Key result
Disruption of core nonsense-mediated decay components PfUPF1 and PfUPF2 using CRISPR-Cas9 showed they are not required for degradation of nonsense transcripts or parasite growth ex vivo.
Why the study?
Plasmodium falciparum exhibits high levels of intron retention, but whether these variant transcripts are functional targets of nonsense-mediated decay was unclear.
The highly conserved NMD proteins do not impact the abundance of nonsense transcripts in the malaria parasite, and efficient CRISPR-Cas9 editing can be achieved using commercial Cas9 nuclease and synthetic guide RNA.
NMD inefficient and non-essential in P. falciparum; leaves open whether intron retention regulates gene expression independently of decay.
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 the P. falciparum orthologs of two core NMD components: Pf UPF1 (PF3D7_1005500) and Pf UPF2 (PF3D7_0925800). We localize both Pf UPF1 and Pf UPF2 to puncta within the parasite cytoplasm and show that these proteins interact with each other and other mRNA-binding proteins. Using RNA-seq, we find that although these core NMD orthologs are expressed and interact in P. falciparum , they are not required for degradation of nonsense transcripts. 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 . IMPORTANCE In many organisms, the process of destroying nonsense transcripts is dependent on a small set of highly conserved proteins. We show that in the malaria parasite, these proteins do not impact the abundance of nonsense transcripts. Furthermore, we demonstrate efficient CRISPR-Cas9 editing of the malaria parasite using commercial Cas9 nuclease and synthetic guide RNA, streamlining genomic modifications in this genetically intractable organism.
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McHugh et al. (2023) studied Plasmodium falciparum (Malaria). CRISPR-Cas9 disruption of PfUPF1 and PfUPF2 was evaluated on Degradation of nonsense transcripts and parasite growth ex vivo. Disruption of core nonsense-mediated decay components PfUPF1 and PfUPF2 using CRISPR-Cas9 showed they are not required for degradation of nonsense transcripts or parasite growth ex vivo.
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