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Enhanced Northern Blot Detection of Small RNA Species in Drosophila Melanogaster
Published on: August 21, 2014
The Drosophila nerfin-1 mRNA requires multiple microRNAs to regulate its spatial and temporal translation dynamics in
Alexander Kuzin1, Mukta Kundu, Thomas Brody
1Neural Cell-Fate Determinants Section, NINDS, NIH, Bethesda, MD, USA. Alex.Kuzin@ninds.nih.gov
Abstract:
The mRNA encoding the Drosophila Zn-finger transcription factor Nerfin-1, required for CNS axon pathfinding events, is subject to post-transcriptional silencing. Although nerfin-1 mRNA is expressed in many neural precursor cells including all early delaminating CNS neuroblasts, the encoded Nerfin-1 protein is detected only in the nuclei of neural precursors that divide just once to generate neurons and then only transiently in nascent neurons. Using a nerfin-1 promoter-controlled reporter transgene, replacement of the nerfin-1 3' UTR with the viral SV-40 3' UTR releases the neuroblast translational block and prolongs reporter protein expression in neurons. Comparative genomics analysis reveals that the nerfin-1 mRNA 3' UTR contains multiple highly conserved sequence blocks that either harbor and/or overlap 21 predicted binding sites for 18 different microRNAs. To determine the functional significance of these microRNA-binding sites and less conserved microRNA target sites, we have studied their ability to block or limit the expression of reporter protein in nerfin-1-expressing cells during embryonic development. Our results indicate that no single microRNA is sufficient to fully inhibit protein expression but rather multiple microRNAs that target different binding sites are required to block ectopic protein expression in neural precursor cells and temporally restrict expression in neurons. Taken together, these results suggest that multiple microRNAs play a cooperative role in the post-transcriptional regulation of nerfin-1 mRNA, and the high degree of microRNA-binding site evolutionary conservation indicates that all members of the Drosophila genus employ a similar strategy to regulate the onset and extinction dynamics of Nerfin-1 expression.
Insights
Multiple microRNAs cooperatively regulate Nerfin-1 protein expression in Drosophila development. This post-transcriptional silencing ensures precise temporal and spatial control of Nerfin-1 during neural development.
Area of Science:
- Developmental Biology
- Neuroscience
- Molecular Biology
Background:
- The transcription factor Nerfin-1 is crucial for central nervous system (CNS) axon pathfinding in Drosophila.
- Nerfin-1 mRNA is widely expressed in neural precursors, but the protein is transiently detected only in specific neural populations.
- Post-transcriptional regulation likely controls Nerfin-1 protein levels during development.
Purpose of the Study:
- To investigate the mechanisms of post-transcriptional silencing of Nerfin-1 mRNA.
- To identify the role of microRNAs in regulating Nerfin-1 protein expression.
- To understand the functional significance of conserved microRNA-binding sites in the Nerfin-1 3' UTR.
Main Methods:
- Utilized a reporter transgene with the nerfin-1 promoter to assess translational regulation.
- Replaced the endogenous nerfin-1 3' untranslated region (UTR) with a viral SV-40 3' UTR.
- Performed comparative genomics to identify conserved microRNA-binding sites in the nerfin-1 3' UTR.
- Studied the impact of microRNA-binding sites on reporter protein expression in vivo.
Main Results:
- Replacing the nerfin-1 3' UTR with SV-40 3' UTR released translational repression and prolonged protein expression.
- Comparative genomics revealed multiple conserved sequence blocks in the nerfin-1 3' UTR with predicted microRNA binding sites.
- No single microRNA was sufficient for complete inhibition; multiple microRNAs targeting different sites were required for repression.
- These microRNAs temporally restricted Nerfin-1 expression in neurons and blocked ectopic expression in neural precursors.
Conclusions:
- Multiple microRNAs cooperatively regulate nerfin-1 mRNA post-transcriptionally.
- The evolutionary conservation of microRNA-binding sites suggests a conserved regulatory strategy across Drosophila species.
- This cooperative microRNA action precisely controls the onset and extinction dynamics of Nerfin-1 expression during neural development.
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