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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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uAUGs are enriched in var 5′ UTRs.For each gene, sequences ranging from bp −500 to −1 relative to the ATG start codon were downloaded from PlasmoDB version 7.2 (www.plasmoDB.org) and the counts of the trinucleotide sequence ‘ATG’ were assessed in sliding windows of 50 bps using custom-made Perl scripts. The average ATG counts for each sequence set were plotted using the statistical analysis package R (www.r-project.org). The var gene set includes 60 sequences, subdivided into groups “upsA”, “upsB”, “upsC” and “others” (upsB/C, upsB/A, upsE) according to the classification by Lavstsen and colleagues [15]. The control set consists of 5′ UTR sequences of 403 genes with peak transcription in ring stages. Selection of these sequences was based on RNASeq data [39] according to the following criteria: timing of maximal expression: 8 hpi and 16 hpi; maximal expression ratio: 8-fold induction; maximum expression percentile: 30th percentile. uAUGs are significantly enriched in var 5′ UTRs compared to the control set of ring stage-specific genes (p = 7.56×10−11; Welch t-test).
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pone-0100183-g005: uAUGs are enriched in var 5′ UTRs.For each gene, sequences ranging from bp −500 to −1 relative to the ATG start codon were downloaded from PlasmoDB version 7.2 (www.plasmoDB.org) and the counts of the trinucleotide sequence ‘ATG’ were assessed in sliding windows of 50 bps using custom-made Perl scripts. The average ATG counts for each sequence set were plotted using the statistical analysis package R (www.r-project.org). The var gene set includes 60 sequences, subdivided into groups “upsA”, “upsB”, “upsC” and “others” (upsB/C, upsB/A, upsE) according to the classification by Lavstsen and colleagues [15]. The control set consists of 5′ UTR sequences of 403 genes with peak transcription in ring stages. Selection of these sequences was based on RNASeq data [39] according to the following criteria: timing of maximal expression: 8 hpi and 16 hpi; maximal expression ratio: 8-fold induction; maximum expression percentile: 30th percentile. uAUGs are significantly enriched in var 5′ UTRs compared to the control set of ring stage-specific genes (p = 7.56×10−11; Welch t-test).

Mentions: At this stage we do not know whether translation initiation at uAUGs and/or translation of uORFs is involved in the inhibitory function of the upsC 5′ UTR. It is plausible that translational inhibition is mediated in a uORF-independent fashion, for instance by secondary mRNA structures and/or sequence-specific RNA/protein interactions that may block PIC recruitment and/or scanning. Notably, however, we observed a prominent enrichment of uAUGs in var 5′ UTRs in general compared to other ring stage-specific transcripts (Figure 5). The investigated upsC sequence in pBKminC is no exception to that rule. In fact, the 519 bp 5′ UTR sequence contains the remarkable number of 33 uAUGs. Moreover, the 101 bp MEE sequence element alone carries six uAUGs that may serve as initiation sites for the translation of 6-11 amino acid (aa) peptides. If uORF-translation indeed plays a role in regulating expression of var genes other than var2csa remains to be investigated. Whereas the similar average size (4-6aa) of uORFs with a predicted function in yeast [67] supports such an assumption, conserved uORF-encoded peptides in Drosophila (70aa) [68] and the var2csa gene (120aa) [49] are much larger. Importantly, however, irrespective of whether translation is initiated within the upsC 5′ UTR or not, uAUGs can lead to a substantial decrease in translation efficiency and they were shown to have important roles in translational control during development and conditions of cell stress [69], [70]. Clearly, P. falciparum must have evolved mechanisms to bypass the “first AUG rule” in order to express PfEMP1. This may be achieved through the well-known mechanisms of leaky uAUG scanning, re-initiation after uORF translation (as demonstrated for VAR2CSA expression [51]), or by using cap-independent strategies to guide ribosomes directly to the regular start site [65]. Although the exact mechanism by which translation of upsC-derived mRNA is inhibited remains to be determined, our findings demonstrate that P. falciparum uses this type of control to modulate expression of PfEMP1 variants. Similar to our observations, the 5′ UTR of a P. falciparum house-keeping gene was recently identified to reduce translation efficiency [71], and a recent study based on polysome profiling suggests the regulation of translation by 5′ UTRs may be a widespread mechanism to control protein expression in P. falciparum[72].


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)

uAUGs are enriched in var 5′ UTRs.For each gene, sequences ranging from bp −500 to −1 relative to the ATG start codon were downloaded from PlasmoDB version 7.2 (www.plasmoDB.org) and the counts of the trinucleotide sequence ‘ATG’ were assessed in sliding windows of 50 bps using custom-made Perl scripts. The average ATG counts for each sequence set were plotted using the statistical analysis package R (www.r-project.org). The var gene set includes 60 sequences, subdivided into groups “upsA”, “upsB”, “upsC” and “others” (upsB/C, upsB/A, upsE) according to the classification by Lavstsen and colleagues [15]. The control set consists of 5′ UTR sequences of 403 genes with peak transcription in ring stages. Selection of these sequences was based on RNASeq data [39] according to the following criteria: timing of maximal expression: 8 hpi and 16 hpi; maximal expression ratio: 8-fold induction; maximum expression percentile: 30th percentile. uAUGs are significantly enriched in var 5′ UTRs compared to the control set of ring stage-specific genes (p = 7.56×10−11; Welch t-test).
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Related In: Results  -  Collection

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pone-0100183-g005: uAUGs are enriched in var 5′ UTRs.For each gene, sequences ranging from bp −500 to −1 relative to the ATG start codon were downloaded from PlasmoDB version 7.2 (www.plasmoDB.org) and the counts of the trinucleotide sequence ‘ATG’ were assessed in sliding windows of 50 bps using custom-made Perl scripts. The average ATG counts for each sequence set were plotted using the statistical analysis package R (www.r-project.org). The var gene set includes 60 sequences, subdivided into groups “upsA”, “upsB”, “upsC” and “others” (upsB/C, upsB/A, upsE) according to the classification by Lavstsen and colleagues [15]. The control set consists of 5′ UTR sequences of 403 genes with peak transcription in ring stages. Selection of these sequences was based on RNASeq data [39] according to the following criteria: timing of maximal expression: 8 hpi and 16 hpi; maximal expression ratio: 8-fold induction; maximum expression percentile: 30th percentile. uAUGs are significantly enriched in var 5′ UTRs compared to the control set of ring stage-specific genes (p = 7.56×10−11; Welch t-test).
Mentions: At this stage we do not know whether translation initiation at uAUGs and/or translation of uORFs is involved in the inhibitory function of the upsC 5′ UTR. It is plausible that translational inhibition is mediated in a uORF-independent fashion, for instance by secondary mRNA structures and/or sequence-specific RNA/protein interactions that may block PIC recruitment and/or scanning. Notably, however, we observed a prominent enrichment of uAUGs in var 5′ UTRs in general compared to other ring stage-specific transcripts (Figure 5). The investigated upsC sequence in pBKminC is no exception to that rule. In fact, the 519 bp 5′ UTR sequence contains the remarkable number of 33 uAUGs. Moreover, the 101 bp MEE sequence element alone carries six uAUGs that may serve as initiation sites for the translation of 6-11 amino acid (aa) peptides. If uORF-translation indeed plays a role in regulating expression of var genes other than var2csa remains to be investigated. Whereas the similar average size (4-6aa) of uORFs with a predicted function in yeast [67] supports such an assumption, conserved uORF-encoded peptides in Drosophila (70aa) [68] and the var2csa gene (120aa) [49] are much larger. Importantly, however, irrespective of whether translation is initiated within the upsC 5′ UTR or not, uAUGs can lead to a substantial decrease in translation efficiency and they were shown to have important roles in translational control during development and conditions of cell stress [69], [70]. Clearly, P. falciparum must have evolved mechanisms to bypass the “first AUG rule” in order to express PfEMP1. This may be achieved through the well-known mechanisms of leaky uAUG scanning, re-initiation after uORF translation (as demonstrated for VAR2CSA expression [51]), or by using cap-independent strategies to guide ribosomes directly to the regular start site [65]. Although the exact mechanism by which translation of upsC-derived mRNA is inhibited remains to be determined, our findings demonstrate that P. falciparum uses this type of control to modulate expression of PfEMP1 variants. Similar to our observations, the 5′ UTR of a P. falciparum house-keeping gene was recently identified to reduce translation efficiency [71], and a recent study based on polysome profiling suggests the regulation of translation by 5′ UTRs may be a widespread mechanism to control protein expression in P. falciparum[72].

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