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Transcriptome-wide identification of coding and noncoding RNA-binding proteins defines the extended RNA interactome of Leishmania mexicana

ABSTRACT: Non-poly(A) RNA molecules including noncoding RNAs (ncRNAs) comprise the major portion of the total transcribed molecules in the cell. In addition to the mRNAs the ncRNAs also function as ribonucleoprotein particles (RNPs) and carry out biological functions including synthesis of new proteins, RNA processing, genome remodelling and regulation of transcription. We therefore envisaged a comprehensive transcriptome-wide identification of coding and non-coding RNA-binding proteins (RBPs) in the Leishmania spp. Towards this we applied the recently reported orthogonal organic phase separation (OOPS) method in combination with tandem mass tag (TMT) labelling-based quantitative proteomic mass spectrometry (MS) and report herein the most comprehensive identification of RBPs in Leishmania mexicana (L. mexicana) parasites. This study identified novel RNA binding property of thousands of L. mexicana proteins, significantly expanding the RBP landscape of the parasite. Furthermore, we showed that the classical Hsp90 inhibitor tanespimycin differentially regulates the RNA-binding property of hundreds of L. mexicana RBPs, shedding light into hitherto unknown large-scale downstream molecular effects of the small molecule inhibitor in the parasite.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Leishmania Mexicana Mhom/gt/2001/u1103

SUBMITTER: Kalesh Karunakaran

LAB HEAD: Kalesh Karunakaran

PROVIDER: PXD028092 | Pride | 2022-01-14

REPOSITORIES: Pride

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Project description:The general role of RNA in the cell beyond protein biosynthesis is only beginning to emerge with little knowledge about the structural or organizational functions of RNA. In turn, functional non-coding RNAs (ncRNAs) are often identified by their regulation or impact on cellular phenotypes. However, the huge number and diversity of ncRNAs highly complicate the task of their molecular characterization and rationalization into cellular processes. For the vast majority of ncRNAs as well as for non-coding functions of known mRNAs, the mechanisms underlying their modes of action as well as their impact on protein complexes are unknown. Thus, the greatest challenge in the field of RNA biology today is the elucidation of molecular mechanisms and functional interaction partners of RNA molecules. Since RNAs do not act alone, but often interact with specific protein partners, proteomic approaches have been developed to study RNA-binding proteins (RBPs) and aim at revealing the molecular mechanisms underlying RNA function. Large-scale studies aiming at globally identifying RNA-binding proteins principally focused on poly(A)-RNA transcripts. A notable limitation of such approaches is that many ncRNAs are not polyadenylated, so that their interaction partners were not detected in these screens. Most importantly however, the overlap between the numerous studies aiming to identify RBPs is limited and their specificity remains unclear. Moreover, the previous studies were based on the identification of RBPs omitting their participation in protein-protein complexes and more interestingly in RNA-dependent protein-protein complexes. Here, we introduce the concept of "RNA dependence" to overcome these challenges. We define a protein as RNA-dependent if its interactome (hence likely its function) depends on RNA without necessarily directly binding to RNA. Based on this new concept, we developed a proteome-wide screening approach to gain mechanistic insight into the function of RNAs - both coding and non-coding - in RNA-protein complexes and their impact on the function of such complexes.

			Action	DRS
	Ama_RBP_TMT6plex.raw	Raw
	Ama_Tan_RBP_TMT6plex.raw	Raw
	Proma_RBP_TMT6plex.raw	Raw
	Proma_Tan_RBP_TMT6plex.raw	Raw
	checksum.txt	Txt

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Transcriptome-wide identification of coding and noncoding RNA-binding proteins defines the extended RNA interactome of Leishmania mexicana

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