CRISPR Interference of a Clonally Variant GC-Rich Noncoding RNA Family Leads to General Repression of var Genes in Plasmodium falciparum

Abstract

The human malaria parasite Plasmodium falciparum uses mutually exclusive expression of the PfEMP1-encoding var gene family to evade the host immune system. Despite progress in the molecular understanding of the default silencing mechanism, the activation mechanism of the uniquely expressed var member remains elusive. A GC-rich noncoding RNA (ncRNA) gene family has coevolved with Plasmodium species that express var genes. Here, we show that this ncRNA family is transcribed in a clonally variant manner, with predominant transcription of a single member occurring when the ncRNA is located adjacent to and upstream of an active var gene. We developed a specific CRISPR interference (CRISPRi) strategy that allowed for the transcriptional repression of all GC-rich members. A lack of GC-rich ncRNA transcription led to the downregulation of the entire var gene family in ring-stage parasites. Strikingly, in mature blood-stage parasites, the GC-rich ncRNA CRISPRi affected the transcription patterns of other clonally variant gene families, including the downregulation of all Pfmc-2TM members. We provide evidence for the key role of GC-rich ncRNA transcription in var gene activation and discovered a molecular link between the transcriptional control of various clonally variant multigene families involved in parasite virulence. This work opens new avenues for elucidating the molecular processes that control immune evasion and pathogenesis in P. falciparum. IMPORTANCE Plasmodium falciparum is the deadliest malaria parasite species, accounting for the vast majority of disease cases and deaths. The virulence of this parasite is reliant upon the mutually exclusive expression of cytoadherence proteins encoded by the 60-member var gene family. Antigenic variation of this multigene family serves as an immune evasion mechanism, ultimately leading to chronic infection and pathogenesis. Understanding the regulation mechanism of antigenic variation is key to developing new therapeutic and control strategies. Our study uncovers a novel layer in the epigenetic regulation of transcription of this family of virulence genes by means of a multigene-targeting CRISPR interference approach.

An ApiAP2 transcription factor influencing virulence gene transcription and sexual development in Plasmodium falciparum

“…During asexual growth, the histone modification H3K4me2 is known to mark promoters temporarily inactive in trophozoites and schizonts for transcription after the next reinvasion 29 . Probably just before transcription, a DNA helicase -RecQ1 -is recruited to the to-be-activated var site 17 , perhaps mediated by an interaction of factors loaded with antisense ncRNAs stemming from the promoter activity localized in the var intron 9,10,35 or with GC-rich ncRNAs 13 . HP1 is found at transcriptionally silenced sites in the genome and is believed to recruit deacetylases and methyl-histone transferases 15 , which then modify varassociated chromatin (H3K9me3), by the action of at least SIR2A/B 33,36 .…”

“…The var intron found in all complete var genes appears to bi-directionally transcribe non-coding RNAs which are involved in var promoter activity 910 , although activation or silencing can also be achieved in the absence of this motif 11 . GC-rich ncRNAs seem to play a major role in the control of var gene expression 12,13 , and the exosome related RNAse PfRrP6 seems to fine-regulate the degradation of these RNAs 14 . Several proteins interact directly or indirectly with var 5' upstream regions:…”

Stage-Specific Long Non-coding RNAs inCryptosporidium parvumas Revealed by Stranded RNA-Seq

“…Another possibility is that lncRNAs function as positive regulators of the neighboring mRNA expression. In P. falciparum, ncRNAs derived from GC-rich elements that are interspersed among the internal chromosomal var gene clusters are hypothesized to play a role in var gene activation while the mechanism is unclear (Guizetti, Barcons-Simon et al 2016, Barcons-Simon, Cordon-Obras et al 2020. lncRNAs have been associated with chromatin remodeling to achieve transcriptional regulation in many studies (Li, Baptista et al 2020).…”

Noncoding RNAs in Apicomplexan Parasites: An Update

“…Overexpression of distinct GC-rich elements resulted in the activation of a specific subset of var genes, escaping MEE control [29]. Transcriptional repression of all GC-rich members by CRISPR interference (CRISPRi) led to downregulation of the entire var gene family in ring-stage parasites [30]. Thus, these GC-rich ncRNAs are hypothesized to play a role in var gene activation.…”