Spatiotemporal mapping of RNA editing in the developing mouse brain using in situ sequencing reveals regional and cell-type-specific regulation

Elin Lundin,Mats Nilsson,Chenglin Wu,Albin Widmark,Marie Öhman,Jens Hjerling-Leffler,Chammiran Daniel,Mikaela Behm

doi:10.1186/s12915-019-0736-3

Abstract

BackgroundAdenosine-to-inosine (A-to-I) RNA editing is a process that contributes to the diversification of proteins that has been shown to be essential for neurotransmission and other neuronal functions. However, the spatiotemporal and diversification properties of RNA editing in the brain are largely unknown. Here, we applied in situ sequencing to distinguish between edited and unedited transcripts in distinct regions of the mouse brain at four developmental stages, and investigate the diversity of the RNA landscape.ResultsWe analyzed RNA editing at codon-altering sites using in situ sequencing at single-cell resolution, in combination with the detection of individual ADAR enzymes and specific cell type marker transcripts. This approach revealed cell-type-specific regulation of RNA editing of a set of transcripts, and developmental and regional variation in editing levels for many of the targeted sites. We found increasing editing diversity throughout development, which arises through regional- and cell type-specific regulation of ADAR enzymes and target transcripts.ConclusionsOur single-cell in situ sequencing method has proved useful to study the complex landscape of RNA editing and our results indicate that this complexity arises due to distinct mechanisms of regulating individual RNA editing sites, acting both regionally and in specific cell types.

Highlights

Adenosine-to-inosine (A-to-I) RNA editing is a process that contributes to the diversification of proteins that has been shown to be essential for neurotransmission and other neuronal functions
Study design and rational We studied the pattern of A-to-I RNA editing during development, by analyzing coronal sections from brain tissues from E15, P0, P7, and adult mice (n = 5) using in situ sequencing (ISS)
Each edited position was targeted by a pair of Padlock probes (PLPs), with one PLP targeting the cDNA of the unedited transcript and the other targeting the cDNA of the edited transcript (Fig. 1a)

Summary

Introduction

Adenosine-to-inosine (A-to-I) RNA editing is a process that contributes to the diversification of proteins that has been shown to be essential for neurotransmission and other neuronal functions. This results in an auto-regulatory loop of ADAR2, where high ADAR2 activity will decrease the expression of active ADAR2 protein until a balance is reached [4, 15] In addition to this auto-regulatory loop, trans-acting regulators of RNA editing have been identified, whose expression likely contributes to temporal, cell type-specific and tissue-specific RNA editing patterns [16]. These include regulators of ADAR expression and stability [14, 17], subcellular localization [18], activity [19], and regulators of editing at specific sites [20]. Given these many mechanisms for regulating editing levels, generally or for specific substrates, there are many ways to generate a diverse editing landscape

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Discussion

Conclusion