Analyzing RNA Localization Using the RNA Proximity Labeling Method OINC-seq

Abstract

Thousands of RNAs are localized to specific subcellular locations, and these localization patterns are often required for optimal cell function. However, the sequences within RNAs that direct their transport are unknown for almost all localized transcripts. Similarly, the RNA content of most subcellular locations remains unknown. To facilitate the study of subcellular transcriptomes, we developed the RNA proximity labeling method OINC-seq. OINC-seq utilizes photoactivatable, spatially restricted RNA oxidation to specifically label RNA in proximity to a subcellularly localized bait protein. After labeling, these oxidative RNA marks are then read out via high-throughput sequencing due to their ability to induce predictable misincorporation events by reverse transcriptase. These induced mutations are then quantitatively assessed for each gene using our software package PIGPEN. The observed mutation rate for a given RNA species is therefore related to its proximity to the localized bait protein. This protocol describes procedures for assaying RNA localization via OINC-seq experiments as well as computational procedures for analyzing the resulting data using PIGPEN. Key features • OINC-seq assays the RNA content of a variety of subcellular locations. • OINC-seq utilizes a photoactivatable, proximity-dependent RNA oxidation reaction to label RNAs. • Oxidative RNA marks are read using high-throughput sequencing without the need for enrichment. • Oxidative RNA marks are identified and quantified using the associated PIGPEN software.

Keywords: Proximity labeling; RNA localization; RNA modifications; RNA oxidation; RNA trafficking.

Figure 1.. Visualization of HaloTag localization with fluorescence microscopy.

(A) A max projected image of HeLa cells expressing cytoplasmically localized HaloTag-P65. Nuclei are counterstained with DAPI. Scale bar = 10 μm. (B) Max projected image of HeLa cells expressing nuclear-localized HaloTag-H2B. Nuclei are counterstained with DAPI. Scale bar = 10 μm.

Figure 2.. Confirmation of HaloTag fusion protein generation.

Two HeLa cell lines expressing doxycycline-inducible HaloTag fusion proteins are visualized on a protein gel. Total protein load is visualized with Coomassie blue (left). Appendage of a HaloTag to the protein of interest is visualized with a fluorescent band (right) at the expected molecular weight of the protein plus the HaloTag (~33 kDa). Fluorescent bands are visible in a doxycycline-inducible manner for the HaloTag fusion proteins P65 (MW = 65 kDa) and ATP5MC1 (MW = 14 kDa).

Figure 3.. Light-induced labeling setup.

(A) Place cell dishes on the bottom light panel and remove the lids. Three 10 cm dishes are shown. (B) Place the second light panel on top of the dishes and turn on the green lights. Protect the cells from external light by closing the cabinet door.