Subcellular sequencing of single neurons reveals the dendritic transcriptome of GABAergic interneurons

Pérez, Julio; Dieck, Susanne tom; Álvarez-Castelao, Beatriz; Chan, I.C.W.; Schuman, Erin M.

doi:10.1101/2020.10.17.341651

Cited by 9 publications

(13 citation statements)

References 56 publications

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“…We also observed near-significant enrichment of Rims1 mRNA, which encodes the active zone protein RIM1 that was recently shown to be critically involved in both axonal and somatodendritic DA release (Liu et al, 2018;Robinson et al, 2019). Many of these mRNAs have also been identified in the dendrites of both glutamatergic and GABAergic hippocampal neurons (Perez et al, 2021) (Figure S7A).…”

Section: Midbrain Synaptosome Ribotag Ip Reveals Dendritic Localization Of Mrnas Encoding Vesicular Release Proteinsmentioning

confidence: 54%

“…The most abundant RNA species in all samples were derived from mitochondrialencoded genes such as Mt-Rnr1, Mt-Rnr2, and Mt-Nd1 (Figure 3I). Although mRNAs encoding ribosomal proteins and nuclear-encoded mitochondrial proteins have been observed in axons (Aschrafi et al, 2016;Briese et al, 2016;Gumy et al, 2011;Shigeoka et al, 2016Shigeoka et al, , 2019Taylor et al, 2009), these mRNAs are also present in dendrites (Fusco et al, 2021;Perez et al, 2021), the synaptic neuropil (Biever et al, 2020;Cajigas et al, 2012), and perisynaptic astrocyte processes (Mazaré et al, 2020). Given the ambiguous cellular and subcellular contribution to the mRNA content of our FASS samples, it is unclear whether any FASS-enriched mRNAs are derived from DA synaptosomes.…”

Section: Lack Of Evidence For Axonal Translation In Striatal Ribotag Ipsmentioning

confidence: 94%

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Subcellular and regional localization of mRNA translation in midbrain dopamine neurons

Hobson

Kong

Angelo

et al. 2021

Preprint

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Local translation within excitatory and inhibitory neurons is involved in neuronal development and synaptic plasticity. Despite the extensive dendritic and axonal arborizations of central monoaminergic neurons, the subcellular localization of protein synthesis is not well-characterized in these populations. Here, we investigated mRNA localization and translation in midbrain dopaminergic (mDA) neurons, cells with enormous axonal and dendritic projections, both of which exhibit stimulation-evoked dopamine (DA) release. Using highly-sensitive ribosome-bound RNA-sequencing and imaging approaches in mDA axons, we found no evidence for axonal mRNA localization or translation. In contrast, mDA neuronal dendritic projections into the substantia nigra reticulata (SNr) contain ribosomes and mRNAs encoding the major components of DA synthesis, release, and reuptake machinery. Surprisingly, we also observed dendritic localization of mRNAs encoding synaptic vesicle-related proteins, including those involved in vesicular exocytic fusion. Our results are consistent with a role for local translation in the regulation of DA release from dendrites, but not from axons. Our translatome data further defined a molecular signature of the sparse mDA neurons resident in the SNr, including enrichment of Atp2a3/SERCA3, an ER calcium pump previously undescribed in mDA neurons.

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Section: Midbrain Synaptosome Ribotag Ip Reveals Dendritic Localization Of Mrnas Encoding Vesicular Release Proteinsmentioning

confidence: 54%

Section: Lack Of Evidence For Axonal Translation In Striatal Ribotag Ipsmentioning

confidence: 94%

Subcellular and regional localization of mRNA translation in midbrain dopamine neurons

Hobson

Kong

Angelo

et al. 2021

Preprint

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“…Many experimental methods for transcriptome profiling by next-generation sequencing (RNA-seq) have been developed to cover various scales of input samples, ranging from bulk samples [1,2] to single cells [3][4][5] or even subcellular components [6,7]. High-quality single-cell RNA-seq (scRNA-seq) data can be used to reveal the kinetic details of gene expression and transitions between cell states or types [8][9][10].…”

Section: Introductionmentioning

confidence: 99%

Rapid and sensitive single-cell RNA sequencing with SHERRY2

et al. 2022

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Background Prevalent single-cell transcriptomic profiling (scRNA-seq) methods are mainly based on the synthesis and enrichment of full-length double-stranded complementary DNA. These approaches are challenging to generate accurate quantification of transcripts when their abundance is low or their full-length amplifications are difficult. Results Based on our previous finding that Tn5 transposase can directly cut-and-tag DNA/RNA hetero-duplexes, we present SHERRY2, a specifically optimized protocol for scRNA-seq without second-strand cDNA synthesis. SHERRY2 is free of pre-amplification and eliminates the sequence-dependent bias. In comparison with other widely used scRNA-seq methods, SHERRY2 exhibits significantly higher sensitivity and accuracy even for single nuclei. Besides, SHERRY2 is simple and robust and can be easily scaled up to high-throughput experiments. When testing single lymphocytes and neuron nuclei, SHERRY2 not only obtained accurate countings of transcription factors and long non-coding RNAs, but also provided bias-free results that enriched genes in specific cellular components or functions, which outperformed other protocols. With a few thousand cells sequenced by SHERRY2, we confirmed the expression and dynamics of Myc in different cell types of germinal centers, which were previously only revealed by gene-specific amplification methods. Conclusions SHERRY2 is able to provide high sensitivity, high accuracy, and high throughput for those applications that require a high number of genes identified in each cell. It can reveal the subtle transcriptomic difference between cells and facilitate important biological discoveries.

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“…Fractionation-based approaches allowed for the characterization of the entire pool of dendritically localized mRNAs (Gumy et al, 2011;Taylor et al, 2009), revealing the richness of the local transcriptome and subsequently proteome (Cajigas et al, 2012;Zappulo et al, 2017) as well as isoform-specific regulation (Ciolli Mattioli et al, 2019). Recent advances in spatial transcriptomics have enabled the study of dendritically localized RNAs at single cell resolution (Perez et al, 2021), with massively parallel hybridization approaches (Wang et al, 2020), and with expansion sequencing (Alon et al, 2021). This body of work reveals a wealth of mRNA localization patterns.…”

Section: Introductionmentioning

confidence: 99%

A massively parallel reporter assay reveals focused and broadly encoded RNA localization signals in neurons

Mikl

Eletto

Lee

et al. 2021

Preprint

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Asymmetric subcellular localization of mRNA is a common cellular phenomenon that is thought to contribute to spatial gene regulation. In highly polar neurons, subcellular transcript localization and translation are thought to enhance cellular efficiency and timely responses to external cues. Although mRNA localization has been observed in many tissues and numerous examples of the functional importance of this process exist, we still lack a systematic understanding of how the transcript sorting machinery works in a sequence-specific manner. Here, we addressed these gaps by combining subcellular transcriptomics and rationally designed sequence libraries. We developed a massively parallel reporter assay (MPRA) for mRNA localization and tested ~50,000 sequences for their ability to drive RNA localization to neurites of neuronal cell lines. By scanning the 3'UTR of >300 genes we identified many previously unknown localization regions and mapped the localization potential of endogenous sequences. Our data suggest two ways the localization potential can be encoded in the 3'UTR: focused localization motifs and broadly encoded localization potential based on small contributions. We identified sequence motifs enriched in dendritically localized transcripts and tested the potential of these motifs to affect the localization behavior of an mRNA. This assay revealed sequence elements with the ability to bias localization towards neurite as well as soma. Depletion of RNA binding proteins predicted or experimentally shown to bind these motifs abolished the effect on localization, suggesting that these motifs act by recruiting specific RNA-binding proteins. Based on our dataset we developed machine learning models that accurately predict the localization behavior of novel sequences. Testing this predictor on native mRNA sequencing data showed good agreement between predicted and observed localization potential, suggesting that the rules uncovered by our MPRA also apply to the localization of native transcripts. Applying similar systematic high-throughput approaches to other cell types will open the door for a comparative perspective on RNA localization across tissues and reveal the commonalities and differences of this crucial regulatory mechanism.

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Subcellular sequencing of single neurons reveals the dendritic transcriptome of GABAergic interneurons

Cited by 9 publications

References 56 publications

Subcellular and regional localization of mRNA translation in midbrain dopamine neurons

Subcellular and regional localization of mRNA translation in midbrain dopamine neurons

Rapid and sensitive single-cell RNA sequencing with SHERRY2

A massively parallel reporter assay reveals focused and broadly encoded RNA localization signals in neurons

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