Department of Molecular and Cell Biology, Quantitative and Systems Biology Graduate Program, University of California, Merced, CA, United States of America.
Department of Pharmaceutical Sciences and Department of Chemistry, University of California, Irvine, CA, United States of America.
PLoS One. 2020 Dec 2;15(12):e0240386. doi: 10.1371/journal.pone.0240386. eCollection 2020.
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.
获取神经元转录组具有挑战性；由于其复杂的形态和相互连接的微环境，在不改变基因表达的情况下分离神经元是很困难的。果蝇幼虫的多树突感觉神经元 (md 神经元) 通常用于研究外周神经系统生物学，特别是树突分枝。我们试图测试 EC 标记是否可以用于发现新的 md 神经元树突分枝调节因子，EC 标记是一种合成 RNA 标记和纯化方法，可避免物理分离的缺点。我们的目标有两个：发现新的 md 神经元转录本并测试 EC 标记的灵敏度。通过在 md 神经元中表达 CD:UPRT（一种核碱基转换融合酶）并向幼虫喂食 5-乙炔胞嘧啶 (EC) 来生物合成标记 RNA。只有表达 CD:UPRT 的细胞有能力将 EC 转化为 5-乙炔尿嘧啶 - 单磷酸，随后将其掺入新生 RNA 转录本中。标记的 RNA 被纯化并用于 RNA-seq。使用类似的方法用 5-乙炔尿嘧啶 (EUd) 标记所有细胞中的 RNA 来制备参考 RNA，并对“模拟标记”样品进行阴性对照 RNA-seq 以鉴定非特异性纯化的转录本。差异表达分析鉴定了 md 神经元富集和耗尽的转录本。对编码 RNA 结合蛋白 (RBP) 的三个候选基因进行了测试，以研究它们在 md 神经元树突分枝中的作用。m6A 结合因子 Ythdc1 的功能丧失不会导致任何树突分枝缺陷，而另外两个候选基因，多聚 (A) 聚合酶 Hiiragi 和翻译调节因子 Hephaestus 的 RNAi 导致树突分枝缺陷显著。这项工作提供了 md 神经元转录的扩展视图，并为结合 EC 标记和 RNA-seq 来分析可能不易进行物理分离的细胞中的转录提供了技术框架。
