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Transcriptome-wide discovery of coding and noncoding RNA-binding proteins.

ABSTRACT: Transcriptome-wide identification of RNA-binding proteins (RBPs) is a prerequisite for understanding the posttranscriptional gene regulation networks. However, proteomic profiling of RBPs has been mostly limited to polyadenylated mRNA-binding proteins, leaving RBPs on nonpoly(A) RNAs, including most noncoding RNAs (ncRNAs) and pre-mRNAs, largely undiscovered. Here we present a click chemistry-assisted RNA interactome capture (CARIC) strategy, which enables unbiased identification of RBPs, independent of the polyadenylation state of RNAs. CARIC combines metabolic labeling of RNAs with an alkynyl uridine analog and in vivo RNA-protein photocross-linking, followed by click reaction with azide-biotin, affinity enrichment, and proteomic analysis. Applying CARIC, we identified 597 RBPs in HeLa cells, including 130 previously unknown RBPs. These newly discovered RBPs can likely bind ncRNAs, thus uncovering potential involvement of ncRNAs in processes previously unknown to be ncRNA-related, such as proteasome function and intermediary metabolism. The CARIC strategy should be broadly applicable across various organisms to complete the census of RBPs.

SUBMITTER: Huang R

PROVIDER: S-EPMC5924899 | biostudies-other | 2018 Apr

REPOSITORIES: biostudies-other

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Transcriptome-wide discovery of coding and noncoding RNA-binding proteins.

Huang Rongbing R Han Mengting M Meng Liying L Chen Xing X

Proceedings of the National Academy of Sciences of the United States of America 20180410 17

Transcriptome-wide identification of RNA-binding proteins (RBPs) is a prerequisite for understanding the posttranscriptional gene regulation networks. However, proteomic profiling of RBPs has been mostly limited to polyadenylated mRNA-binding proteins, leaving RBPs on nonpoly(A) RNAs, including most noncoding RNAs (ncRNAs) and pre-mRNAs, largely undiscovered. Here we present a click chemistry-assisted RNA interactome capture (CARIC) strategy, which enables unbiased identification of RBPs, indepe ...[more]

PMID: 29636419

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Project description: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. IMPORTANCE Advances in proteomics and mass spectrometry have revealed the mRNA-binding proteins in many eukaryotic organisms, including the protozoan parasites Leishmania 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 Leishmania, 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 Leishmania. 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 Leishmania life cycle stages. In addition, we characterize for the first time the quantitative changes in RNA-protein interactions in Leishmania 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 Leishmania, thus significantly expanding our knowledge of the emergence of RNA-protein interactions in Leishmania biology.

Dataset Information

Transcriptome-wide discovery of coding and noncoding RNA-binding proteins.

Publications

Transcriptome-wide discovery of coding and noncoding RNA-binding proteins.

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