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Spatiotemporal mapping of RNA editing in the developing mouse brain using in situ sequencing reveals regional and cell-type-specific regulation
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).ORCID iD: 0000-0002-4827-0208
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).ORCID iD: 0000-0002-0635-1122
Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.ORCID iD: 0000-0002-0362-923X
Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.ORCID iD: 0000-0002-3272-1377
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Background: 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. 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.

Results: We 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.

Conclusions: Our 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.

Keywords [en]
Single cell resolution, RNA editing, spatially resolved transcriptomics, brain development
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-177099OAI: oai:DiVA.org:su-177099DiVA, id: diva2:1379455
Funder
Swedish Research CouncilSwedish Cancer SocietyAvailable from: 2019-12-17 Created: 2019-12-17 Last updated: 2019-12-17Bibliographically approved
In thesis
1. RNA-based spatial characterization of cell and tissue heterogeneity
Open this publication in new window or tab >>RNA-based spatial characterization of cell and tissue heterogeneity
2020 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Technical advances in cell biology have revolutionized the field of cell biology. With new technology it is now possible to address scientific questions in cell biology at the molecular level. Single-cell RNA-sequencing can reveal transcriptomic information for single cells and spatially resolved transcriptomic technology can visualize thousands or millions of cells and transcripts for spatial molecular profiling. The work in this thesis describes the technological development from traditional in situ hybridization to the current state-of-the-art technology for spatial multiplexed gene expression analysis. This development has enabled RNA-based molecular characterization of cells and tissues with the spatial dimension maintained. The work included in the thesis highlights the potential and the advantages of padlock-probe-based technology for spatial RNA-based profiling of cells and tissues. Furthermore, it demonstrates the possibilities arising from the inherent ability of padlock probes to distinguish between transcripts based on differences in single nucleotides.

The study in paper I investigates the prevalence of Enterovirus species B in patients with Crohn’s disease by a chromogenic in situ hybridization assay combined with immunohistochemistry to detect viral RNA and proteins directly in tissue samples.

In paper II, padlock probes were used to study the spatial gene expression of gene homologs from the X and Y chromosome in human embryonic nervous tissue. Furthermore, a strategy was devised to visualize and evaluate spatial expression patterns.

The padlock probe-based approach for multiplexed spatial transcriptional profiling, in situ sequencing, was applied in paper III to study the regional and cell-type-specific dynamics of A-to-I RNA editing in the developing mouse brain.

In paper IV, a technical characterization of padlock probes was performed with the aim of determining how to design a padlock probe to obtain optimal detection efficiency.

The work in this thesis demonstrates the dramatic shift in how biological questions in cell and tissue biology can be addressed, enabled by the technological evolution of traditional in situ hybridization assays into high-throughput, multiplexed spatial transcription profiling.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2020. p. 65
Keywords
Padlock probes, in situ sequencing, single cell resolution, single nucleotide variant resolution, spatial transcription profiling
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-177114 (URN)978-91-7797-958-6 (ISBN)978-91-7797-959-3 (ISBN)
Public defence
2020-02-14, Air & Fire, Science for Life Laboratory, Tomtebodavägen 23 A, Solna, 10:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript.

Available from: 2020-01-22 Created: 2019-12-17 Last updated: 2020-05-25Bibliographically approved

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