UC Riverside Metabolomics Core - Karine Le Roch LAB

Functional genomics of RAP proteins and their role in mitoribosome regulation in Plasmodium falciparum.
  • Organism: Plasmodium falciparum
  • Instrument: Xevo TQ-XS,Xevo G2-XS Tof
  • SpikeIn: No
  • Keywords: Malaria; Plasmodium; RAP; RNA-binding proteins
  • Lab head: Karine Le Roch Submitter: Karine Le Roch
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.
Experiment Description
Tightly synchronized parasites (5 x10^9 parasites at 56 h and 9 x10^8 parasites at 72 h and 80 h), were lysed with saponin, flash frozen and stored at -80°C. Lipids and polar metabolites were extracted from malaria pellets using a biphasic approach. To each sample, 1 mL of ice cold 3:2 methyl tert-butyl ether:80% methanol was added. To break up malaria pellets, samples were vortexed 2 min, sonicated for 15 min, vortexed for 2 min, sonicated for 15 min, then vortexed for 30 min at 4°C. All sonication was performed in an ice bath. 200 µL of water was added to induce phase separation, followed by a 5 min vortex. After centrifugation for 15 min at 4 °C at 16,000 x g, 200 µL of the top, nonpolar layer was transferred to a 2 mL glass vial and the bottom, polar layer was transferred to a new 2 mL glass vial then analyzed by LC-MS. The nonpolar fraction was dried under a gentle stream of nitrogen at room temperature then resuspended in 400 µL of 9:1 methanol:toluene and analyzed by LC-MS. LC-MS Lipidomics LC-MS lipidomics analysis was performed at the UC Riverside Metabolomics Core Facility as described previously61, with minor modifications. Briefly, analysis was performed on a Waters G2-XS quadrupole time-of-flight mass spectrometer coupled to a Waters Acquity I-class UPLC system. Separations were carried out on a Waters CSH C18 column (2.1 x 100 mm, 1.7 µM). The mobile phases were (A) 60:40 acetonitrile:water with 10 mM ammonium formate and 0.1% formic acid and (B) 90:10 isopropanol:acetonitrile with 10 mM ammonium formate and 0.1% formic acid. The flow rate was 400 µL/min and the column was held at 65 ᴼC. The injection volume was 2 µL. The gradient was as follows: 0 min, 10% B; 1 min, 10% B; 3 min, 20% B; 5 min, 40% B; 16 min, 80% B; 18 min, 99% B; 20 min 99% B; 20.5 min, 10% B. The MS was scan range was (50 to 1600 m/z) with a 100 ms scan time. MS/MS was acquired in data dependent fashion. Source and desolvation temperatures were 150 ᴼC and 600 ᴼC, respectively. Desolvation gas was set to 1100 L/hr and cone gas to 150 L/hr. All gases were nitrogen except the collision gas, which was argon. Capillary voltage was 1 kV in positive ion mode. A quality control sample, generated by pooling equal aliquots of each sample, was analyzed periodically to monitor system stability and performance. Samples were analyzed in random order. Leucine enkephalin was infused and used for mass correction. LC-MS Metabolomics – polar metabolites Targeted metabolomics of polar, primary metabolites was performed on a TQ-XS triple quadrupole mass spectrometer (Waters) coupled to an I-class UPLC system (Waters). Separations were carried out on a ZIC-pHILIC column (2.1 x 150 mm, 5 µM) (EMD Millipore, 150460). The mobile phases were (A) water with 15 mM ammonium bicarbonate adjusted to pH 9.6 with ammonium hydroxide and (B) acetonitrile. The flow rate was 200 µL/min and the column was held at 50°C. The injection volume was 2 µL. The gradient was as follows for PfRAP01: 0 min, 90% B; 1.5 min, 90% B; 16 min, 20% B; 18 min, 20% B; 20 min, 90% B; 28 min, 90% B, and as follows for PfRAP21: 0 min, 90% B; 1.5 min, 90% B; 16 min, 10% B; 18 min, 10% B; 20 min, 90% B; 28 min, 90% B. The MS was operated in selected reaction monitoring mode62. Source and desolvation temperatures were 150°C and 600°C respectively. Desolvation gas was set to 1100 L/hr and cone gas to 150 L/hr. Collision gas was set to 0.15 mL/min. All gases were nitrogen except the collision gas, which was argon. Capillary voltage was 1 kV in positive ion mode and 2 kV in negative ion mode. System stability was monitored by analyzing a quality control sample (generated by pooling together equal volumes of all sample extracts) every 3 injections. Samples were analyzed in random order.
Sample Description
Synchronized rings of Plasmodium falciparum were cultured in the presence or absence of aTc and triplicate samples were collected at 56 h, 72 h, and 80 h in the second cycle, corresponding to just before and during the death phenotype. In the skyline and raw data, PfRAP01 and PfRAP21 may be annotated as protein 01 and 14, respectively.
Created on 2/3/22, 10:08 AM
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20210927_LeRoch Karine Batch 2 PfRAP01_72_80hrs_2022-02-05_18-12-10.sky.zip2022-02-07 17:24:141011511922216
20210926 LeRoch Karine Batch-1 PfRAP21_72_80hrs_2022-02-05_18-11-03.sky.zip2022-02-07 17:24:0910767614516
20190729Malaria_Protein_PfRAP01_and_PfRAP21_56hrs_pos_2022-02-05_18-10-29.sky.zip2022-02-07 17:24:04650656512720
20190729Malaria_Protein_PfRAP01_and_PfRAP21_56hrs_neg_2022-02-05_18-09-41.sky.zip2022-02-07 17:24:0330037375820
20190726Malaria_Protein_PfRAP01_56hrs_pos_2022-02-05_18-08-46.sky.zip2022-02-07 17:24:01720727214110
20190726Malaria_Protein_PfRAP01_56hrs_neg_2022-02-05_18-07-24.sky.zip2022-02-07 17:24:0032039396210