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Author Spotlight: Identifying Compensatory Pathways in Malaria Parasites Containing Hypomorphic Allele of Essential Protein Kinases
Published on: November 22, 2024
Nonsense-mediated decay machinery in Plasmodium falciparum is inefficient and non-essential
Emma McHugh1, Michaela S Bulloch1, Steven Batinovic2
1Department of Biochemistry and Pharmacology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne , Parkville, Victoria, Australia.
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 the P. falciparum orthologs of two core NMD components: PfUPF1 (PF3D7_1005500) and PfUPF2 (PF3D7_0925800). We localize both PfUPF1 and PfUPF2 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.
Insights
Nonsense-mediated decay (NMD) proteins are not essential for degrading faulty transcripts or parasite growth in Plasmodium falciparum. This study also demonstrates efficient CRISPR-Cas9 gene editing in malaria parasites.
Area of Science:
- Molecular Biology
- Genetics
- Parasitology
Background:
- Nonsense-mediated decay (NMD) is a critical mRNA quality control pathway conserved across eukaryotes, eliminating transcripts with premature termination codons.
- In metazoans, NMD also regulates gene expression through programmed intron retention, a phenomenon observed at high levels in Plasmodium falciparum.
Purpose of the Study:
- To investigate the role of NMD in Plasmodium falciparum, specifically examining its involvement in the degradation of nonsense transcripts and regulation of intron retention.
- To assess the requirement of core NMD factors, PfUPF1 and PfUPF2, for parasite growth and viability ex vivo.
- To establish and validate CRISPR-Cas9 gene editing efficiency in Plasmodium falciparum.
Main Methods:
- CRISPR-Cas9 gene editing was employed to disrupt and epitope-tag the Plasmodium falciparum orthologs of UPF1 and UPF2 (PfUPF1 and PfUPF2).
- Immunofluorescence microscopy was used to localize PfUPF1 and PfUPF2 within the parasite cytoplasm.
- Co-immunoprecipitation assays were performed to analyze protein interactions.
- RNA sequencing (RNA-seq) was conducted to assess transcript abundance and identify potential targets of NMD and intron retention.
Main Results:
- PfUPF1 and PfUPF2 were localized to cytoplasmic puncta and shown to interact with each other and other mRNA-binding proteins.
- Despite their expression and interaction, PfUPF1 and PfUPF2 were found not to be required for the degradation of nonsense-containing transcripts in P. falciparum.
- The study suggests that most intron retention events in P. falciparum lack functional significance and that NMD is dispensable for parasite growth ex vivo.
- Efficient CRISPR-Cas9 editing was demonstrated, simplifying genomic modifications in this challenging organism.
Conclusions:
- The canonical nonsense-mediated decay pathway is not essential for degrading aberrant transcripts or for the growth of Plasmodium falciparum.
- Programmed intron retention in Plasmodium falciparum appears largely non-functional and not regulated by NMD.
- The developed CRISPR-Cas9 system provides a streamlined approach for genetic manipulation of Plasmodium falciparum, facilitating future research.
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