한빛사 논문
Yongwoo Na1,2, Hyunjoon Kim1,2, Yeon Choi1,2, Sanghee Shin1,2, Jae Hun Jung3, S. Chul Kwon1,2, V. Narry Kim1,2,* and Jong-Seo Kim1,2,*
1Center for RNA Research, Institute for Basic Science, Seoul 08826, Korea,
2School of Biological Sciences, Seoul National University, Seoul 08826, Korea and
3Department of Applied Chemistry, Kyung Hee University, Yongin 17104, Korea
*To whom correspondence should be addressed
Abstract
RNA–protein interaction is central to post-transcriptional gene regulation. Identification of RNA-binding proteins relies mainly on UV-induced crosslinking (UVX) followed by the enrichment of RNA–protein conjugates and LC-MS/MS analysis. However, UVX has limited applicability in tissues of multicellular organisms due to its low penetration depth. Here, we introduce formaldehyde crosslinking (FAX) as an alternative chemical crosslinking for RNA interactome capture (RIC). Mild FAX captures RNA–protein interaction with high specificity and efficiency in cell culture. Unlike UVX-RIC, FAX-RIC robustly detects proteins that bind to structured RNAs or uracil-poor RNAs (e.g. AGO1, STAU1, UPF1, NCBP2, EIF4E, YTHDF proteins and PABP), broadening the coverage. Applied to Xenopus laevis oocytes and embryos, FAX-RIC provided comprehensive and unbiased RNA interactome, revealing dynamic remodeling of RNA–protein complexes. Notably, translation machinery changes during oocyte-to-embryo transition, for instance, from canonical eIF4E to noncanonical eIF4E3. Furthermore, using Mus musculus liver, we demonstrate that FAX-RIC is applicable to mammalian tissue samples. Taken together, we report that FAX can extend the RNA interactome profiling into multicellular organisms.
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