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.

An essential pentatricopeptide repeat protein in the apicomplexan remnant chloroplast

“…This suggests a UUAU binding motif is preferred by Pf PPR1. This sequence motif is the same as the UUAU apicoplast RNA processing site previously identified (Nisbet et al, ).…”

“…Based on the results of Nisbet et al (), we transcribed apicoplast RNA in vitro . One transcript spanned the tufA to clpC region of the apicoplast genome as this shows two clearly defined processing sites (at the tRNA‐Phe and tRNA‐Trp genes), and a second transcript spanned the LSUrRNA to rpoB region, which includes a known processing site at tRNA‐Thr.…”

“…We show that Pf PPR1 binds RNA at a UUAU‐type motif. This motif is found at known cleavage sites for Plasmodium apicoplast transcripts, which are often immediately adjacent to tRNA molecules (Nisbet et al, ). We show that the protein binds in vitro to apicoplast RNA transcripts containing known cleavage sites and can protect RNA from degradation by RNase A.…”

“…The apicoplast genome encodes 30 proteins, two rRNAs, and 25 tRNAs (Wilson et al, ). Primary RNA transcripts are polycistronic, and there is extensive RNA processing to produce individual tRNA, rRNA, and mRNA molecules (Nisbet, Kurniawan, Bowers, & Howe, ; Nisbet & McKenzie, ). RNA processing must be controlled by nuclear‐encoded proteins that are targeted to the organelle, because no RNA processing proteins are encoded on the apicoplast genome.…”

Transcription of the apicoplast genome

“…High levels of antisense transcription often, but not always corresponded with high levels of sense transcription [31]. These results have been confirmed by the discovery of antisense transcripts in Plasmodium [23], including the presence of long, polycistronic antisense transcripts. These transcripts cover protein-encoding, tRNA and rRNA genes, and may be processed at the same sites as the corresponding sense transcripts, as shown in Fig.…”