Structural RNAs of known and unknown function identified in malaria parasites by comparative genomics and RNA analysis

Abstract

As the genomes of more eukaryotic pathogens are sequenced, understanding how molecular differences between parasite and host might be exploited to provide new therapies has become a major focus. Central to cell function are RNA-containing complexes involved in gene expression, such as the ribosome, the spliceosome, snoRNAs, RNase P, and telomerase, among others. In this article we identify by comparative genomics and validate by RNA analysis numerous previously unknown structural RNAs encoded by the Plasmodium falciparum genome, including the telomerase RNA, U3, 31 snoRNAs, as well as previously predicted spliceosomal snRNAs, SRP RNA, MRP RNA, and RNAse P RNA. Furthermore, we identify six new RNA coding genes of unknown function. To investigate the relationships of the RNA coding genes to other genomic features in related parasites, we developed a genome browser for P. falciparum (http://areslab.ucsc.edu/cgi-bin/hgGateway). Additional experiments provide evidence supporting the prediction that snoRNAs guide methylation of a specific position on U4 snRNA, as well as predicting an snRNA promoter element particular to Plasmodium sp. These findings should allow detailed structural comparisons between the RNA components of the gene expression machinery of the parasite and its vertebrate hosts.

FIGURE 1.

Telomerase RNA in P. falciparum. (a) Northern hybridization with P. falciparum telomerase RNA specific oligonucleotides (probe 12 and probe 16) and a complementary RNA (cRNA) probe (probe 5-4, representing genomic coordinates chr9:763,006–763,193). (M) RNA molecular weight marker. Positions of LSU and SSU rRNAs were determined by staining the blot with methylene blue after Northern transfer (see Materials and Methods). (b) Multiple sequence alignment of the highly conserved regions in Plasmodium telomerase RNA. (*) invariant residues; (+) pairs of compensatory changes; (0) a single base change that converts a Watson–Crick base apposition to or from a G-U apposition (this is technically not covariation). Corresponding color-coded regions are proposed to pair. (c) Secondary structure model of P. falciparum telomerase RNA. A, B, C, D, E, Pk-1 and Pk-2 are stem forming regions, color coded to match the corresponding boxes from multiple sequence alignment, described above. (Chr) chromosome; (TBE) template boundary element.

FIGURE 2.

Expression, mapping, and secondary structure models of methylation and pseudouridylation guide snoRNAs from P. falciparum. (a) Northern blots of P. falciparum RNA run on denaturing polyacrylamide gels. (Lane 1) snoR09; (lane 2) snoR15; (lane 3) snoR21; (lane 5) snoR27; (lane 6) snoR11; (lanes 4,7) 10-bp ladder molecular weight marker. (b) Primer extension analysis to map the 5′ end of snoRNA transcripts: (Lane 1) 10-bp ladder; (lane 2) snoR09; (lane 3) snoR15; (lane 4) snoR11; (lane 5) snoR21; (lane 6) snoR27. (c) SnoR15 is a C/D box RNA that may guide modification on U4 spliceosomal snRNA. (d) SnoR21 is a C/D box snoRNA that contains two guide sequences for the large subunit (LSU) ribosomal RNA. (e) Mapping 2′-O-methylation sites on U4 snRNA . End-labeled snRNA specific primer was extended with Reverse Trascriptase (RT) in the presence of different amounts of dNTPs (100 μM and 10 μM) using either P. falciparum total RNA (lanes 5,6, in vivo) or in vitro transcribed U4 RNA (lanes 7,8). Lanes 1–4 show a sequencing ladder generated by the dideoxy termination method using P. falciparum U4 RNA. Modification site at A66 is marked with *. RT halts one nucleotide before this modification site in the presence of low dNTPs. The sequence of snoR15 paired with U4 snRNA is shown alongside sequencing lanes. (f) snoR27 is an H/ACA type Plasmodium snoRNAs that has an ortholog in yeast (snR34) and human (U65). H and ACA boxes are underlined. The proposed target LSU rRNA sequence is shown in the second hairpin loop near ACA box.

FIGURE 3.

Northern analysis and secondary structure models of the RNAs of unknown function (RUFs). Northern hybridization data for (a) RUF1, (b) RUF2, (c) RUF3, (d) RUF4, (e) RUF5, and (f) RUF6. Ten-base-pair DNA ladders were used as molecular weight markers. Secondary structure models for (g) RUF1 (presumptive H and ACA boxes underlined, 100% conserved residues from multiple sequence alignments are shown in gray) and (h) putative secondary structure of RUF6 RNA.

FIGURE 4.

Spliceosomal RNAs from P. falciparum. (a) Northern blot showing U small nuclear RNAs in the malaria parasite. U1snRNA, 170 nt; U2snRNA, 201 nt; U4snRNA, 144 nt; U5snRNA, 127 nt; and U6snRNA, 105 nt. (M) 10-bp ladder. (b) Primer extension reaction to map the snRNA transcripts. Lane C represents a negative control reaction containing yeast total RNA. (M) 10-bp molecular weight marker. (c) Secondary structure models of U1 snRNA. (d) U4 and U6 snRNA interaction—the phylogenetically invariant U6 region A41 to A47 (P. falciparum numbering) is underlined. Base pairing interaction in the internal asymmetric stem of U4 helix I is shown by squiggly lines. (e) U5 snRNA secondary structure and (f) U2 and U6 snRNA interaction—native folding pattern of the 5′ region of U2 snRNA preceding branchpoint (BP) sequence is shown in inset. The Sm protein binding sites are underlined. (SS) Complementary region to splice site sequences. Plasmodium U2 stem IIa terminal loop (nucleotides U55 to A59) has conserved complementarity to downstream single stranded sequence (U85 to A89), shown by line and arc. Alternatively, G54 to A57 in this loop could also pair with downstream residues U93 to C96. (g) 5′ splice site and (h) 3′ splice site conservation in P. falciparum and human (2000 constitutive GT-AG introns were analyzed from human genome database, http://genome.ucsc.edu). Also see Senapathy et al. (1990).

FIGURE 5.

U3 snoRNA and SNPE. (a) Secondary structure model of P. falciparum U3 snoRNA. 5′ and 3′ hinge region has sequence complementarity to the 5′ upstream region of P. falciparum 18S rRNA. The C′/D box kink-turn motif is shaded in gray. (b) Northern hybridization of U3 snoRNA from P. falciparum asexual blood stage. One hundred- and 200-nt bands are shown in the marker lane from a 10-bp ladder marker. (c) Sequence logo showing snRNA promoter-like element (SNPE), identified upstream of U1, U2, U3, U4, and U5 RNAs. This logo is generated after aligning multiple sequences from available Plasmodium sp. for respective U snRNAs (see Supplemental Fig. 8).

FIGURE 6.

Expression data and secondary structure models for P. falciparum RNAse P and MRP. (a) Secondary structure model of P. falciparum RNAse P. In this structure, helices P designates paired regions that are common to archeal, bacterial, and eukaryotic RNAse P RNAs and eP for eukaryotic paired regions only. (CR) Conserved regions. Two possible arrangements of helix eP8 and eP9 are shown in insets I and II. (b) Secondary structure model of P. falciparum RNAse MRP RNA. The conserved regions of the loop internal to the P3 helix, common to RNAse P and MRP RNA, are marked with brackets. (c) Northern hybridization with RNAse MRP (lane 1) and RNAse P (lane 2) specific probes on total RNA isolated from P. falciparum.