Analysis of subcellular transcriptomes by RNA proximity labeling with Halo-seq

Author:

Engel Krysta L1,Lo Hei-Yong G12,Goering Raeann12,Li Ying3,Spitale Robert C45ORCID,Taliaferro J Matthew12ORCID

Affiliation:

1. Department of Biochemistry and Molecular Genetics, University of Colorado Anschutz Medical Campus, Aurora, CO, USA

2. RNA Bioscience Initiative, University of Colorado Anschutz Medical Campus, Aurora, CO, USA

3. Department of Chemistry, Hong Kong University

4. Department of Pharmaceutical Sciences, University of California Irvine, Irvine, CA, USA

5. Department of Chemistry, University of California Irvine, Irvine, CA, USA

Abstract

Abstract Thousands of RNA species display nonuniform distribution within cells. However, quantification of the spatial patterns adopted by individual RNAs remains difficult, in part by a lack of quantitative tools for subcellular transcriptome analysis. In this study, we describe an RNA proximity labeling method that facilitates the quantification of subcellular RNA populations with high spatial specificity. This method, termed Halo-seq, pairs a light-activatable, radical generating small molecule with highly efficient Click chemistry to efficiently label and purify spatially defined RNA samples. We compared Halo-seq with previously reported similar methods and found that Halo-seq displayed a higher efficiency of RNA labeling, indicating that it is well suited to the investigation of small, precisely localized RNA populations. We then used Halo-seq to quantify nuclear, nucleolar and cytoplasmic transcriptomes, characterize their dynamic nature following perturbation, and identify RNA sequence features associated with their composition. Specifically, we found that RNAs containing AU-rich elements are relatively enriched in the nucleus. This enrichment becomes stronger upon treatment with the nuclear export inhibitor leptomycin B, both expanding the role of HuR in RNA export and generating a comprehensive set of transcripts whose export from the nucleus depends on HuR.

Funder

National Institutes of Health

University of Colorado Anschutz Medical Campus

Predoctoral Training Grant in Molecular Biology

Publisher

Oxford University Press (OUP)

Subject

Genetics

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