mRNAs encoding neurodevelopmental regulators have equal N6-methyladenosine stoichiometry in Drosophila neuroblasts and neurons

Abstract

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.

Orb2 enables rare-codon-enriched mRNA expression during Drosophila neuron differentiation

“…As our data with GFP reporters implicated mRNA stability as an important mechanism of codon-dependent Orb2 regulation, we again used actD treatment and quantitative RT-PCR to examine the role of Orb2 in mGluR mRNA stability. Relative to ND-18 , a common-codon-enriched mRNA with a CAI of 0.862 shown previously to be highly stable in the nervous system 100 , mGluR mRNA is less stable after an hour of ActD treatment (Fig. 5J ).…”

“…Courtship behavior was measured as described 100 . Briefly, male flies of the desired genotype were aged 4 days in isolation from female flies.…”

Dynamically regulated transcription factors are encoded by highly unstable mRNAs in theDrosophilalarval brain

“…Current measurements of RNA stability have largely been derived from single-celled organisms or cultured mammalian cells, and so do not directly address in vivo mechanisms of cell-state changes in developing tissues. Very few studies have estimated RNA stability in multicellular organisms 11,12 , and we are unaware of any data describing RNA stability across an entire brain. In this paper, we estimate synthesis and decay rates in the Drosophila larval brain genome wide by analysing the metabolic labeling kinetics of more than 7,000 RNAs.…”