Summary
Chemical modification on mRNA can recruit specific binding proteins (readers/partners) to determine post-transcriptional gene regulation. However, the identification of the reader is extremely limited owing to the rather weak and highly dynamic non-covalent interactions between mRNA modification and reader, and therefore the sensitive and robust approaches are desirable. Here, we report a DNA-guided photoactivatable-based chemical proteomic approach for profiling the readers of mRNA methylation. By use of N
6
-methyladenosine (m
6
A), we illustrated that this method can be successfully utilized for labelling and enriching the readers of mRNA modification, as well as for the discovery of new partners. Thus we applied this strategy to a new modification 2′-O-methyladenosine. As a result, DDX1 was identified and verified as a potential binding protein. Our study therefore provides a powerful chemical proteomics tool for identifying the binding factors of mRNA modification and reveals the underlying function of mRNA modification.
Anaphylaxis due to autoimmune progesterone dermatitis is a rare but severe allergic disease in women. The clinical manifestations of APD are diverse, and a proper understanding of the disease can help even diagnose and treat it. A case of autoimmune progesterone dermatitis related in our department is reported as follows. She developed a rash with severe pruritus that was highly consistent with her menstrual cycle without any trigger 10 years ago. Laboratory tests were unremarkable. But all the symptoms disappeared during her pregnancy and resurfaced after the miscarriage. Two years ago, after a positive progesterone intradermal test confirmed the diagnosis of PH, she was given mifepristone, contraceptives(OCPs), and skin embedding treatment, and her symptoms improved.
Deciphering the endogenous interactors of histone post-translational modifications (hPTMs, also called histone marks) is essential to understand the mechanisms of epigenetic regulation. However, most of the analytical methods to determine hPTM interactomes are in vitro settings, lacking interrogating native chromatin. Although lysine crotonylation (Kcr) has recently been considered an important hPTM for the regulation of gene transcription, the interactors of Kcr still remain to be explored. Herein, we present a general approach relying upon a genetic code expansion system, APEX2 (engineered peroxidase)-mediated proximity labeling, and quantitative proteomics to profile interactomes of the selected hPTMs in living cells. We genetically fused APEX2 to the recombinant histone H3 with a crotonyl lysine inserted site specifically to generate APEX2− H3K9cr that incorporated into native chromatin. Upon activation, APEX2 triggered in vivo biotin labeling of H3K9cr interactors that can then be enriched with streptavidin beads and identified by mass spectrometry. Proteomic analysis further revealed the endogenous interactomes of H3K9cr and confirmed the reliability of the method. Moreover, DPF2 was identified as a candidate interactor, and the binding interaction of DPF2 to H3K9c was further characterized and verified. This study provides a novel strategy for the identification of hPTM interactomes in living cells, and we envision that this is key to elucidating epigenetic regulatory pathways.
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