Abstract
SummaryChemical 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 N6-methyladenosine (m6A), 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.
Highlights
The catalog of posttranscriptional chemical modifications on lowly expressed messenger RNA has recently been expanded greatly, including N6-methyladenosine (m6A), 5-methylcytosine (m5C), N1methyladenosine (m1A), pseudouridine (J), 5-hydroxymethylcytosine, 20-O-methylation (Nm) and so on (Boccaletto et al, 2018)
Analysis strategy and preparation of DNA-guided photoactivatable RNA probes To profile binding partners of mRNA modification, a dual probe is designed. (i) RNA oligonucleotide bearing mRNA modification acted as a recognizing group is conjugated to a single-stranded DNA to generate a ‘‘binding probe’’ (BP)
(ii) a ‘‘capture probe’’ (CP) is prepared by conjugating a photo-reactive group to another complementary single-stranded DNA that is modified by a biotin or fluorescein (FAM) for detection
Summary
The catalog of posttranscriptional chemical modifications on lowly expressed messenger RNA (mRNAs) has recently been expanded greatly, including N6-methyladenosine (m6A), 5-methylcytosine (m5C), N1methyladenosine (m1A), pseudouridine (J), 5-hydroxymethylcytosine (hm5C), 20-O-methylation (Nm) and so on (Boccaletto et al, 2018). For m6A readers, YTH domain family 1 (YTHDF1) increases mRNA translation by facilitating ribosome loading, whereas YTHDF2 locates bound mRNA to decay site, a different group of proteins, such as HNRNPC, prefer an ‘‘m6A-switch’’ (Liu et al, 2015a), which remodels local RNA structure and modulates RNA-protein interactions around or nearby. Identifying these binding factors will provide us a profound insight into our understanding of the fine-tune regulation by epigenetic modification temporally and spatially, and will contribute further to our explanation of the regulation mechanisms and physiological function of mRNA. It is still challenging to detect such interactions in transcriptomes, the sensitive and robust technologies are urgently desirable
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