Two long non-coding RNAs generated from subtelomeric regions accumulate in a novel perinuclear compartment in Plasmodium falciparum
Graphical abstract
The lncRNAs define a novel subdomain in the P. falciparum nucleus.
Highlights
► Telomeric and TARE regions are transcribed as sense RNAs by RNA polymerase II. ► The ncRNAs define a novel subdomain in the P. falciparum nucleus. ► TARE-6 lncRNA binds to P. falciparum histone H3.
Introduction
In recent years, heterochromatin at chromosome ends has been the focus of many studies because it plays an important role in regulating gene expression in both yeast and humans [1], [2]. Similarly, in the protozoan Plasmodium falciparum, which is the most virulent human malaria parasite, subtelomeres exert a silencing effect on the clonally variant virulence gene families. The chromosome ends in this pathogen are organized into heterochromatin, as defined by the presence of high levels of the repressive histone H3 lysine 9 tri-methylation (H3K9me3) [3], [4] and heterochromatin protein 1 (PfHP-1) [5], [6]. This heterochromatin is associated with a 1.2-kb non-coding telomere repeat region (GGGTTT/CA) and an adjacent non-coding region that together form a mosaic of six different blocks of repetitive sequences known as telomere-associated repetitive elements (TAREs) 1–6 [7]. Adjacent to TARE-6 are members of several gene families that encode variant surface proteins, such as var, rif, stevor and pfmc-2tm. These gene families are, by default, epigenetically repressed and undergo phenotypic variation through in vitro switches. Expression in a single cell occurs either by mono-allelic expression (var gene members) or by co-transcription of several members (stevor, rif and pfmc-2tm) [7], [8]. The overall chromatin and DNA organization of the chromosome ends is highly conserved among all the chromosomes of the malaria parasite [3], [7], [8]. In addition to the identified heterochromatin factors H3K9me3 and PfHP-1, a number of histone-modifying enzymes and other factors with unknown functions, such as PfSir2, PfOrc1 and PfKMT1, are recruited to subtelomeric regions [3], [6], [9]. Experimental evidence suggests that interactions between these proteins and the telomeric and subtelomeric regions generate a repressive epigenetic center [3], [10]. This repressive center may extend to adjacent virulence gene families to create a molecular platform for the phenotypic variation of virulence factors.
Specialized nuclear compartments are crucial for the function of the Plasmodium nucleus [11], and they are particularly relevant for the mutually exclusive expression of var genes when an active var gene is placed in the var expression site. However, the model that describes this expression site as the limiting factor for the expression of a single var gene needs to be reconsidered because this expression site can harbor more than one transcriptionally active var gene [12]. Finally, fluorescence in situ hybridization assays with DNA (DNA-FISH) have shown that an active episomal rifin promoter colocalizes with a transcriptionally active var promoter, indicating that these gene families utilize the same subnuclear expression site [13]. Therefore, specialized nuclear compartments are important for the regulation of expression of several multi-copy gene families and for rDNA in distinct perinuclear compartments [3], [10], [14], [15].
Although multiple epigenetic factors contribute to the coordinated expression of virulence genes, the role of non-coding RNAs (ncRNAs) remains, for the most part, unexplored in P. falciparum. Initial evidence indicating a role for ncRNAs in malaria parasites was provided by Kyes et al. in 2003, who described var-associated ncRNAs [16]. Later, Deitsch and colleagues identified non-coding transcripts in P. falciparum from centromeric repeats [17]. In addition, the same group showed that an intron-derived ncRNA is enriched in the chromatin of var genes, but no functional data were presented [18]. More recently, a genome-wide study in P. falciparum revealed the presence of short transcripts derived from non-coding subtelomeric regions [19], [20], suggesting that the heterochromatic chromosome ends can be transcribed. A more recent study using microarray analysis suggested that long ncRNA (lncRNA) are produced from TARE-3 repeats [21].
In the present study, we used northern blot, FISH and run-on analyses to demonstrate that P. falciparum telomeres and non-coding subtelomeric repeat regions are transcribed as heterogeneous long ncRNAs (lncRNAs) by RNA polymerase II. We found that only the sense strand is transcribed in asexual blood stages. Transcription peaks occur during the schizont stage and produce two lncRNAs: a ∼4 kb lncRNA that contains TARE-3 and telomere sequences (lncTARE-3-Telomere) and a >6 kb lncRNA that consists of 21-bp repeats from TARE-6 (lncTARE-6). After transcription, both lncRNAs relocate to a single novel nuclear compartment that is distinct from the chromosome ends. Furthermore, the data from gel shift, super gel shift and northwestern blot assays suggest that this lncRNA may form large networks of interlinked RNA–protein complexes whose functions are unknown.
Section snippets
Parasites
The P. falciparum strain FCR3 was maintained according to standard culture conditions [22]. Panning assays for the selection of FCR3 parasites that transcribe var genes associated with chondroitin-sulfate A (CSA) binding were performed as previously described [23].
Nuclear and total RNA purification and northern blotting
Nuclear RNA was prepared from asynchronous cultures of FCR3 P. falciparum. Parasites were isolated from infected erythrocytes by saponin lysis, resuspended in 1 ml of lysis buffer (10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1
Telomeric and TARE regions are transcribed as sense RNAs by RNA polymerase II
To determine whether the chromosome end regions of P. falciparum are transcribed as long non-coding RNAs, we performed run-on assays. The resulting de novo RNAs were hybridized with positive and negative single-stranded M13 DNA probes from the telomere and the TARE-3 and TARE-6 regions. The polarity of the single-stranded DNA was established with respect to the subtelomeric var gene (PFI1830c) located on chromosome 9 (Fig. 1A). A hybridization signal was observed with three negative
Discussion
In this work, we characterized a novel class of long non-coding RNAs that are transcribed from the repetitive non-coding subtelomeric and telomeric regions of P. falciparum chromosomes. We also showed that these long non-coding RNAs have several potentially interesting properties, including RNA polymerase II-mediated transcription, recruitment to a novel perinuclear subcompartment and specific binding to histone H3.
The northern blot results suggest that multiple transcription start sites
Author contribution
R.H.-R designed research; M.S.-M. and D.M.D performed research, L.M-S and M.V. help in the RNA-FISH assays, S.M-C help in the run-on assays, N.V-S performed RNA secondary structure, A.S and R.H.-R. analyzed data and wrote the paper.
Conflict of interest statement
The authors declare no conflict of interest.
Acknowledgments
We would like to thank to Nicolai Siegel for their critical comments. This work was supported by the Consejo Nacional de Ciencia y Tecnología [45687/A-1] and French-Mexican collaborative program [ANR-CONACyT Paractin 140364] to RHR and by the French Agency for Research (ANR Blanc 027401), and European Research Council Executive Agency Advanced Grant (PlasmoEscape 250320) to AS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the
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