Rrp6 Regulates Heterochromatic Gene Silencing via ncRNA RUF6 Decay in Malaria Parasites

Abstract

The heterochromatin environment plays a central role in silencing genes associated with the malaria parasite’s development, survival in the host, and transmission to the mosquito vector. However, the underlying mechanism regulating the dynamic chromatin structure is not understood yet. Here, we have uncovered that Plasmodium falciparum Rrp6, an orthologue of eukaryotic RNA exosome-associated RNase, controls the silencing of heterochromatic genes. PfRrp6 knockdown disrupted the singular expression of the GC-rich ncRNA RUF6 family, a known critical regulator of virulence gene expression, through the stabilization of the nascent transcripts. Mechanistic investigation showed that the accumulation of the multiple RUF6 ncRNAs triggered local chromatin remodeling in situ, which activated their adjacent var genes. Strikingly, chromatin isolation by RNA purification analysis (ChIRP-seq) revealed that a remarkable RUF6 ncRNA had interacted with distal heterochromatin regions directly and stimulated a global derepression effect on heterochromatic genes, including all variant gene families and the sexual commitment-associated regulator ap2-g gene. Collectively, Rrp6 appears to conduct the epigenetic surveillance of heterochromatic gene expression through controlling RUF6 levels, thereby securing antigenic variation and sexual commitment of malaria parasites during the infection of the host. IMPORTANCE Malaria remains a major public health and economic burden. The heterochromatin environment controls the silencing of genes associated with the fate of malaria parasites. Previous studies have demonstrated that a group of GC-rich ncRNAs (RUF6) is associated with the mutually exclusive expression of var genes, but the underlying mechanisms remain elusive. Here, through a series of genetic manipulation and genome-wide multiomics analysis, we have identified the plasmodial orthologue of RNA exosome-associated Rrp6 as an upstream regulator of RUF6 expression and revealed that the dysregulation of RUF6 upon Rrp6 knockdown triggered local chromatin alteration, thereby activating most heterochromatic genes via direct interaction of RUF6 and distal gene loci. This finding not only uncovered the in-depth mechanism of RUF6-mediated regulation of heterochromatic genes but also identified Rrp6 as a novel regulator of gene expression in human malaria parasites, which provides a new target for developing intervention strategies against malaria.

The Architectural Factor HMGB1 Is Involved in Genome Organization in the Human Malaria Parasite Plasmodium falciparum

“…S3A ). We examined the changes in transcriptome upon Pfhmgb1 KO at ring, trophozoite, and schizont stages and found that most differentially expressed genes (DEGs) were structured noncoding RNAs (ncRNAs) such as tRNA, rRNA, and snoRNA and heterochromatin-associated genes ( 10 , 11 ), such as genes encoding exported proteins, variant genes of var , rifin , stevor , and Pfmc-2tm , and var -associated noncoding ruf6 genes ( 14 , 17 ) ( Fig. S3B and C and Table S1B ).…”

“…Collectively, these data are supportive of the speculation that the loss of PfHMGB1 releases the centromere superdomain, which, to some extent, disrupts the strictly organized centromere/telomere-dependent nuclear architecture. Such a defect may trigger alteration of local chromatin environment and gene expression, particularly for those genes which are highly dependent on the local chromatin structure, i.e., variant genes ( 13 , 14 ).…”

“…For example, HP1-dependent heterochromatin marked by trimethylation of lysine 9 on histone 3 (H3K9me3) characterizes transcriptionally repressed regions, whereas euchromatin marked by H3K4me2/3, acetylated lysine 9 of histone 3 (H3K9ac), or H2A.Z is usually linked to active transcription ( 10 – 12 ). In particular, local chromatin alterations occurring within the promoter regions lead to silencing or activation of variant genes, suggesting that the dynamics of local chromatin structure control the transcriptional state of variant genes ( 13 , 14 ). In addition, other mechanisms involving agents such as transcription factor (TF) ApiAP2, long noncoding RNAs (lncRNAs), and nascent RNAs are also involved in the singular expression of members of the variant gene family ( 15 – 18 ).…”

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

“…For a number of upsA var loci, a special RNAse, PfRNAse II, plays a role in silencing upsA derived transcripts 19 . The concentration of ncRNA RUF6 from different loci seems to play a general role in silencing heterochromatic genes such as var, rif and others, given that the knockdown of the RNAse PfRrP6 leads to generalized derepression of heterochromatin 14 . We hypothesized that by using temporary knockdown of factors putatively participating in these events, the hierarchy of factors playing a role in maintenance or modification of epigenetic signatures of var transcription might be elucidated.…”

“…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:…”