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

PLoS One. 2020 Dec 2;15(12):e0240386. doi: 10.1371/journal.pone.0240386. eCollection 2020.

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Animals, Genetically Modified
  • Cytosine / administration & dosage
  • Cytosine / analogs & derivatives
  • Cytosine / metabolism
  • Dendrites / physiology*
  • Deoxyuracil Nucleotides / chemistry
  • Deoxyuracil Nucleotides / metabolism
  • Drosophila Proteins / genetics
  • Drosophila Proteins / metabolism*
  • Drosophila melanogaster / genetics
  • Drosophila melanogaster / growth & development
  • Drosophila melanogaster / metabolism
  • Gene Expression Regulation, Developmental*
  • Larva / genetics
  • Larva / growth & development
  • Larva / metabolism
  • Loss of Function Mutation
  • Neurogenesis / genetics*
  • Nuclear Proteins / genetics
  • Nuclear Proteins / metabolism
  • Polynucleotide Adenylyltransferase / genetics
  • Polynucleotide Adenylyltransferase / metabolism*
  • Polypyrimidine Tract-Binding Protein / genetics
  • Polypyrimidine Tract-Binding Protein / metabolism*
  • RNA / chemistry
  • RNA / metabolism
  • RNA Interference
  • RNA-Seq
  • Sensory Receptor Cells / cytology
  • Sensory Receptor Cells / physiology*
  • Staining and Labeling / methods

Substances

  • 5-ethynylcytosine
  • Deoxyuracil Nucleotides
  • Drosophila Proteins
  • Heph protein, Drosophila
  • Nuclear Proteins
  • Ythdc1 protein, Drosophila
  • Polypyrimidine Tract-Binding Protein
  • RNA
  • 5-ethynyl-2'-deoxyuridylic acid
  • Cytosine
  • Polynucleotide Adenylyltransferase
  • hrg protein, Drosophila