Antigenic variation, which is an important strategy used by the malaria parasite Plasmodium falciparum to evade the human immune system, results from switches in the expression of members of the multicopy var gene family. The var genes encode erythrocyte membrane protein 1 (PfEMP1), which is expressed on the surface of infected erythrocytes, with only a single PfEMP1 variant being expressed at any given time. The regulatory mechanisms controlling var gene expression are not fully understood, but Dzikowski and colleagues now show that antisense long noncoding RNAs (lncRNAs) contribute to the mutually exclusive activation pattern of these genes.
var loci exhibit a distinctive genetic architecture, in which an upstream promoter controls mRNA transcription and an internal promoter directs bidirectional transcription of two lncRNAs. The sense lncRNA is postulated to have a role in regulating epigenetic memory by imprinting var genes for silencing, but little is known about the antisense lncRNA, other than that its sequence is complementary to the variable region of its cognate var gene, which led the authors to reason that it might have a role in var gene activation.
Using reverse transcription quantitiative PCR on parasite cell lines, the authors demonstrated that the cognate antisense lncRNA of the active var gene was expressed, whereas no antisense transcripts from silenced var gene loci were detectable. Furthermore, lncRNAs were found to be produced from late ring-stage parasites during the intraerythrocytic cycle when the upstream promoter of their corresponding var gene was active, which shows that the antisense lncRNA is associated exclusively with the active var gene. Moreover, chromatin immunoprecipitation experiments showed that the antisense lncRNAs are incorporated into chromatin, providing additional evidence for a potential role in gene regulation.
Next, the authors tested whether delivery of an antisense lncRNA could activate expression of its cognate var gene. Indeed, transfection of the antisense transcript into cells in which the corresponding var gene was silent resulted in var gene activation in a sequence- and dose-dependent manner. By contrast, knockdown of the antisense lncRNA using peptide nucleic acids silenced the active var gene and induced antigen switching.
lncRNAs contribute to var gene activation and antigenic variation in P. falciparum
Together, these data provide compelling evidence that antisense lncRNAs contribute to var gene activation and antigenic variation in P. falciparum. Although the precise mechanistic details of var gene activation remain elusive, Dzikowski and colleagues suggest that antisense lncRNAs may regulate gene expression through the recruitment of chromatin-remodelling enzymes or transcription factors, or by preventing the binding of gene-silencing insulator proteins.
Amit-Avraham, I. et al. Antisense long noncoding RNAs regulate var gene activation in the malaria parasite Plasmodium falciparum. Proc. Natl Acad. Sci. USA http://dx.doi.org/10.1073/pnas.1420855112 (2015)
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Attar, N. The antisense antigen switch. Nat Rev Microbiol 13, 189 (2015). https://doi.org/10.1038/nrmicro3455