Transcripts in the Plasmodium Apicoplast Undergo Cleavage at tRNAs and Editing, and Include Antisense Sequences

Abstract

The apicoplast, an organelle found in Plasmodium and many other parasitic apicomplexan species, is a remnant chloroplast that is no longer able to carry out photosynthesis. Very little is known about primary transcripts and RNA processing in the Plasmodium apicoplast, although processing in chloroplasts of some related organisms (chromerids and dinoflagellate algae) shows a number of unusual features, including RNA editing and the addition of 3' poly(U) tails. Here, we show that many apicoplast transcripts are polycistronic and that there is extensive RNA processing, often involving the excision of tRNA molecules. We have identified major RNA processing sites, and have shown that these are associated with a conserved sequence motif. We provide the first evidence for the presence of RNA editing in the Plasmodium apicoplast, which has evolved independently from editing in dinoflagellates. We also present evidence for long, polycistronic antisense transcripts, and show that in some cases these are processed at the same sites as sense transcripts. Together, this research has significantly enhanced our understanding of the evolution of chloroplast RNA processing in the Apicomplexa and dinoflagellate algae.

Keywords: Apicoplast; Plasmodium; RNA editing.; RNA processing; antisense RNA; chloroplast.

Figure 1

Plasmodium falciparum apicoplast genome. Purple indicates protein-coding genes, blue indicates tRNA genes and red indicates rRNA genes. Genome drawn using OrganellarGenomeDRAW (Lohse et al. 2013).

Figure 2

Sense transcription of LSU/SSU rRNA and tRNA locus. Arrows represent protein coding genes, letters represent tRNA genes named as per standard single-letter tRNA convention. Note that tRNAs shown above the line show genes transcribed in one strand (i.e. left to right, same orientation as the SSU-A rRNA gene in the middle of the figure), while those below the line show those transcribed from the other strand (i.e. right to left, same orientation as the LSU rRNA gene). RNA transcripts are shown in red; genomic DNA is shown in black. Transcript a shows the extent of linear RT-PCR products identified with a reverse primer placed within the 5′ region of SSU-A rRNA and forward primers at intervals towards LSU rRNA. Transcript b shows maximum length of sense transcripts identified by circular RT-PCR using primers within SSU rRNA. Red arrows show major RNA processing sites. The black arrows within the transcript indicated the region from which primers were designed. Not to scale. The agarose gel analysis of the circular RT-PCR reaction is shown above. Lane H1: Promega hyperladder 1 marker, with sizes to the left. Lane 1: RNA circularization experiment, lane 2: no reverse transcriptase control.

Figure 3

Sense and antisense transcription of the LSU/tRNA/ribosomal protein locus. Genes are shown as Figure 2. Numbered arrows 1-3 represent annealing site of primers for cDNA synthesis to identify sense transcripts, and the test sites for PCRs are labelled with black dashed lines. The associated agarose gel is shown above, with lanes corresponding to the cDNA synthesis reactions below. (Some lanes have been removed for clarity). Note that the test PCR is the same for each cDNA, and thus all PCR products should be the same length. Lane H1 is Bioline Hyperladder 1 marker, with sizes of DNA fragments indicated in bp. Arrow 4 below the genes represent the annealing site of the primer to identify antisense transcripts, and the test PCR region is shown. Although representative, the figure is not to scale.

Figure 4

Sense and antisense transcription of sufB locus. Genes and transcripts are shown as Figure 2. Transcript a shows maximum length of sense transcripts identified by circular RT-PCR using primers within sufB. Arrows show major processing sites, further processing sites indicated by dotted vertical lines. The block within the transcript indicates the region from which primers were designed. Transcript b shows maximum extent of linear antisense RT-PCR products across the region. Not to scale. The gel shows results of circular RT-PCR for sufB. Lane H1, hyperladder 1 kb (Bioline) with size markers indicated in bp, lane 2, internal (control) sufB PCR, lane 3 sufB circular PCR, lane 3 no RT-control sufB inwards PCR.

Figure 5

‘Transcription of rps2-orf105-clpC-tufA’ locus. Genes and transcripts are shown as Figure 2. Transcripts are shown above or below the genome depending on the DNA strand from which they were transcribed (note that sense and antisense are gene-specific, and genes are encoded on both DNA strands). Red arrows show major processing sites; further processing sites are indicated by dotted black vertical lines. The black arrows within each transcript indicates the region from which primers were designed. Not to scale, although processing sites shown to be conserved across multiple genes are aligned. The gel shows results of circular RT-PCR for clpC. Lane H1, hyperladder 1 kb (Bioline) with size markers indicated in bp, lane 1, internal (control) clpC PCR, lane 2 clpC circular PCR, lane 3 no RT-control clpC inwards PCR.

Figure 6

Sense and antisense transcription of rps2. Agarose gel showing transcription of both sense (S) and antisense (AS) transcripts from rps2, together with control reactions with no reverse transcriptase (RT). H1: hyperladder 1. All lanes are from one gel.

Figure 7

Summary of post-transcriptional processing. Schematic diagram. RNA editing event is indicated with a star.