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Transcriptome-Wide Identification of Coding and Noncoding RNA-Binding Proteins Defines the Comprehensive RNA Interactome of Leishmania mexicana

Kalesh, K.; Wei, W.; Mantilla, B.S.; Roumeliotis, T.I.; Choudhary, J.; Denny, P.W.

Transcriptome-Wide Identification of Coding and Noncoding RNA-Binding Proteins Defines the Comprehensive RNA Interactome of Leishmania mexicana Thumbnail


Authors

K. Kalesh

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Dr Wenbin Wei wenbin.wei2@durham.ac.uk
Chief Experimental Officer (Bioinformatics)

B.S. Mantilla

T.I. Roumeliotis

J. Choudhary



Abstract

Proteomic profiling of RNA-binding proteins in Leishmania is currently limited to polyadenylated mRNA-binding proteins, leaving proteins that interact with nonadenylated RNAs, including noncoding RNAs and pre-mRNAs, unidentified. Using a combination of unbiased orthogonal organic phase separation methodology and tandem mass tag-labeling-based high resolution quantitative proteomic mass spectrometry, we robustly identified 2,417 RNA-binding proteins, including 1289 putative novel non-poly(A)-RNA-binding proteins across the two main Leishmania life cycle stages. Eight out of 20 Leishmania deubiquitinases, including the recently characterized L. mexicana DUB2 with an elaborate RNA-binding protein interactome were exclusively identified in the non-poly(A)-RNA-interactome. Additionally, an increased representation of WD40 repeat domains were observed in the Leishmania non-poly(A)-RNA-interactome, thus uncovering potential involvement of this protein domain in RNA-protein interactions in Leishmania. We also characterize the protein-bound RNAs using RNA-sequencing and show that in addition to protein coding transcripts ncRNAs are also enriched in the protein-RNA interactome. Differential gene expression analysis revealed enrichment of 142 out of 195 total L. mexicana protein kinase genes in the protein-RNA-interactome, suggesting important role of protein-RNA interactions in the regulation of the Leishmania protein kinome. Additionally, we characterize the quantitative changes in RNA-protein interactions in hundreds of Leishmania proteins following inhibition of heat shock protein 90 (Hsp90). Our results show that the Hsp90 inhibition in Leishmania causes widespread disruption of RNA-protein interactions in ribosomal proteins, proteasomal proteins and translation factors in both life cycle stages, suggesting downstream effect of the inhibition on protein synthesis and degradation pathways in Leishmania. This study defines the comprehensive RNA interactome of Leishmania and provides in-depth insight into the widespread involvement of RNA-protein interactions in Leishmania biology.

Citation

Kalesh, K., Wei, W., Mantilla, B., Roumeliotis, T., Choudhary, J., & Denny, P. (2022). Transcriptome-Wide Identification of Coding and Noncoding RNA-Binding Proteins Defines the Comprehensive RNA Interactome of Leishmania mexicana. Microbiology Spectrum, 10(1), e02422-21. https://doi.org/10.1128/spectrum.02422-21

Journal Article Type Article
Acceptance Date Jan 13, 2022
Online Publication Date Feb 9, 2022
Publication Date 2022-02
Deposit Date Apr 2, 2022
Publicly Available Date Jul 5, 2022
Journal Microbiology Spectrum
Publisher American Society for Microbiology
Peer Reviewed Peer Reviewed
Volume 10
Issue 1
Article Number e0242221
Pages e02422-21
DOI https://doi.org/10.1128/spectrum.02422-21

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