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Identification of novel regulators of dendrite arborization using cell type-specific RNA metabolic labeling

Obtaining neuron transcriptomes is challenging; their complex morphology and interconnected microenvironments make it difficult to isolate neurons without potentially altering gene expression. Multidendritic sensory neurons (md neurons) of Drosophila larvae are commonly used to study peripheral nerv...

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Autores principales: Aboukilila, Mohamed Y., Sami, Josephine D., Wang, Jingtian, England, Whitney, Spitale, Robert C., Cleary, Michael D.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7710095/
https://www.ncbi.nlm.nih.gov/pubmed/33264304
http://dx.doi.org/10.1371/journal.pone.0240386
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author Aboukilila, Mohamed Y.
Sami, Josephine D.
Wang, Jingtian
England, Whitney
Spitale, Robert C.
Cleary, Michael D.
author_facet Aboukilila, Mohamed Y.
Sami, Josephine D.
Wang, Jingtian
England, Whitney
Spitale, Robert C.
Cleary, Michael D.
author_sort Aboukilila, Mohamed Y.
collection PubMed
description Obtaining neuron transcriptomes is challenging; their complex morphology and interconnected microenvironments make it difficult to isolate neurons without potentially altering gene expression. Multidendritic sensory neurons (md neurons) of Drosophila larvae are commonly used to study peripheral nervous system biology, particularly dendrite arborization. We sought to test if EC-tagging, a biosynthetic RNA tagging and purification method that avoids the caveats of physical isolation, would enable discovery of novel regulators of md neuron dendrite arborization. Our aims were twofold: discover novel md neuron transcripts and test the sensitivity of EC-tagging. RNAs were biosynthetically tagged by expressing CD:UPRT (a nucleobase-converting fusion enzyme) in md neurons and feeding 5-ethynylcytosine (EC) to larvae. Only CD:UPRT-expressing cells are competent to convert EC into 5-ethynyluridine-monophosphate which is subsequently incorporated into nascent RNA transcripts. Tagged RNAs were purified and used for RNA-sequencing. Reference RNA was prepared in a similar manner using 5-ethynyluridine (EUd) to tag RNA in all cells and negative control RNA-seq was performed on “mock tagged” samples to identify non-specifically purified transcripts. Differential expression analysis identified md neuron enriched and depleted transcripts. Three candidate genes encoding RNA-binding proteins (RBPs) were tested for a role in md neuron dendrite arborization. Loss-of-function for the m6A-binding factor Ythdc1 did not cause any dendrite arborization defects while RNAi of the other two candidates, the poly(A) polymerase Hiiragi and the translation regulator Hephaestus, caused significant defects in dendrite arborization. This work provides an expanded view of transcription in md neurons and a technical framework for combining EC-tagging with RNA-seq to profile transcription in cells that may not be amenable to physical isolation.
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spelling pubmed-77100952020-12-03 Identification of novel regulators of dendrite arborization using cell type-specific RNA metabolic labeling Aboukilila, Mohamed Y. Sami, Josephine D. Wang, Jingtian England, Whitney Spitale, Robert C. Cleary, Michael D. PLoS One Research Article Obtaining neuron transcriptomes is challenging; their complex morphology and interconnected microenvironments make it difficult to isolate neurons without potentially altering gene expression. Multidendritic sensory neurons (md neurons) of Drosophila larvae are commonly used to study peripheral nervous system biology, particularly dendrite arborization. We sought to test if EC-tagging, a biosynthetic RNA tagging and purification method that avoids the caveats of physical isolation, would enable discovery of novel regulators of md neuron dendrite arborization. Our aims were twofold: discover novel md neuron transcripts and test the sensitivity of EC-tagging. RNAs were biosynthetically tagged by expressing CD:UPRT (a nucleobase-converting fusion enzyme) in md neurons and feeding 5-ethynylcytosine (EC) to larvae. Only CD:UPRT-expressing cells are competent to convert EC into 5-ethynyluridine-monophosphate which is subsequently incorporated into nascent RNA transcripts. Tagged RNAs were purified and used for RNA-sequencing. Reference RNA was prepared in a similar manner using 5-ethynyluridine (EUd) to tag RNA in all cells and negative control RNA-seq was performed on “mock tagged” samples to identify non-specifically purified transcripts. Differential expression analysis identified md neuron enriched and depleted transcripts. Three candidate genes encoding RNA-binding proteins (RBPs) were tested for a role in md neuron dendrite arborization. Loss-of-function for the m6A-binding factor Ythdc1 did not cause any dendrite arborization defects while RNAi of the other two candidates, the poly(A) polymerase Hiiragi and the translation regulator Hephaestus, caused significant defects in dendrite arborization. This work provides an expanded view of transcription in md neurons and a technical framework for combining EC-tagging with RNA-seq to profile transcription in cells that may not be amenable to physical isolation. Public Library of Science 2020-12-02 /pmc/articles/PMC7710095/ /pubmed/33264304 http://dx.doi.org/10.1371/journal.pone.0240386 Text en © 2020 Aboukilila et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Article
Aboukilila, Mohamed Y.
Sami, Josephine D.
Wang, Jingtian
England, Whitney
Spitale, Robert C.
Cleary, Michael D.
Identification of novel regulators of dendrite arborization using cell type-specific RNA metabolic labeling
title Identification of novel regulators of dendrite arborization using cell type-specific RNA metabolic labeling
title_full Identification of novel regulators of dendrite arborization using cell type-specific RNA metabolic labeling
title_fullStr Identification of novel regulators of dendrite arborization using cell type-specific RNA metabolic labeling
title_full_unstemmed Identification of novel regulators of dendrite arborization using cell type-specific RNA metabolic labeling
title_short Identification of novel regulators of dendrite arborization using cell type-specific RNA metabolic labeling
title_sort identification of novel regulators of dendrite arborization using cell type-specific rna metabolic labeling
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7710095/
https://www.ncbi.nlm.nih.gov/pubmed/33264304
http://dx.doi.org/10.1371/journal.pone.0240386
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