A global view of the nonprotein-coding transcriptome in Plasmodium falciparum

Carsten A. Raabe 2 Cecilia P. Sanchez 1 Gerrit Randau 2 Thomas Robeck 2 Boris V. Skryabin 2 Suresh V. Chinni 2 Michael Kube 0 Richard Reinhardt 0 Guey Hooi Ng 3 Ravichandran Manickam 3 Vladimir Y. Kuryshev 2 Michael Lanzer 1 Juergen Brosius 2 Thean Hock Tang 3 Timofey S. Rozhdestvensky 2 0 MPI Molecular Genetics , Ihnestrasse 63-73, 14195 Berlin-Dahlem, Germany 1 Abteillung Parasitologie Hygiene Institut , Im Neuenheimer Feld 324, 69120 Heidelberg 2 Institute of Experimental Pathology , ZMBE, University of Muenster , Von-Esmarch-Str. 56, 48149 Muenster 3 Advanced Medical and Dental Institute (AMDI), Institute for Molecular Research (INFORMM) , USM 11800 Malaysia Nonprotein-coding RNAs (npcRNAs) represent an important class of regulatory molecules that act in many cellular pathways. Here, we describe the experimental identification and validation of the small npcRNA transcriptome of the human malaria parasite Plasmodium falciparum. We identified 630 novel npcRNA candidates. Based on sequence and structural motifs, 43 of them belong to the C/D and H/ACA-box subclasses of small nucleolar RNAs (snoRNAs) and small Cajal body-specific RNAs (scaRNAs). We further observed the exonization of a functional H/ACA snoRNA gene, which might contribute to the regulation of ribosomal protein L7a gene expression. Some of the small npcRNA candidates are from telomeric and subtelomeric repetitive regions, suggesting their potential involvement in maintaining telomeric integrity and subtelomeric gene silencing. We also detected 328 cis-encoded antisense npcRNAs (asRNAs) complementary to P. falciparum protein-coding genes of a wide range of biochemical pathways, including determinants of virulence and pathology. All cisencoded asRNA genes tested exhibit lifecyclespecific expression profiles. For all but one of the respective sense-antisense pairs, we deduced concordant patterns of expression. Our findings have important implications for a better understanding of gene regulatory mechanisms in P. falciparum, revealing an extended and sophisticated npcRNA network that may control the expression housekeeping genes and virulence factors. - Malaria is a devastating, life-threatening parasitic disease causing an estimated 515 million clinical cases and 1.0 million deaths annually (1). More than half of the worlds human population lives in areas where malaria is endemic. The protozoan parasite Plasmodium falciparum is the etiological agent of the most virulent form of malaria in humans. Plasmodium falciparum has acquired resistance to many antimalarial drugs and the mosquito vectors have developed resistance to available insecticides. The P. falciparum genome was recently sequenced (2), and consists of 23 Mb distributed over 14 chromosomes with sizes ranging from 0.643 to 3.29 Mb. In addition to the nuclear genome, P. falciparum contains a 5.9 kb mitochondrial and a 35 kb apicoplast genome. Approximately 5300 protein-coding genes have been identified. The (A + T) content ranges from 80.6% to 90% in introns and intergenic regions. Genomes typically encode two different types of RNA molecules: messenger RNAs (mRNAs) and various classes of nonprotein-coding RNAs (npcRNAs). The mRNAs provide templates for protein synthesis, whereas the npcRNAs do not code for proteins but rather perform various regulatory functions exerted by the RNA itself or in complexes with proteins (RNPs). npcRNAs participate in the regulation of diverse biochemical pathways, including chromosome modification, transcription and translation, splicing, developmental timing, cell differentiation, proliferation, apoptosis and organ development [for reviews see (37)]. Although the small npcRNA transcriptome has been experimentally identified and successfully verified in several model organisms, very few systematic attempts to uncover the diversity and functions of small npcRNAs in pathogenic organisms have been undertaken to date. Recently, and based mainly on comparative genomics, Chakrabarti et al. (8) detected a number of structural RNAs, e.g. telomerase RNA, 35 small nucleolar RNAs (snoRNAs), spliceosomal small nuclear RNAs (snRNAs), MRP RNA and RNase P RNA, in Plasmodium. In addition, the authors described six new npcRNAs, as of yet, unknown functions. Similarly, Mourier et al. (9) performed computational screens in intronic and intergenic regions of P. falciparum to identify conserved RNA secondary structures between distantly related Plasmodium species, yielding a set of 604 putative npcRNAs; 33 of which they verified experimentally. Notably, 29 RNAs from this dataset overlapped with those of the aforementioned set of Chakrabarti et al. (8). Li et al. (10) reported centromeric expression of small npcRNA candidates (75 and 175 nt) in P. falciparum. Their data suggested bidirectional promoter activities within the centromers of the parasite. This subclass of npcRNA candidates is localized within the nucleus and appears to associate with the centromeric chromatin (10). Taken together, these observations support the hypothesis that these npcRNAs function in the coordinated organizational assembly of chromatin. Interestingly and consistent with the absence of genes encoding argonaute and dicer proteins (11), so far there were no bona fide miRNAs experimentally verified in Plasmodium. However, there might be different and evolutionarily distinct classes of small npcRNAs that compensate for this apparent lack of RNA interference machinery. Despite the aforementioned studies, the total number of small npcRNAs in the P. falciparum genome, their importance and the variety of functions they serve are still largely unknown. This is partly due to the inherent computational limitations of the algorithms used for RNA predictions. Here we report the experimental identification and analysis of the global small npcRNA transcriptome in P. falciparum and provide its functional annotation. Our data point to the potential regulation of P. falciparum gene expression and virulence by small stable npcRNAs. Conceptual similarities to the RNome of Saccharomyces cerevisiae are discussed. Moreover, given the broad mechanistic spectrum within which npcRNAs act and the low degree of host parasite conservation, small npcRNAs might be considered as potential drug targets. MATERIALS AND METHODS cDNA library construction Total RNA extracted from all human erythrocyte stages of P. falciparum 3D7 strain was size fractionated (1060 nt and 60500 nt) on an 8% (w/v) denaturing polyacrylamide gel (7 M urea, 1 TBE buffer). Passive elution was performed in 0.3 M NaOAc (pH 5.3) overnight at 4 C. Subsequently, 5 mg of size-fractionated RNA was treated with tobacco acid pyrophosphatase (Epicenter) for 1 h at 37 C and C-tailed with poly-A polymerase (Invitrogen) for 2 h at 37 C (12). A 50-DNA SalI adapter (50-CAAC GCGTCGACTACGTGAGATTTGAGGTTC-30) was then ligated to the 50-end of the RNA using T4 RNA ligase (Fermentas) at 4 C overnight. First-strand cDNA synthesi (...truncated)