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.

N6-methyladenosine (m6A) is the most prevalent internal mRNA modification in metazoans and is particularly abundant in the central nervous system. The extent to which m6A is dynamically regulated and whether m6A contributes to cell type-specific mRNA metabolism in the nervous system, however, is largely unknown. To address these knowledge gaps, we mapped m6A and measured mRNA decay in neural progenitors (neuroblasts) and neurons of the Drosophila melanogaster larval brain. We identified 867 m6A targets; 233 of these are novel and preferentially encode regulators of neuroblast proliferation, cell fate-specification and synaptogenesis. Comparison of the neuroblast and neuron m6A transcriptomes revealed that m6A stoichiometry is largely uniform; we did not find evidence of neuroblast-specific or neuron-specific m6A modification. While m6A stoichiometry is constant, m6A targets are significantly less stable in neuroblasts than in neurons, potentially due to m6A-independent stabilization in neurons. We used in vivo quantitative imaging of m6A target proteins in Mettl3 methyltransferase null brains and Ythdf m6A reader overexpressing brains to assay metabolic effects of m6A. Target protein levels decreased in Mettl3 null brains and increased in Ythdf overexpressing brains, supporting a previously proposed model in which m6A enhances translation of target mRNAs. We conclude that m6A does not directly regulate mRNA stability during Drosophila neurogenesis but is rather deposited on neurodevelopmental transcripts that have intrinsic low stability in order to augment protein output.

N6-methyladenosine (m6A) is the most prevalent internal mRNA modification in metazoans and is particularly abundant in the central nervous system. The extent to which m6A is dynamically regulated and whether m6A contributes to cell type-specific mRNA metabolism in the nervous system, however, is largely unknown. To address these knowledge gaps, we mapped m6A and measured mRNA decay in neural progenitors (neuroblasts) and neurons of the Drosophila melanogaster larval brain. We identified 867 m6A targets; 233 of these are novel and preferentially encode regulators of neuroblast proliferation, cell fate-specification and synaptogenesis. Comparison of the neuroblast and neuron m6A transcriptomes revealed that m6A stoichiometry is largely uniform; we did not find evidence of neuroblast-specific or neuron-specific m6A modification. While m6A stoichiometry is constant, m6A targets are significantly less stable in neuroblasts than in neurons, potentially due to m6A-independent stabilization in neurons. We used in vivo quantitative imaging of m6A target proteins in Mettl3 methyltransferase null brains and Ythdf m6A reader overexpressing brains to assay metabolic effects of m6A. Target protein levels decreased in Mettl3 null brains and increased in Ythdf overexpressing brains, supporting a previously proposed model in which m6A enhances translation of target mRNAs. We conclude that m6A does not directly regulate mRNA stability during Drosophila neurogenesis but is rather deposited on neurodevelopmental transcripts that have intrinsic low stability in order to augment protein output.

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. RNAs were biosynthetically tagged by expressing CD:UPRT (a nucleobase-converting fusion enzyme) in md neurons and feeding 5-ethynylcytosine (EC) to larvae. Tagged RNAs were subsequently 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.