In vivo AGO-APP identifies a module of microRNAs cooperatively preserving neural progenitors
Karine Narbonne-Reveau, Andrea Erni, Norbert Eichner, Shobana Sankar, Surbhi Kapoor, Gunter Meister, Harold Cremer, Cédric Maurange, Christophe Beclin
Abstract
MicroRNAs (miRNAs) are small RNAs that silence messenger RNAs (mRNAs) by binding to partially complementary sequences. They play a crucial role in cell type specification during development. Computational models suggest that miRNAs can target many mRNAs, and individual mRNAs can be regulated by multiple miRNAs. This promiscuity raises the questions of how target specificity and silencing efficiency are controlled. To address this, we used in vivo AGO-APP, a new technique to isolate active miRNAs in specific cell types within tissues.
Introduction
MicroRNAs (miRNA) generally suppress protein production post-transcriptionally by binding to a short sequence motif in the 3’UTR of target mRNAs. Bioinformatic tools predict that a single miRNA can target hundreds of mRNAs and, vice versa, a single mRNA can be regulated by several miRNAs [1–3], indicating that miRNA/mRNA interactions are intrinsically promiscuous. Paradoxically, the literature mostly describes interactions between a single miRNA and a privileged mRNA [4,5]. Notably, some in vitro and in silico studies have also proposed that miRNAs may control more complex biological process, regulating large gene networks through additive, synergistic, or even combinatorial interactions [6–9].
Materials and method
Fly lines
Drosophila stocks were maintained at room temperature on standard medium (8% cornmeal, 8% yeast, 1% agar). Experiments were performed at 29 °C. Crosses to yw (Bloomington #1495) line are used as controls. For generating prospero-/-, nerfin-1-/- and miR-1KO clones, we used the mosaic analysis with a repressible cell marker (MARCM) technique [68]. The following MARCM stocks were used: tub-GAL4, UAS-nGFP-myc, hs-FLP; FRT82B, tub-GAL80/TM6c, FRT82B, pros17/TM6b (from Bloomington #5458), tub-GAL4, UAS-nGFP-myc hsFLP122; tub-GAL80, FRT2A/TM6b and Df(3L) nerfin-1159, FRT2A/TM6. For generating clones mis-expressing miRNAs or sponges, we used the Flp-out (Flpout) technique. Flpout clones were generated using hs-FLP; Act5c>CD2 > GAL4, UAS-GFP or hs-FLP; Act5c>CD2 > GAL4, UAS-RFP/TM3 (from Bloomington #7 and #30558).
Results
Ago-APP identifies cell type specific miRNAs in larval neurogenesis
The AGO-APP miRNA isolation approach relies on the expression of the T6B peptide tagged with FLAG-HA-YFP (T6B-FHY) (Fig 1A). Binding of T6B to AGO disrupts the RISC by displacing GW182 proteins. Subsequently, the remaining miRNA:Ago:T6B-FHY complex can be pulled down with anti-GFP nanobodies, the miRNA is released and can be analyzed by RT-qPCR or sequencing (Fig 1A).
Discussion
Most studies describe miRNA function in the light of their interaction with a given mRNA. However, each miRNA has the potential to bind dozens, sometimes hundreds, of different mRNAs in each cell, raising questions about how specificity is achieved. Here, using a new technology for RNA isolation in vivo, we identify in Drosophila neural progenitors a module of miRNAs able to target a gene network that promotes neuronal differentiation. Using simultaneous knockdown of multiple miRNAs, we show that miRNAs of the module act cooperatively to maintain a specific subtype of neural progenitors.
Acknowledgments
Stocks obtained from the Bloomington Drosophila Stock Center (NIH P40OD018537), Vienna Drosophila Resource Center (VDRC) and the Drosophila Genomics Resource Center were used in this study. We also thank Developmental Studies Hybridoma Bank (DSHB) and the fly community for strains and reagents. We are grateful to the imaging facility at IBDM, member of the National Infrastructure France-BioImaging (https://ror.org/01y7vt929) supported by the French National Research Agency (ANR-24-INSB-0005 FBI BIOGEN) as well as the IBDM animal facilities.
Citation: Narbonne-Reveau K, Erni A, Eichner N, Sankar S, Kapoor S, Meister G, et al. (2025) In vivo AGO-APP identifies a module of microRNAs cooperatively preserving neural progenitors. PLoS Genet 21(4): e1011680. https://doi.org/10.1371/journal.pgen.1011680
Editor: Shuo Gu, National Cancer Institute Center for Cancer Research, UNITED STATES OF AMERICA
Received: April 17, 2024; Accepted: April 7, 2025; Published: April 29, 2025
Copyright: © 2025 Narbonne-Reveau et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: The sequencing datasets generated and analyzed during this study are available at the functional genomics data collection ArrayExpress (https://www.ebi.ac.uk/biostudies/arrayexpress/studies/E-MTAB-14970) under accession number E-MTAB-14970.
Funding: 4. HC was supported by Agence Nationale de la Recherche (ANR) (MicroRNAct, ANR-17-CE16-0025; Uncoding, ANR-21-CE16-0034; Miniature, ANR-21-CE13-0003; Goligo, ANR-21-CE16-0023) and the Fondation pour la Recherche Médicale (Program Equipe FRM). AE received a postdoc fellowship from the Swiss National Science Foundation (SNSF). SK received a PhD fellowship from Neuroschool Marseille from Aix Marseille University. CM was funded by ANR (Miniature, ANR-21-CE13-0003) and the Ligue Contre le Cancer (programme Equipe Labellisée). SS received a PhD salary from ANR (Miniature, ANR-21-CE13-0003). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
