Profiling of nucleus localized RNAs by HoeDBF-seq

Abstract

<p>In eukaryotic cells, RNAs can be recruited to various cell compartments to perform their functions. Thus, RNA subcellular localization plays a vital role in maintaining normal biological functions. Various kinds of methods have been developed to study RNA subcellular localization including proximity labeling. Proximity labeling takes advantage of modified enzymes or artificial catalysts to generate activated intermediates, which then spread to the surroundings and react with proximal biomolecules including RNAs to realize proximity labeling (Choi and Rhee 2022). Although many proximity labeling methods have been developed, only a few were applied to RNA localization, including APEX-seq, Cap-seq and Halo-seq. However, all these methods need to express fusion proteins in cells to direct the modified enzymes into locations of interest and generating cell lines stably expression these fusions, which is strenuous. Furthermore, the fusion proteins stably expressed in cells may influence some biological process of them. Here, we developed a novel and convenient small molecule-based proximity labeling approach, HoeDBF-seq, which can be used to characterize nucleus-localized RNAs without expressing fusion proteins in cells (Li, Liang et al. 2019). HoeDBF is a small molecule that combine a bisbenzimidazole dye, Hoechst 33342, with a dibromofluorescein (DBF) moiety via a PEG linker. Hoechst 33342 serves as a nucleus-targeting moiety because of its high affinity to double-strand DNAs in nucleus and thus the DBF moiety can be guided into nucleus without introducing fusion constructs into cells. In nucleus, upon green light irradiation, DBF can generate singlet oxygen by which guanosines in proximal RNAs can be oxidized to 8-oxoguanosine (8oxoG) (Li, Aggarwal et al. 2017). As 8oxoG is susceptible to attack by a nucleophile propargyl amine (PA) because of its higher oxidation state (Li, Aggarwal et al. 2017), proximity RNAs can be alkylated, i.e. labeled. Next, via copper(I)-catalyzed azide alkyne cycloaddition (CuAAC), biotins can be ligated onto alkylated RNAs. Thus, biotinylated RNAs, i.e. labeled RNAs, can be enriched by streptavidin-pulldown followed by RNA-seq for nuclear transcriptome profiling.</p>