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PMID: 29636419 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

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

Proceedings of the National Academy of Sciences of the United States of America ·Vol. 115 ·No. 17 ·2018-00-24 ·Pages E3879-E3887

Huang R, Han M, Meng L, Chen X

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.

Keywords
RNA RNA–protein interactions bioorthogonal chemistry noncoding RNA proteomics
MeSH Terms
Gene Expression Profiling HEK293 Cells HeLa Cells Humans RNA-Binding Proteins/genetics,metabolism Transcriptome/physiology
Chemicals
RNA-Binding Proteins
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Huang Rongbing
College of Chemistry and Molecular Engineering, Peking University, 100871 Beijing, China. | Beijing National Laboratory for Molecular Sciences, 100871 Beijing, China.
Han Mengting
College of Chemistry and Molecular Engineering, Peking University, 100871 Beijing, China. | Beijing National Laboratory for Molecular Sciences, 100871 Beijing, China.
Meng Liying
College of Chemistry and Molecular Engineering, Peking University, 100871 Beijing, China. | Peking-Tsinghua Center for Life Sciences, Peking University, 100871 Beijing, China.
Chen Xing ORCID
College of Chemistry and Molecular Engineering, Peking University, 100871 Beijing, China; [email protected]. | Beijing National Laboratory for Molecular Sciences, 100871 Beijing, China. | Peking-Tsinghua Center for Life Sciences, Peking University, 100871 Beijing, China. | Synthetic and Functional Biomolecules Center, Peking University, 100871 Beijing, China. | Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, Peking University, 100871 Beijing, China.
Conflict of Interest

The authors declare no conflict of interest.

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Article Info
Journal
Proceedings of the National Academy of Sciences of the United States of America
Abbr.
Proc Natl Acad Sci U S A
ISSN
1091-6490
DOI
10.1073/pnas.1718406115
Email
[email protected]
Published
2018-00-24
Epub
2018-00-10
Pages
E3879-E3887
Language
English
Region
United States
NLM ID
7505876
PMCID
PMC5924899
Subset
IM
Corrections
CommentIn
PMID: 29855583
External Links
PubMed source DOI: 10.1073/pnas.1718406115 PMC Full Text View journal details
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