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

Proteomic profiling of RNA-binding proteins in 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 life cycle stages. Eight out of 20 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 non-poly(A)-RNA-interactome, thus uncovering potential involvement of this protein domain in RNA-protein interactions in . 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 protein kinome. Additionally, we characterize the quantitative changes in RNA-protein interactions in hundreds of proteins following inhibition of heat shock protein 90 (Hsp90). Our results show that the Hsp90 inhibition in 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 . This study defines the comprehensive RNA interactome of and provides in-depth insight into the widespread involvement of RNA-protein interactions in biology. Advances in proteomics and mass spectrometry have revealed the mRNA-binding proteins in many eukaryotic organisms, including the protozoan parasites spp., the causative agents of leishmaniasis, a major infectious disease in over 90 tropical and subtropical countries. However, in addition to mRNAs, which constitute only 2 to 5% of the total transcripts, many types of non-coding RNAs participate in crucial biological processes. In , RNA-binding proteins serve as primary gene regulators. Therefore, transcri