The fragmented mitochondrial ribosomal RNAs of Plasmodium falciparum

Abstract

Background: The mitochondrial genome in the human malaria parasite Plasmodium falciparum is most unusual. Over half the genome is composed of the genes for three classic mitochondrial proteins: cytochrome oxidase subunits I and III and apocytochrome b. The remainder encodes numerous small RNAs, ranging in size from 23 to 190 nt. Previous analysis revealed that some of these transcripts have significant sequence identity with highly conserved regions of large and small subunit rRNAs, and can form the expected secondary structures. However, these rRNA fragments are not encoded in linear order; instead, they are intermixed with one another and the protein coding genes, and are coded on both strands of the genome. This unorthodox arrangement hindered the identification of transcripts corresponding to other regions of rRNA that are highly conserved and/or are known to participate directly in protein synthesis.

Principal findings: The identification of 14 additional small mitochondrial transcripts from P. falciparum and the assignment of 27 small RNAs (12 SSU RNAs totaling 804 nt, 15 LSU RNAs totaling 1233 nt) to specific regions of rRNA are supported by multiple lines of evidence. The regions now represented are highly similar to those of the small but contiguous mitochondrial rRNAs of Caenorhabditis elegans. The P. falciparum rRNA fragments cluster on the interfaces of the two ribosomal subunits in the three-dimensional structure of the ribosome.

Significance: All of the rRNA fragments are now presumed to have been identified with experimental methods, and nearly all of these have been mapped onto the SSU and LSU rRNAs. Conversely, all regions of the rRNAs that are known to be directly associated with protein synthesis have been identified in the P. falciparum mitochondrial genome and RNA transcripts. The fragmentation of the rRNA in the P. falciparum mitochondrion is the most extreme example of any rRNA fragmentation discovered.

Figure 1. Schematic map of the P. falciparum mt genome.

A schematic map of the 6 kb element is shown, with genes above or below the line depending on direction of transcription (orange arrows: left to right above the line and right to left below). Because the P. falciparum mt genome is tandemly repeated, the endpoints shown are the endpoints of Genbank submission M76611, rather than actual structure. Protein coding genes are indicated by white boxes, small mt rDNA sequences are shown with blue (SSU rRNA) and green (LSU rRNA) boxes and labels, and the locations of RNA23t-RNA27t are indicated with red arrows and labels. Gene abbreviations: cox1 and cox3, cytochrome c oxidase subunits I and III; cob, cytochrome b; LA-LG, LSU rRNA fragments; SA-SF, SSU rRNA fragments; 1–22, RNA1-RNA22; 23t-27t, RNA23t-RNA27t.

Figure 2. SSU rRNA secondary structure.

The P. falciparum mt SSU rRNA secondary structure model is superimposed onto the E. coli SSU rRNA secondary structure model diagram. Regions where P. falciparum has no structure equivalent to E. coli are shown using gray circles and lines. Colored nucleotides compare the P. falciparum sequence to the Three Phylogenetic Domains/Two Organelles (aka 3P2O) consensus sequence from the Gutell lab’s Comparative RNA Web (CRW) Site [URL for CRW Site: http://www.rna.ccbb.utexas.edu/. URL for 16S rRNA conservation diagram: http://www.rna.ccbb.utexas.edu/SAE/2B/ConsStruc/Diagrams/cons.16.3.3DOM.pdf]. Upper case colored nucleotides are conserved in at least 98% of the sequences used. For the P. falciparum mt rRNA fragments: red nucleotides match the 3P2O consensus sequence, light blue nucleotides differ, and black nucleotides cannot be compared to the consensus (which is conserved at less than 90%). Base pair symbols are colored when both of the paired nucleotides have the same color. Each fragment is labeled at its 5′ and 3′ ends. Each helix present in P. falciparum is labeled with its helix number in green (e.g., H500), based on the CRW Site’s helix numbering system [URL: 16S rRNA, http://www.rna.ccbb.utexas.edu/CAR/1A/Structures/h.16.b.E.coli.hlxnum.pdf]. Inset: C. elegans mt SSU rRNA secondary structure model.

Figure 3. LSU rRNA secondary structure (5′ half.)

The P. falciparum mt LSU rRNA (5′ half) secondary structure model is superimposed onto the E. coli LSU rRNA (5′ half) secondary structure model diagram. Regions where P. falciparum has no structure equivalent to E. coli are shown using gray circles and lines. Colored nucleotides compare the P. falciparum sequence to the Three Phylogenetic Domains/Two Organelles (aka 3P2O) consensus sequence from the Gutell lab’s Comparative RNA Web (CRW) Site [URL for CRW Site: http://www.rna.ccbb.utexas.edu/. URL for 23S rRNA (5′ half) conservation diagram: http://www.rna.ccbb.utexas.edu/SAE/2B/ConsStruc/Diagrams/cons.23.3.3DOM.5.pdf]. Upper case colored nucleotides are conserved in at least 98% of the sequences used. For the P. falciparum mt rRNA fragments: red nucleotides match the 3P2O consensus sequence, light blue nucleotides differ, and black nucleotides cannot be compared to the consensus (which is conserved at less than 90%). Base pair symbols are colored when both of the paired nucleotides have the same color. Each fragment is labeled at its 5′ and 3′ ends. Each helix present in P. falciparum is labeled with its helix number in green (e.g., H500), based on the CRW Site’s helix numbering system [URL: 23S rRNA 5′ half, http://www.rna.ccbb.utexas.edu/CAR/1A/Structures/h.235.b.E.coli.hlxnum.pdf]. Inset: C. elegans mt LSU rRNA (5′ half) secondary structure model.

Figure 4. LSU rRNA secondary structure (3′ half.)

The P. falciparum mt LSU rRNA (3′ half) secondary structure model is superimposed onto the E. coli LSU rRNA (3′ half) secondary structure model diagram. Regions where P. falciparum has no structure equivalent to E. coli are shown using gray circles and lines. Colored nucleotides compare the P. falciparum sequence to the Three Phylogenetic Domains/Two Organelles (aka 3P2O) consensus sequence from the Gutell lab’s Comparative RNA Web (CRW) Site [URL for CRW Site: http://www.rna.ccbb.utexas.edu/. URL for 23S rRNA (3′ half) conservation diagram: http://www.rna.ccbb.utexas.edu/SAE/2B/ConsStruc/Diagrams/cons.23.3.3DOM.3.pdf]. Upper case colored nucleotides are conserved in at least 98% of the sequences used. For the P. falciparum mt rRNA fragments: red nucleotides match the 3P2O consensus sequence, light blue nucleotides differ, and black nucleotides cannot be compared to the consensus (which is conserved at less than 90%). Base pair symbols are colored when both of the paired nucleotides have the same color. Each fragment is labeled at its 5′ and 3′ ends. Each helix present in P. falciparum is labeled with its helix number in green (e.g., H500), based on the CRW Site’s helix numbering system [URL: 23S rRNA 3′ half, http://www.rna.ccbb.utexas.edu/CAR/1A/Structures/h.233.b.E.coli.hlxnum.pdf]. Inset: C. elegans mt LSU rRNA (3′ half) secondary structure model.

Figure 5. rRNA tertiary structure.

The P. falciparum mt rRNAs are superimposed on a space-filling model of the three dimensional rRNA structure. Each individual P. falciparum rRNA fragment is colored and labeled; regions of the model with no P. falciparum equivalent are colored gray. Functional regions of the rRNAs are labeled in black. Full-page versions of each panel are available as Figures S12–S16. (A) P. falciparum SSU rRNA superimposed on Thermus thermophilus (PDB ID 1J5E; left  =  front/interface side, right  =  back); (B) P. falciparum LSU rRNA superimposed on Haloarcula marismortui (PDB ID 1S72; left  =  crown/interface side, right  =  back); (C–E) secondary structure diagrams for SSU, 5′ LSU, and 3′ LSU rRNAs, respectively, with each P. falciparum mt rRNA fragment color-coordinated with the fragment colors in the three-dimensional structure.