Functional characterization of the m⁶A-dependent translational modulator PfYTH.2 in the human malaria parasite
Posttranscriptional regulation of gene expression is central to the development and replication of the malaria parasite, Plasmodium falciparum, within its human host. The timely coordination of RNA maturation, homeostasis, and protein synthesis relies on the recruitment of specific RNA-binding prote...
| Main Authors: | , , , , , , , , , , , , |
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| Format: | Journal Article |
| Language: | English |
| Published: |
2022
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| Online Access: | https://hdl.handle.net/10356/154051 |
| _version_ | 1811689211173011456 |
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| author | Sinha, Ameya Baumgarten, Sebastian Distiller, Amy McHugh, Emma Chen, Patty Singh, Meetali Bryant, Jessica M. Liang, Jiaqi Cecere, Germano Dedon, Peter C. Preiser, Peter Rainer Ralph, Stuart A. Scherf, Artur |
| author2 | School of Biological Sciences |
| author_facet | School of Biological Sciences Sinha, Ameya Baumgarten, Sebastian Distiller, Amy McHugh, Emma Chen, Patty Singh, Meetali Bryant, Jessica M. Liang, Jiaqi Cecere, Germano Dedon, Peter C. Preiser, Peter Rainer Ralph, Stuart A. Scherf, Artur |
| author_sort | Sinha, Ameya |
| collection | NTU |
| description | Posttranscriptional regulation of gene expression is central to the development and replication of the malaria parasite, Plasmodium falciparum, within its human host. The timely coordination of RNA maturation, homeostasis, and protein synthesis relies on the recruitment of specific RNA-binding proteins to their cognate target mRNAs. One possible mediator of such mRNA-protein interactions is the N6-methylation of adenosines (m6A), a prevalent mRNA modification of parasite mRNA transcripts. Here, we used RNA protein pulldowns, RNA modification mass spectrometry, and quantitative proteomics to identify two P. falciparum YTH domain proteins (PfYTH.1 and PfYTH.2) as m6A-binding proteins during parasite blood-stage development. Interaction proteomics revealed that PfYTH.2 associates with the translation machinery, including multiple subunits of the eukaryotic initiation factor 3 (eIF3) and poly(A)-binding proteins. Furthermore, knock sideways of PfYTH.2 coupled with ribosome profiling showed that this m6A reader is essential for parasite survival and is a repressor of mRNA translation. Together, these data reveal an important missing link in the m6A-mediated mechanism controlling mRNA translation in a unicellular eukaryotic pathogen.IMPORTANCE Infection with the unicellular eukaryotic pathogen Plasmodium falciparum causes malaria, a mosquito-borne disease affecting more than 200 million and killing 400,000 people each year. Underlying the asexual replication within human red blood cells is a tight regulatory network of gene expression and protein synthesis. A widespread mechanism of posttranscriptional gene regulation is the chemical modification of adenosines (m6A), through which the fate of individual mRNA transcripts can be changed. Here, we report on the protein machinery that "reads" this modification and "translates" it into a functional outcome. We provide mechanistic insight into one m6A reader protein and show that it interacts with the translational machinery and acts as a repressor of mRNA translation. This m6A-mediated phenotype has not been described in other eukaryotes as yet, and the functional characterization of the m6A interactome will ultimately open new avenues to combat the disease. |
| first_indexed | 2024-10-01T05:44:29Z |
| format | Journal Article |
| id | ntu-10356/154051 |
| institution | Nanyang Technological University |
| language | English |
| last_indexed | 2024-10-01T05:44:29Z |
| publishDate | 2022 |
| record_format | dspace |
| spelling | ntu-10356/1540512023-02-28T17:10:05Z Functional characterization of the m⁶A-dependent translational modulator PfYTH.2 in the human malaria parasite Sinha, Ameya Baumgarten, Sebastian Distiller, Amy McHugh, Emma Chen, Patty Singh, Meetali Bryant, Jessica M. Liang, Jiaqi Cecere, Germano Dedon, Peter C. Preiser, Peter Rainer Ralph, Stuart A. Scherf, Artur School of Biological Sciences Singapore-MIT Alliance for Research and Technology Science::Biological sciences Plasmodium Falciparum m6A mRNA Methylation Posttranscriptional regulation of gene expression is central to the development and replication of the malaria parasite, Plasmodium falciparum, within its human host. The timely coordination of RNA maturation, homeostasis, and protein synthesis relies on the recruitment of specific RNA-binding proteins to their cognate target mRNAs. One possible mediator of such mRNA-protein interactions is the N6-methylation of adenosines (m6A), a prevalent mRNA modification of parasite mRNA transcripts. Here, we used RNA protein pulldowns, RNA modification mass spectrometry, and quantitative proteomics to identify two P. falciparum YTH domain proteins (PfYTH.1 and PfYTH.2) as m6A-binding proteins during parasite blood-stage development. Interaction proteomics revealed that PfYTH.2 associates with the translation machinery, including multiple subunits of the eukaryotic initiation factor 3 (eIF3) and poly(A)-binding proteins. Furthermore, knock sideways of PfYTH.2 coupled with ribosome profiling showed that this m6A reader is essential for parasite survival and is a repressor of mRNA translation. Together, these data reveal an important missing link in the m6A-mediated mechanism controlling mRNA translation in a unicellular eukaryotic pathogen.IMPORTANCE Infection with the unicellular eukaryotic pathogen Plasmodium falciparum causes malaria, a mosquito-borne disease affecting more than 200 million and killing 400,000 people each year. Underlying the asexual replication within human red blood cells is a tight regulatory network of gene expression and protein synthesis. A widespread mechanism of posttranscriptional gene regulation is the chemical modification of adenosines (m6A), through which the fate of individual mRNA transcripts can be changed. Here, we report on the protein machinery that "reads" this modification and "translates" it into a functional outcome. We provide mechanistic insight into one m6A reader protein and show that it interacts with the translational machinery and acts as a repressor of mRNA translation. This m6A-mediated phenotype has not been described in other eukaryotes as yet, and the functional characterization of the m6A interactome will ultimately open new avenues to combat the disease. Ministry of Education (MOE) National Research Foundation (NRF) Singapore-MIT Alliance for Research and Technology (SMART) Published version This work was supported by a European Research Council Advanced Grant (PlasmoSilencing 670301) and the French Parasitology consortium ParaFrap (ANR-11- LABX0024) awarded to A. Scherf. S.B. is supported by an EMBO Advanced Long-Term Fellowship (aALTF 632-2018). P.R.P., P.C.D., J.L., and A. Sinha were supported by the National Research Foundation Singapore under its Singapore-MIT Alliance for Research and Technology (SMART) Centre, Antimicrobial Resistance IRG and Singapore Ministry of Education Academic Research Fund Tier 2 (MOE2018-T2-2-131). A. Sinha and J.L. acknowledge support from the Singapore-MIT Alliance (SMA) Graduate Fellowship. Proteomics work was performed in part in the Center for Environmental Health Sciences BioCore, which is supported by Center grant P30-ES002109 from the National Institute of Environmental Health Sciences. We also acknowledge Peiying Ho and Tan Tse Mien for providing support at SMART laboratories in Singapore. G.C. and M.S. were funded by a European Research Council (ERC) Starting Grant (ERC-StG-679243). Work on this project in the laboratory of S.A.R. is funded by a grant from the Australian National Health and Medical Research Council (1165354) and by a grant from the Australian Research Council (DP160100389). 2022-02-09T08:34:46Z 2022-02-09T08:34:46Z 2021 Journal Article Sinha, A., Baumgarten, S., Distiller, A., McHugh, E., Chen, P., Singh, M., Bryant, J. M., Liang, J., Cecere, G., Dedon, P. C., Preiser, P. R., Ralph, S. A. & Scherf, A. (2021). Functional characterization of the m⁶A-dependent translational modulator PfYTH.2 in the human malaria parasite. MBio, 12(2), e00661-21-. https://dx.doi.org/10.1128/mBio.00661-21 2150-7511 https://hdl.handle.net/10356/154051 10.1128/mBio.00661-21 33906926 2-s2.0-85104700403 2 12 e00661-21 en MOE2018-T2-2-131 mBio © 2021 Sinha et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license. application/pdf |
| spellingShingle | Science::Biological sciences Plasmodium Falciparum m6A mRNA Methylation Sinha, Ameya Baumgarten, Sebastian Distiller, Amy McHugh, Emma Chen, Patty Singh, Meetali Bryant, Jessica M. Liang, Jiaqi Cecere, Germano Dedon, Peter C. Preiser, Peter Rainer Ralph, Stuart A. Scherf, Artur Functional characterization of the m⁶A-dependent translational modulator PfYTH.2 in the human malaria parasite |
| title | Functional characterization of the m⁶A-dependent translational modulator PfYTH.2 in the human malaria parasite |
| title_full | Functional characterization of the m⁶A-dependent translational modulator PfYTH.2 in the human malaria parasite |
| title_fullStr | Functional characterization of the m⁶A-dependent translational modulator PfYTH.2 in the human malaria parasite |
| title_full_unstemmed | Functional characterization of the m⁶A-dependent translational modulator PfYTH.2 in the human malaria parasite |
| title_short | Functional characterization of the m⁶A-dependent translational modulator PfYTH.2 in the human malaria parasite |
| title_sort | functional characterization of the m⁶a dependent translational modulator pfyth 2 in the human malaria parasite |
| topic | Science::Biological sciences Plasmodium Falciparum m6A mRNA Methylation |
| url | https://hdl.handle.net/10356/154051 |
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