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Identification of Clustered Organellar Short (Cos) Rnas and of A Published online 8 August 2018 Nucleic Acids Research, 2018, Vol. 46, No. 19 10417–10431 doi: 10.1093/nar/gky710 Identification of clustered organellar short (cos) RNAs and of a conserved family of organellar RNA-binding proteins, the heptatricopeptide repeat proteins, in the malaria parasite Arne Hillebrand1, Joachim M. Matz2, Martin Almendinger1,KatjaMuller¨ 2, Kai Matuschewski2 and Christian Schmitz-Linneweber1,* Downloaded from https://academic.oup.com/nar/article/46/19/10417/5068241 by guest on 26 September 2021 1Humboldt University Berlin, Molecular Genetics, Berlin, Germany and 2Humboldt University, Department of Molecular Parasitology, Berlin, Germany Received December 12, 2017; Revised July 20, 2018; Editorial Decision July 23, 2018; Accepted July 24, 2018 ABSTRACT malaria globally, resulting in almost half a million deaths (1). While some antimalarial treatments are available, para- Gene expression in mitochondria of Plasmodium site resistance is a continuing challenge. Plasmodium spp. falciparum is essential for parasite survival. The belong to the family of apicomplexan parasites, most of molecular mechanisms of Plasmodium organellar which contain a non-photosynthetic plastid called the api- gene expression remain poorly understood. This coplast (2). As a remnant of a red algae chloroplast, the api- includes the enigmatic assembly of the mitochon- coplast contains its own DNA, as does the Plasmodium mi- drial ribosome from highly fragmented rRNAs. Here, tochondrion. The Plasmodium nuclear genome has a size we present the identification of clustered organellar of 24 MB and contains >5000 genes; by contrast, the api- short RNA fragments (cosRNAs) that are possible coplast and mitochondrial genomes are small––35 and 6 kb, footprints of RNA-binding proteins (RBPs) in Plas- respectively. modium organelles. In plants, RBPs of the pentatri- In keeping with the descendance of the apicoplast from cyanobacteria, the apicoplast genome is organized similarly copeptide repeat (PPR) class produce footprints as a to bacterial chromosomes. Genes are organized into oper- consequence of their function in processing organel- ons and transcribed as polycistronic precursors, which are lar RNAs. Intriguingly, many of the Plasmodium cos- heavily processed post-transcriptionally (3,4). Blocking api- RNAs overlap with 5 -ends of rRNA fragments. We hy- coplast gene expression with inhibitors of transcription or pothesize that these are footprints of RBPs involved translation leads to an immediate or a ‘delayed’ death phe- in assembling the rRNA fragments into a functioning notype, depending on the drug used (5). ribosome. A bioinformatics search of the Plasmod- The mitochondrial genome organization in Apicomplexa ium nuclear genome identified a hitherto unrecog- is unique. With a very small genome size of 6 kb and only nized organellar helical-hairpin-repeat protein family three protein-coding genes, it is one of the smallest genomes that we term heptatricopeptide repeat (HPR) proteins. discoveredtodate(6). In addition to the protein-coding We demonstrate that selected HPR proteins are tar- genes, the genome contains highly fragmented rRNA genes instead of full-length rRNA genes. There is strong evidence geted to mitochondria in P. berghei and that one of for translational activity in Plasmodium mitochondria (7,8), them, PbHPR1, associates with RNA, but not DNA in suggesting that these rRNA fragments are assembled into vitro. A phylogenetic search identified HPR proteins functional ribosomes. This inference is based on the es- in a wide variety of eukaryotes. We hypothesize that sential nature of the electron transport chain in mitochon- HPR proteins are required for processing and stabi- dria (7) and the finding that mutations underlying resis- lizing RNAs in Apicomplexa and other taxa. tance to atovaquone, a specific inhibitor of the respiratory chain, map to a mitochondrial protein-coding gene (9). Fur- INTRODUCTION thermore, tRNAs are imported into mitochondria in Toxo- plasma gondii and P.falciparum, providing indirect evidence The protozoan parasite Plasmodium spp. is the causative for mitochondrial translation (10,11). agent of malaria in humans. A 2016 report by the World Health Organization showed 214 million infections with *To whom correspondence should be addressed. Tel: +49 30 2093 49700; Fax: +49 30 2093 49701; Email: [email protected] C The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected] 10418 Nucleic Acids Research, 2018, Vol. 46, No. 19 Transcription of the mitochondrial genome has been As a first step toward understanding RNA degrada- shown to lead to polycistronic transcripts, with mRNAs tion in P. falciparum, we searched for potential binding and rRNA fragments transcribed together as one precur- sites of helical-hairpin-repeat proteins in Plasmodium or- sor molecule (12,13). Even transcripts representing the en- ganelle RNA metabolism by creating small-RNA sequenc- tire genome have been detected (12). This expression organi- ing libraries. This effort was complemented by a search zation suggests that post-transcriptional regulatory events, for helical-hairpin-repeat proteins. We discovered a novel such as RNA processing, RNA stabilization, and RNA helical-hairpin-repeat protein family distinct from PPR and degradation, play an important role in mitochondria, simi- OPR proteins, which we termed heptatricopeptide repeat lar to post-transcriptional gene regulation in the apicoplast (HPR) proteins, and demonstrated that individual HPRs (3). Indeed, monocistronic mRNAs have been found for are targeted to Plasmodium mitochondria. Although these CYTB and COXI (14), which are likely derived from poly- proteins seem particularly abundant in apicomplexan para- cistronic precursors via RNA processing. Notably, mito- sites, they were also found in other species within the Alve- chondrial mRNAs directly abut their neighboring mRNAs olata. In fact, while they were not dected in bacteria, HPR Downloaded from https://academic.oup.com/nar/article/46/19/10417/5068241 by guest on 26 September 2021 or rRNAs, a relationship that has been taken as evidence proteins are found in most eukaryotic groups analyzed, in- that polycistronic precursors are precisely cleaved to yield cluding humans. individual mature transcripts (12,13). These processes are expected to require a large number of RNA-binding pro- teins (RBPs), but most of the components and mechanisms MATERIALS AND METHODS of the RNA-processing machinery remain unknown even Ethical statement in human mitochondria (15,16). All organellar RBPs must originate from the nuclear genome and be transferred to This study was carried out in strict accordance with the Ger- man ‘Tierschutzgesetz in der Fassung vom 22. Juli 2009’ the organelles post-translationally. Few nuclear factors for / / Plasmodium RNA processing have been characterized to and the Directive 2010 63 EU of the European Parliament date, and how RNA metabolism is regulated remains largely and Council ‘On the protection of animals used for scien- unknown. tific purposes’. The protocol was approved by the ethics Because Plasmodium, with its apicoplast, has also au- committee of the Berlin state authority (Landesamt f¨ur Gesundheit und Soziales Berlin, permit numbers G0469/09 totrophic organisms among its ancestry, a comparison with / organellar gene expression in plants and algae can shed light and G0294 15). Female NMRI mice were used for all ex- on RNA-processing mechanisms in the organelles of this periments and purchased from Charles River Laboratories parasite. RNA metabolism in plant organelles is character- (Sulzfeld, Germany). ized by a massive expansion of nuclear-encoded RBPs. The largest class of organellar RBPs is helical-hairpin-repeat Cultivation and harvest of Plasmodium falciparum asexual proteins called pentatricopeptide repeat proteins (PPRs) stages (17). These proteins contain a tandem-repeat motif with up to 35 repetitions. Structurally, each repeat forms two Plasmodium falciparum NF54 was cultivated as described alpha-helical elements that fold back onto each other. Al- by Trager and Jensen (23). Cultures were split every 2–3 pha helices from consecutive repeats are stacked to form days. Cultures were not synchronized. an extended RNA-interacting surface. Within this surface, each repeat is responsible for binding one base in a single- Total RNA extraction from Plasmodium falciparum asexual stranded RNA molecule. Two dedicated amino acids are stages key for specific RNA base recognition (18). Of the ∼450 predicted PPR proteins in Arabidopsis, approximately two- Asexual stages of P.falciparum NF54 in red blood cells were thirds are localized to the mitochondria, with the remainder harvested as described above. Cell pellets were resuspended are found in the plastid (19). PPR proteins play an impor- in AIM-Buffer (24) containing 0.1% saponin. After incu- tant role in RNA processing and transcript stabilization. bation for 5 minutes on ice, cells were centrifuged at 2500 Binding of a number of PPR proteins to mRNAs acts as × g for 10 min. Pelleted parasites were washed twice with a roadblock against exonucleolytic
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