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, transcriptome-wide identification of RNA-binding proteins is necessary for deciphering the distinctive posttranscriptional mechanisms of gene regulation in . Using a combination of highly efficient orthogonal organic phase separation method and tandem mass tag-labeling-based quantitative proteomic mass spectrometry, we provide unprecedented comprehensive molecular definition of the total RNA interactome across the two main life cycle stages. In addition, we characterize for the first time the quantitative changes in RNA-protein interactions in following inhibition of heat shock protein 90, shedding light into hitherto unknown large-scale downstream molecular effect of the protein inhibition in the parasite. This work provides insight into the importance of total RNA-protein interactions in , thus significantly expanding our knowledge of the emergence of RNA-protein interactions in biology.