Functional genomics of RAP proteins and their role in mitoribosome regulation in Plasmodium falciparum

Abstract

The RAP (RNA-binding domain abundant in Apicomplexans) protein family has been identified in various organisms. Despite expansion of this protein family in apicomplexan parasites, their main biological functions remain unknown. In this study, we use inducible knockdown studies in the human malaria parasite, Plasmodium falciparum, to show that two RAP proteins, PF3D7_0105200 (PfRAP01) and PF3D7_1470600 (PfRAP21), are essential for parasite survival and localize to the mitochondrion. Using transcriptomics, metabolomics, and proteomics profiling experiments, we further demonstrate that these RAP proteins are involved in mitochondrial RNA metabolism. Using high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (eCLIP-seq), we validate that PfRAP01 and PfRAP21 are true RNA-binding proteins and interact specifically with mitochondrial rRNAs. Finally, mitochondrial enrichment experiments followed by deep sequencing of small RNAs demonstrate that PfRAP21 controls mitochondrial rRNA expression. Collectively, our results establish the role of these RAP proteins in mitoribosome activity and contribute to further understanding this protein family in malaria parasites.

Decoding The Nuclear Genome of The Human Pathogen Babesia duncani Shed Light on its Virulence, Drug Susceptibility and Evolution among Apicomplexa

“…Half of the proteins were of unknown function and nucleotide binding domain was the most predicted function. This sub-group of proteins includes one DNA binding proteins of the AP2 class, one RAD51-like protein, and a putative RNA-binding RAP protein associated with Pfam domain PF08373 that could be targeted to the mitochondria as was shown in P. falciparum (41). The Babesidiae cluster is composed of 554 OrthoMCL IDs (Fig.…”

Characterization of Plasmodium falciparum RAP domain proteins-RAP291 and RAP070, and their association with ribosomal RNAs

“…Like-wise knock-out of FASTK4 has also suggested its involvement in mitochondrial RNA processing [21]. Recently using eCLIP-seq methodology in Plasmodium, two RAP proteins; PfRAP01 (PF3D7_0105200) and PfRAP21 (PF3D7_1470600) have been shown to bind distinct mitochondrial rRNA transcripts associated with the small and large mitoribosome subunits [22]. To provide evidence for PfRAP291 or PfRAP070 protein binding to RNAs, if any, we performed RNA-protein interaction assay in P. falciparum parasites using orthogonal organic-phase separation (OOPS) method [32][33][34], as well as CLIP-seq analysis of specific immunoprecipitates of crosslinked parasite lysates after phase separation [34].…”

“…Although the biological significance of the RAP domain proteins in eukaryotes, in particular among apicomplexans, is yet to be established, nevertheless presence of RAP domain in proteins such as C. reinhardtii Raa3 that binds to tscRNA as a part of Ribonucleoprotein complex and Fas-activated serine/threonine kinase (FASTK) that interacts with TIA-1, a downstream effector of eIF2 pathway predict RNA binding function for RAP domains [20,21]. Recently, two Plasmodium RAP proteins have been validated as mitochondrial rRNAs binder and have been suggested to play role(s) in mitoribosome regulation [22].…”

How RNases Shape Mitochondrial Transcriptomes

“…In addition, FASTKD2 was shown to target 16S rRNA [ 91 ]. This is not the sole RAP domain protein to target ribosomal RNA, as in plant chloroplasts where the sole RAP domain protein is implicated in 16S ribosomal RNA maturation [ 92 ], and in Plasmodium , PfRAP01 and PfRAP21 knock-down proteins affect mitoribosomes through their interaction with rRNA fragments [ 93 ]. These last authors advance the hypothesis that the multiplication of RAP proteins in Apicomplexa and Chlorophytes is linked to the highly fragmented rRNA encoded in their genomes.…”