Hello everyone, this week I am sharing an article published in JACS titled “Proteomic Tracking Extracellular Vesicle RNA Interactors in Recipient Immune Cells through Orthogonal Labelings.” The corresponding author is Professor Andy Tao from Purdue University, whose research group focuses on the development and application of functional proteomics and mass spectrometry.
RNA-binding proteins (RBPs) are involved in mediating every stage of the life cycle. Extracellular vesicles (EVs) are secreted by almost all cells and can facilitate intercellular communication by delivering RNA. Currently, there is a lack of a high-throughput method for analyzing EV RBPs.
The authors propose a chemical proteomics strategy called ERICOL, which can selectively capture EV-derived RNA interacting proteins in recipient cells. Specifically, they use 4-thiouridine (4SU) to metabolically label RNA, then separate EVs by ultracentrifugation and incubate them with SILAC-labeled recipient cells. At different time points, they crosslink with UV light, purify RBPs through orthogonal organic phase separation, and ultimately identify them by releasing them with RNase.
The authors first validated that ERICOL can effectively capture EV RBPs in recipient cells (Jurkat T cells). They then co-incubated tumor-derived EVs (TEVs) with Jurkat T cells for different durations (1, 3, 5 hours), revealing the dynamic patterns of EV RNA uptake and RBP binding.
Finally, the authors applied ERICOL to analyze the changes in primary human CD8+ T cells after receiving wild-type (CCLP-EVs) or IDH1 mutant (RBE-EVs) intrahepatic cholangiocarcinoma (ICC)-derived EVs. The analysis suggests that IDH1 mutant EVs may suppress immune responses and promote T cell death. The authors also validated the specific interactions of key candidate RBPs (such as EP300, CNOT1, BRD3) with EV RNA through RNA immunoprecipitation (RIP).
In summary, this article developed ERICOL and achieved dynamic analysis of EV RNA interacting proteins (RBPs) at the whole proteome level in recipient cells.
Author: YDP
Editor: LYC
DOI: 10.1021/jacs.5c07631
Original link: https://doi.org/10.1021/jacs.5c07631
