Secondary structure of Pf mt rRNA fragments and their likely positions in the modeled SSU and LSU.

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PfRSM22 and PfMRPL23 are localized to the mitochondrion and are essential for parasite survival.

(A) Images from fluorescence microscopy showing colocalization of PfRSM22-3HA (green) with Mitotracker (red). (B) Colocalization of PfMRPL23-3HA (green) with Mitotracker (red). In A and B, the parasite nucleus is stained using DAPI (blue). (C) Western blot showing expression of PfRSM22-3HA protein (62 KDa) in the presence or absence of 250 nM aTc over 6 days (3 IDCs). (D) Western blot showing expression of PfMRPL23-3HA protein (32 KDa) in the presence or absence of 250 nM aTc over 4 days (2 IDCs). Pf-Exp2 (33 KDa) was used as a loading control. Representative images of Giemsa-stained D10-PfRSM22-3HA parasites (E) and D10-PfMRPL23-3HA parasites (F) at the trophozoite stage in the presence (aTc ON) and absence of aTc (aTc OFF). Quantification of D10-PfRSM22-3HA parasite growth (G) and D10-PfMRPL23-3HA parasite growth (H) over 8 days in the presence and absence of aTc. Growth index was calculated by multiplication of parasitemia with splitting factors over the time course. Data shown here is mean ± SD of n = 3.</p

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Late effects of PfRSM22 and PfMRPL23 KDs on transcripts of mitoribosomal components.

(A) Log2 fold change of putative PfMRPs on day 6 after PfRSM22 KD (orange) and on day 4 after PfMRPL23 KD (blue). (B) Log2 fold change of mt rRNA fragments on day 6 after PfRSM22 KD (orange) and on day 4 after PfMRPL23 KD (blue).</p

PfRSM22 and PfMRPL23 KD increased sensitivity to antimalarials targeting the parasite <i>bc1</i> complex.

(A) The schematic represents key steps of growth inhibition assays measured by [3H] hypoxanthine incorporation. The diagram was created using BioRender.com. (B and C) Atovaquone hypersensitivity upon PfRSM22 and PfMRPL23 KD respectively. (D and E) ELQ-300 hypersensitivity upon PfRSM22 and PfMRPL23 KD respectively. Reduction in IC50 values and Hill Slope of the curve were reported for respective parasite lines. Data shown are the mean ± S.D. of triplicates from n = 3 independent experiments. (F and G) KD of PfRSM22 or PfMRPL23 did not show hypersensitivity to chloroquine.</p

PfMRPL23 protein domain and amino acid sequence alignment.

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List of primers and oligoes used in this study.

(DOCX)</p

PfRSM22 protein domain and amino acid sequence alignment.

(DOCX)</p

Genes regulated in common after KD of PfRSM22 and PfMRPL23 in the late phase.

(A) Venn diagrams depict the relationship of downregulated gene transcripts in the PfRSM22 KD line on day 6 (orange) and in the PfMRPL23 KD line on day 4 (blue) to the 517 mitochondrial related transcripts (green). Transcripts downregulated ≥ 2-fold are included in this analysis. (B) Heat map of commonly downregulated (68 genes) and upregulated genes (2 genes) between PfRSM22 KD and PfMRPL23 KD at the late phase. (C) Venn diagrams depict the relationship of upregulated gene transcripts in the PfRSM22 KD line on day 6 (orange) and in the PfMRPL23 KD line on day 4 (blue) to the 517 mitochondrial related transcripts (green). Transcripts upregulated ≥ 2-fold are included in this analysis.</p

RT-qPCR of representative SSU and LSU mt rRNA upon protein KD.

(DOCX)</p