Antisense long noncoding RNAs regulate var gene activation in the malaria parasite Plasmodium falciparum

Abstract

The virulence of Plasmodium falciparum, the causative agent of the deadliest form of human malaria, is attributed to its ability to evade human immunity through antigenic variation. These parasites alternate between expression of variable antigens, encoded by members of a multicopy gene family named var. Immune evasion through antigenic variation depends on tight regulation of var gene expression, ensuring that only a single var gene is expressed at a time while the rest of the family is maintained transcriptionally silent. Understanding how a single gene is chosen for activation is critical for understanding mutually exclusive expression but remains a mystery. Here, we show that antisense long noncoding RNAs (lncRNAs) initiating from var introns are associated with the single active var gene at the time in the cell cycle when the single var upstream promoter is active. We demonstrate that these antisense transcripts are incorporated into chromatin, and that expression of these antisense lncRNAs in trans triggers activation of a silent var gene in a sequence- and dose-dependent manner. On the other hand, interference with these lncRNAs using complement peptide nucleic acid molecules down-regulated the active var gene, erased the epigenetic memory, and induced expression switching. Altogether, our data provide evidence that these antisense lncRNAs play a key role in regulating var gene activation and mutually exclusive expression.

Keywords: Plasmodium falciparum; exclusive expression; malaria; noncoding RNA; var genes.

Fig. 1.

Antisense lncRNA is associated with the active var gene. The antisense lncRNA of var genes is transcribed simultaneously with var mRNA. (A) Schematic of the active var genes in the DC-J–off and DC-J–on lines (Upper and Lower, respectively), indicating the locations of the specific primers used for strand-specific cDNA production and RT-qPCR. Steady-state mRNA levels of the entire var gene family measured by RT-qPCR from DC-J–off (B, Left) and DC-J–on (C, Left). (B and C, Right) Strand-specific cDNA levels of mRNA (black) and antisense transcripts (gray) measured by RT-qPCR. RNA was extracted from tightly synchronized ring-stage parasites (∼18 h postinvasion). (D) Specificity of the strand-specific antisense detection. Schematic of the PFB1055c-bsd transgene indicating primer location (Upper) and RT-PCR (Lower), showing the specific detection of the antisense transcript only with the 3′BSD primer set. Amplification in the absence of reverse transcriptase with each of the forward or reverse primers [No RT (P.F) and No RT (P.R) respectively], no primers, and no template (−) were used as negative controls, whereas arginyl-tRNA synthetase was used as the positive control. Error bars represent SEs. The PbDT 3′ UTR is marked with a black rectangle, and the DC-J integration site at the 5′ UTR is marked with a bold gray line.

Fig. 2.

Antisense lncRNA is associated with chromatin. RNA immunoprecipitation of var transcripts was performed on tightly synchronized ring-stage parasites (∼18 h postinvasion) using antibodies against the core histone H3 or with antibodies against C-myc as a negative control. (A) Schematic of the active var genes in each clone indicating the location of the specific primer sets that were used for strand-specific cDNA synthesis and RT-qPCR. The PbDT 3′ UTR is marked with a black rectangle. (B) Western blot of the fractions that were immunoprecipitated with α-H3 and α–C-myc antibodies from DC-J–on and DC-J–off populations and detected by the α-H3 antibody. (C) Levels of sense and antisense var transcripts detected by RT-qPCR from the IP samples (α-H3 or α–C-myc) and non-IP samples (input). Different primer sets presented on top of each column were used differentially to detect mRNA (black), antisense (gray), and sense ncRNA (white). (D) Enrichment levels of var sense and antisense transcripts detected by RT-qPCR and calculated as the relative percentile of the levels detected in the IP samples (α-H3 or α–C-myc) relative to the input. The levels of strand-specific cDNA were measured by RT-qPCR relative to arginyl-tRNA synthetase (PFL0900c). The active gene in each parasite line is marked with a red arrowhead. Each value is the average of two biological replicates and three technical triplicates. Error bars represent SEs. Samples differences were tested using the Student’s t test at P < 0.05.

Fig. 3.

Silent var gene is activated by expression of its specific antisense transcript. (A) Schematics of the PFB1055-bsd recombinant locus in the DC-J parasite line (Upper); the plasmid pBbI₁₀₅₅Dh, which was used to express the specific antisense lncRNA of PFB1055-bsd (Middle Upper); the plasmid phLI₁₀₅₅Dh, which expresses an unrelated sequence (luciferase) that was used as a negative control (Middle Lower); and the plasmid p3′BbIDh, which expresses the antisense lncRNA of PFB1055-bsd fused to an unrelated 3′ UTR (Lower). The locations of the differential primers used for RT-qPCR are indicated by arrows. The PbDT 3′ UTR is marked with a black rectangle. (B, Left) Steady-state mRNA levels of the entire var gene family measured by RT-qPCR from DC-J–off (Upper), DC-J–off transfected with the control plasmid phLI₁₀₅₅Dh expressing an unrelated sequence (Middle Upper), DC-J–off transfected with the plasmid pBbI₁₀₅₅Dh expressing the var-specific antisense transcript (Middle Lower), and DC-J–off transfected with the plasmid p3′BbI₁₀₅₅Dh expressing the var-specific antisense transcript terminated by an unrelated 3′ UTR (Lower). RNA was extracted from tightly synchronized ring-stage parasites (∼18 h postinvasion). (B, Right) Plasmid copy number measured by qPCR of genomic DNA is presented. (C) Growth curves of parasite populations grown under selection with 2 μg/mL blasticidin. Growth curves were calculated for untransfected DC-J–on parasites that were preselected using blasticidin (brown); untransfected DC-J–off, where the bsd is predominantly silent (green); DC-J–off transfected with the mock plasmid phLI₁₀₅₅Dh (purple); DC-J–off transfected with the plasmid expressing the var-specific antisense pBbI₁₀₅₅Dh (light blue); DC-J–off transfected with the plasmid expressing the var-specific antisense fused with the 3′ UTR p3′BbI₁₀₅₅Dh (gray); and WT NF54 clone (A3) transfected with the plasmid expressing the var-specific antisense pBbI₁₀₅₅Dh (orange). Error bars represent SEs.

Fig. 4.

Activation of var genes by the antisense lncRNA is sequence-specific and independent of PolII transcription. The var antisense lncRNA expressed by a PolI promoter activates a silent var gene. (A) Schematics of the PFB1055-bsd recombinant locus in the DC-J parasite line. (Upper) Location of the PEs in the var intron is indicated by a red circle. Two plasmids were used to express the antisense lncRNA of PFB1055-bsd by a PolI promoter: the plasmid pBbRDh (Middle) and the plasmid pBb_I5RDh, where the PolI promoter was fused to the var PEs (Lower). The PbDT 3′ UTR is marked with a black rectangle. (B, Left) Steady-state mRNA levels of the entire var gene family measured by RT-qPCR from DC-J–off (Upper), DC-J–off transfected with the plasmid pBbRDh expressing the var-specific (PFB1055-bsd) antisense transcript (Middle), and DC-J–off transfected with the plasmid pBb_I5RDh expressing the var-specific antisense transcript fused to the PEs (Lower). RNA was extracted from tightly synchronized ring-stage parasites (∼18 h postinvasion). (B, Right) Plasmid copy number measured by qPCR of genomic DNA is presented. (C) Growth curves of parasite populations growing under selection by 2 μg/mL blasticidin. Growth curves were calculated for untransfected DC-J–on parasites after preselection with blasticidin (brown), untransfected DC-J–off in which the bsd gene is predominantly silent (green), DC-J–off transfected with the plasmid pBbRDh (purple), and DC-J–off transfected with the plasmid pBb_I5RDh with the PEs (light blue). Error bars represent SEs.

Fig. 5.

Interference with the antisense lncRNAs down-regulates the active var gene, erases epigenetic memory, and induces var gene switching. PNAs complementary to the antisense transcript of the active var gene (PFB1055c-bsd) in the DC-J–on parasite population (growing without selection) were used to block its activity by hybridization. (A) Steady-state mRNA levels of the entire var gene family measured by RT-qPCR from DC-J–on with no PNA treatment (Upper), DC-J–on incubated with 10 μM scrambled PNA (Middle), and DC-J–on incubated with 10 μM specific α–PFB1055c-bsd antisense PNA (Lower). RNA was extracted from tightly synchronized ring-stage parasites (∼18 h postinvasion). (B) Levels of down-regulation of PFB1055c-bsd were calculated as the ratio between the levels of steady-state mRNA in PNA-treated parasites relative to nontreated parasites. These levels are presented as a percentage of down-regulation. All values presented are the average of three biological replicates and three technical triplicates. Error bars represent SEs.