A divergent nonsense-mediated decay machinery inPlasmodium falciparumis inefficient and non-essential

Abstract

Nonsense-mediated decay (NMD) is a conserved mRNA quality control process that eliminates transcripts bearing a premature termination codon. In addition to its role in removing erroneous transcripts, NMD is involved in post-transcriptional regulation of gene expression via programmed intron retention in metazoans. The apicomplexan parasite Plasmodium falciparum shows relatively high levels of intron retention, but it is unclear whether these variant transcripts are functional targets of NMD. In this study, we use CRISPR-Cas9 to disrupt and epitope-tag two core NMD components: PfUPF1 (PF3D7_1005500) and PfUPF2 (PF3D7_0925800). Using RNA-seq, we find that NMD in P. falciparum is highly derived and requires UPF2, but not UPF1 for transcript degradation. Furthermore, our work suggests that the majority of intron retention in P. falciparum has no functional role and that NMD is not required for parasite growth ex vivo. We localise both PfUPF1 and PfUPF2 to puncta within the parasite cytoplasm, which may represent processing bodies - ribonucleoparticles that are sites of cytoplasmic mRNA decay. Finally, we identify a number of mRNA-binding proteins that co-immunoprecipitate with the NMD core complex and propose a model for a divergent NMD that does not require PfUPF1 and incorporates novel accessory proteins to elicit mRNA decay.

Sulfonylpiperazine compounds prevent Plasmodium falciparum invasion of red blood cells through interference with actin-1/profilin dynamics

“…The 5′ homology flank encompassed a synthetic recodonised region containing the WT or nonsynonymous drug-resistant mutations and synonymous shield mutations to prevent recleavage with Cas9 after recombination into the desired loci ( Fig 2Ai ). To direct Cas9 cleavage, a homologous synthetic guide RNA (gRNA) was mixed with a tracrRNA and recombinant Cas9 enzyme and electroporated into blood stage parasites with the donor plasmid [ 57 ]. After chromosomal integration was selected with WR99210, viable parasites for both the mutant and WT parasites were confirmed to contain the donor cassette using integration PCRs ( Fig 2Aii ).…”

“…The gRNA sequences were designed with a protospacer adjacent motif (PAM) site using the online program https://chopchop.cbu.uib.no/ [ 95 ] (sequences in S1 Table ). The donor constructs were linearized and transfected into WT 3D7 ring-stage parasites along with recombinant Cas9 enzyme and annealed gRNA and tracrRNA (Integrated DNA Technologies) as described in [ 57 ]. Chromosomal integration of the construct, which includes the human dihydrofolate resistance gene (hDHFR), was selected for with 2.5 nM WR99210.…”

The sulfonylpiperazine MMV020291 prevents red blood cell invasion by the malaria parasitePlasmodium falciparumthrough interference with actin-1/profilin dynamics

“…2Ai). To direct Cas9 cleavage, a homologous synthetic guide RNA (gRNA) was mixed with a tracrRNA and recombinant Cas9 enzyme and electroporated into blood stage parasites with the donor plasmid (McHugh et al, 2021). After chromosomal integration was selected with WR99210, viable parasites for both the mutant and wildtype parasites were confirmed to contain the donor cassette using integration PCRs (Fig.…”

“…The guide RNA (gRNA) sequences were designed with a protospacer adjacent motif (PAM) site using the online program https://chopchop.cbu.uib.no/ (Labun et al, 2019) (sequences in Table S1). The donor constructs were linearized and transfected into WT 3D7 ring-stage parasites along with recombinant Cas9 enzyme and annealed gRNA and tracrRNA (Integrated DNA technologies) as described in (McHugh et al, 2021). Chromosomal integration of the construct, which includes the human dihydrofolate resistance gene (hDHFR), was selected for with 2.5 nM WR99210.…”

Transcriptomic complexity of the human malaria parasite Plasmodium falciparum revealed by long-read sequencing

“…Several novel isoforms terminate within CDS, such that they contain ORFs but lack a stop codon. The eukaryotic non-stop RNA decay mechanism recognizes such mRNAs as aberrant and destroys them [53]; however, the machinery for removing aberrant mRNAs in P. falciparum is inefficient because it lacks key non-stop and no-go decay components [50,54]. Novel isoforms lacking a stop codon may be translated if the downstream poly(A) tail functions as a poly-lysine stop to terminate translation.…”