Two long non-coding RNAs generated from subtelomeric regions accumulate in a novel perinuclear compartment in Plasmodium falciparum

Abstract

Chromosome ends have been implicated in the default silencing of clonally variant gene families in the human malaria parasite Plasmodium falciparum. These chromosome regions are organized into heterochromatin, as defined by the presence of a repressive histone H3 lysine 9 trimethylated marker and heterochromatin protein 1. Here, we show that the non-coding subtelomeric region adjacent to virulence genes forms facultative heterochromatin in a cell cycle-dependent manner. We demonstrate that telomere-associated repeat elements (TAREs) and telomeres are transcribed as long non-coding RNAs (lncRNAs) during schizogony. Northern blot assays revealed two classes of lncRNAs: a ~4-kb transcript composed of telomere sequences and a TARE-3 element, and a >6-kb transcript composed of 21-bp repeats from TARE-6. These lncRNAs are transcribed by RNA polymerase II as single-stranded molecules. RNA-FISH analysis showed that these lncRNAs form several nuclear foci during the schizont stage, whereas in the ring stage, they are located in a single perinuclear compartment that does not co-localize with any known nuclear subcompartment. Furthermore, the TARE-6 lncRNA is predicted to form a stable and repetitive hairpin structure that is able to bind histones. Consequently, the characterization of the molecular interactions of these lncRNAs with nuclear proteins may reveal novel modes of gene regulation and nuclear function in P. falciparum.

Fig. 1

Long non-coding telomeric and subtelomeric RNAs are transcribed as single strands with positive polarity by a DNA-dependent RNA polymerase II. (A) Schematic representation of the telomeric and subtelomeric regions of P. falciparum. In this parasite, the chromosome ends are composed of the telomere plus telomere-associated repetitive elements (TAREs 1–6). The lines indicate the probes used in this work. (B) Run-on. RNA was radiolabeled, extracted and hybridized to dot blots of ssM13 DNA (3 μg) containing both the positive and negative strands of the telomeric, TARE-3 and TARE-6 regions respect to var gene PFI1830c. The control DNA included the gene for 28S rRNA and the vector pUC18. (C) Nuclear run-on experiments were performed with asynchronous FCR3 parasites that were incubated in the absence or presence of 0.1 mg/ml α-amanitin. Nascent [α-³²P] dUTP-labeled RNA was obtained from the nuclei of P. falciparum and hybridized with double-stranded DNA fragments (3 μg each) corresponding to the telomere, TARE-3, TARE-6, the DBL-1 domain of the var1CSA gene (RNA polymerase II control), 28S rRNA (RNA polymerase I control) and pUC18 (negative control).

Fig. 2

Telomeric and subtelomeric regions are transcribed as long non-coding RNAs and are expressed in a stage-specific manner. (A) RNA from different asexual developmental stages of FCR3 parasites was dotted on a nylon membrane and hybridized to radiolabeled telomeric, TARE-3 and TARE-6 DNA probes. Seryl tRNA synthetase, which is expressed constitutively throughout all stages, was used as a loading control. R, rings; T, trophozoites; S, schizonts. (B) RNA from ring-, trophozoite- and schizont-stage P. falciparum parasites was treated with DNase I, fractionated in a 1% denaturing agarose gel and transferred to a Hybond N+ membrane. Each membrane was hybridized to a radiolabeled telomeric, TARE-3 or TARE-6 probe. As a positive control for RNA integrity, 28S rRNA was used. Bottom, nuclear RNA was stained with ethidium bromide (EtBr) after agarose gel electrophoresis to control for loading and RNA integrity. The top schematic is a representation of the chromosome ends of P. falciparum. The arrows indicate the putative transcription start sites of the TARE-6 and TARE3-Telomere lncRNAs. Positive and negative strands of lncRNA studied in this work were stablished respect to subtelomeric var gene PFI1830c located on chromosome 9.

Fig. 3

The non-coding telomeric and subtelomeric RNAs colocalize at the nuclear periphery. (A) Telomere, TARE-3 and TARE-6 transcripts were detected by using RNA-FISH assays in parasites infected in the early-schizont (30 h) and late-schizont (36 h) stage. (Top) Early-schizont stage. First row: antisense TARE-6 ss ncRNA was labeled with biotin-16-UTP (red), and antisense Telomere ss ncRNA was labeled with Alexa Fluor 488-5-UTP (green). Second row: Anti-sense TARE-6 ss ncRNA was labeled with biotin-16-UTP (red), and antisense TARE-3 ncRNA was labeled with Alexa Fluor 488-5-UTP (green). Third row: antisense Telomere ss ncRNA was labeled with biotin-6-UTP (red), and antisense TARE-3 ss ncRNA was labeled with Alexa Fluor 488-5-UTP (green). DAPI staining is shown in blue. (Bottom) Late-schizont stage. First row: Antisense TARE-6 ss ncRNA was labeled with biotin-16-UTP (red), and antisense Telomere ss ncRNA was labeled with Alexa Fluor 488-5-UTP (green). Second row: antisense Telomere ss ncRNA was labeled with biotin-16-UTP (red), and antisense TARE-3 ss ncRNA was labeled with Alexa Fluor 488-5-UTP (green). DAPI staining is shown in blue. In each case, the merged images (yellow) show co-localization of the three small ncRNAs at the nuclear periphery. (B) ncRNA telomeric, TARE-3 and TARE-6 transcripts were detected using RNA-FISH assays in parasites infected in the ring stage. In all the case, the merged images (yellow) show co-localization of the three small ncRNAs at the nuclear periphery and only one signal was observed. At least 70 nuclei were examined per experiment two times by duplicate.

Fig. 4

The ncRNAs define a novel subdomain in the P. falciparum nucleus. (A) RNA-FISH signals are shown in red, and DNA-FISH signals are shown in green (TARE-6). The signals of the TARE-6 ncRNA and telomeric DNA do not co-localize in ring stages, but in the late-schizont stage some telomere clusters are transcribed and co-localize with TARE-6 ncRNA probe. (B) P. falciparum nucleus present a complex nuclear architecture with several subcompartments localized in the nuclear periphery as are: telomeric clusters, var gene expression site, nucleolus and non-coding RNA compartment. (C) RNA-FISH signals are shown in red (TARE-6), and 28S rDNA DNA-FISH signals are in green. The signals from 28S rDNA and the non-coding TARE-6 transcript do not co-localize. (D) Two-color RNA-FISH of a var2CSA RNA probe (green) and TARE-6 ss ncRNA transcripts (red) for ring stage parasites (FCR3-CSA parasite population). The signals from the var2CSA transcripts and the TARE-6 ncRNAs do not co-localize. In all images, nuclear DNA was stained with DAPI (blue). Scale bars: 1 μm for the ring and schizont stages. 70 nuclei were analyzed from two independent experiments.

Fig. 5

Predicted secondary structure of the TARE-6 non-coding RNA. (A) Schematic drawing of the secondary structure predicted for 1000 bases of the TARE-6 ncRNA. (B) Predicted secondary structure of the 70 nt of TARE-6 transcript. (C) The primary sequence of the TARE-6 ncRNA used in this work is composed of 70 bases and contains three Rep20 repeats. The free energy of each stem-loop structure at 0 °C and in 3 M NaCl was calculated using RNAfold software.

Fig. 6

TARE-6 lncRNA binds to P. falciparum histone H3. (A) ³² P-labeled TARE-6 RNA. probe was incubated with P. falciparum nuclear extract and three well-defined complexes, denoted RNA–protein complexes C1, C2 and C3 were observed by RNA EMSA assays. The specificity of these complexes was confirmed using a 100-fold molar excess of either homologous (TARE-6) or heterologous (scramble) competitors. (B) Nuclear and cytoplasmic extracts from P. falciparum, as well as total extracts from E. coli and GST protein, were subjected to SDS-PAGE, transferred to a nitrocellulose membrane, renatured in situ and incubated with ³²P-labeled TARE-6 RNA probe. (C) Histones preparations from P. falciparum and calf and GST protein were incubated with radiolabeled TARE-6 ncRNA probe. (D) P. falciparum nuclear proteins were incubated with radiolabeled ncRNA transcript and anti-histone H3 antibody. The RNA–protein-antibody complexes were then analyzed by electrophoresis on a 6% native polyacrylamide gel followed by autoradiography. As a control, the same amount of anti-GST antibody did not alter the formation of these three complexes.