Fine-tuning of the PAX-SIX-EYA-DACH network by multiple microRNAs controls embryo myogenesis

Viaut, Camille, Weldon, Shannon and Münsterberg, Andrea ORCID: (2021) Fine-tuning of the PAX-SIX-EYA-DACH network by multiple microRNAs controls embryo myogenesis. Developmental Biology, 469. pp. 68-79. ISSN 0012-1606

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MicroRNAs (miRNAs), short non-coding RNAs, which act post-transcriptionally to regulate gene expression, are of widespread significance during development and disease, including muscle disease. Advances in sequencing technology and bioinformatics led to the identification of a large number of miRNAs in vertebrates and other species, however, for many of these miRNAs specific roles have not yet been determined. LNA in situ hybridisation has revealed expression patterns of somite-enriched miRNAs, here we focus on characterising the functions of miR-128. We show that antagomiR-mediated knockdown (KD) of miR-128 in developing chick somites has a negative impact on skeletal myogenesis. Computational analysis identified the transcription factor EYA4 as a candidate target consistent with the observation that miR-128 and EYA4 display similar expression profiles. Luciferase assays confirmed that miR-128 interacts with the EYA4 3′UTR. In vivo experiments also suggest that EYA4 is regulated by miR-128. EYA4 is a member of the PAX-SIX-EYA-DACH (PSED) network of transcription factors. Therefore, we identified additional candidate miRNA binding sites in the 3′UTR of SIX1/4, EYA1/2/3 and DACH1. Using the miRanda algorithm, we found sites for miR-128, as well as for other myogenic miRNAs, miR-1a, miR-206 and miR-133a, some of these were experimentally confirmed as functional miRNA target sites. Our results reveal that miR-128 is involved in regulating skeletal myogenesis by directly targeting EYA4 with indirect effects on other PSED members, including SIX4 and PAX3. Hence, the inhibitory effect on myogenesis observed after miR-128 knockdown was rescued by concomitant knockdown of PAX3. Moreover, we show that the PSED network of transcription factors is co-regulated by multiple muscle-enriched microRNAs.

Item Type: Article
Additional Information: Funding Information: CV and SM were funded by the BBSRC Doctoral Training Programme on the Norwich Research Park (NRPDTP) . Research was supported by BBSRC project grants to AM (BB/K003437/1, BB/N007034/1). Acknowledgements: The authors thank Dr Timothy Grocott for help with cloning and luciferase assays and for discussions, Dr Gi Fay Mok for help with microinjection and electroporation, Dr Tracey Swingler for help with qPCR, Dr Simon Moxon for help with bioinformatics.
Uncontrolled Keywords: chicken embryo,eya4,psed network,somite myogenesis,mir-128,myomirs,molecular biology,developmental biology,cell biology ,/dk/atira/pure/subjectarea/asjc/1300/1312
Faculty \ School: Faculty of Science > School of Biological Sciences
UEA Research Groups: Faculty of Science > Research Groups > Cells and Tissues
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Depositing User: LivePure Connector
Date Deposited: 20 Oct 2020 23:58
Last Modified: 21 Apr 2023 00:49
DOI: 10.1016/j.ydbio.2020.10.005

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