the pBKminC concatamer into the endogenous kahrp locus. Restriction sites used in Southern analysis and fragment lengths are indicated and colour-coded. S, StuI; B, BglII. The Southern blot on BglII/StuI-digested gDNA shows integration of pBKminC into the endogenous locus of kahrp. The membrane was hybridised with hdhfr (top) and kahrp (bottom). Fragments are colour-coded according to the integration map. wt, size of the kahrp fragment in 3D7 wild-type parasites. i, integration event; p, plasmid fragment. (C) The upsC 5′ UTR sequence represses kahrp promoter activity. The bars represent the ratio of relative hdhfr-gfp and msp8 transcript levels in 3D7/pBKminC parasites (open bars) compared to the 3D7/pBKmin control (black bars) cultured in absence of WR. Results are the mean +/− s.d. of three independent experiments. Values are normalised for PF3D7_1331700 transcripts.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0100183-g001: Integration of the upsC 5′ upstream sequence into a heterologous context at the kahrp locus.(A) Schematic map of the transfection construct pBKminC. Single-crossover integration was guided by kahrp 5′ homology. The position of the kahrp TSS is indicated [81]. Numbers refer to the nucleotide positions relative to the ATG start codon. The bsd resistance cassette selects for stably transfected parasites. The var intron is indicated by a bold dashed line. hsp86 5′, hsp86 promoter; Pb DT 3′, P. berghei dhfr-thymidylate synthase terminator; rep20, 0.5 kb TARE6 repeat element; hrp2 3′; histidine-rich protein 2 terminator. MEE, location of the 101 bp mutual exclusion element MEE [54]. (B) Genomic situation after integration of the pBKminC concatamer into the endogenous kahrp locus. Restriction sites used in Southern analysis and fragment lengths are indicated and colour-coded. S, StuI; B, BglII. The Southern blot on BglII/StuI-digested gDNA shows integration of pBKminC into the endogenous locus of kahrp. The membrane was hybridised with hdhfr (top) and kahrp (bottom). Fragments are colour-coded according to the integration map. wt, size of the kahrp fragment in 3D7 wild-type parasites. i, integration event; p, plasmid fragment. (C) The upsC 5′ UTR sequence represses kahrp promoter activity. The bars represent the ratio of relative hdhfr-gfp and msp8 transcript levels in 3D7/pBKminC parasites (open bars) compared to the 3D7/pBKmin control (black bars) cultured in absence of WR. Results are the mean +/− s.d. of three independent experiments. Values are normalised for PF3D7_1331700 transcripts.
Mentions: The 101 bp MEE element is located downstream of the transcriptional start site (TSS) in the upsC upstream region and controls inclusion of the locus into the programme of mutually exclusive var activity [54]. Here, we aimed at a more detailed functional characterisation of this regulatory sequence. First, we asked whether an upsC upstream sequence including the MEE is able to modulate gene expression autonomously when placed in a conserved position downstream of the TSS of a heterologous promoter. To achieve this, we used our previously published transfection vector pBKmin as a vehicle to target the endogenous kahrp (knob-associated histidine rich protein) locus [54]. pBKmin contains the blasticidin deaminase (bsd) resistance gene followed by a reporter cassette in which a minimal kahrp promoter (Kmin) controls expression of the hdhfr-gfp (human dihydrofolate reductase fused to green fluorescent protein) reporter gene that confers resistance to the antifolate WR99210 (WR). Here, we replaced the region spanning bps −445 to −1 downstream of the TSS of the minimal kahrp promoter with the upsC sequence (bps −519 to −1) containing the MEE (Figure 1A). Transfected 3D7 parasites were selected on blasticidin-S-HCl (BSD) and the plasmid was integrated into the endogenous kahrp locus by single-crossover homologous recombination (3D7/pBKminC). This event created the kahrp-upsC hybrid upstream sequence kahrpC that drives expression of the hdhfr-gfp gene (Figure 1B). In this context, the wild-type kahrp promoter drives transcription of hdhfr-gfp and produces transcripts in which the 5′ UTR of kahrp had been swapped with that of var upsC. Each of the downstream reporter cassettes on the integrated concatamer is flanked by the minimal KminC 5′ upstream region, whereas the endogenous kahrp gene is controlled by the minimal Kmin sequence. Note that these units are essentially inactive because Kmin has negligible promoter activity [54].

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