miR-206 knockout shows it is critical for myogenesis and directly regulates newly identified target mRNAs.

RNA Biology
Georgiana M. SalantJennifer F. Kugel

Abstract

The muscle specific miRNA, miR-206, is important for the process of myogenesis; however, studying the function of miR-206 in muscle development and differentiation still proves challenging because the complement of mRNA targets it regulates remains undefined. In addition, miR-206 shares close sequence similarity to miR-1, another muscle specific miRNA, making it hard to study the impact of miR-206 alone in cell culture models. Here we used CRISPR/Cas9 technology to knockout miR-206 in C2C12 muscle cells. We show that knocking out miR-206 significantly impairs and delays differentiation and myotube formation, revealing that miR-206 alone is important for myogenesis. In addition, we use an experimental affinity purification technique to identify new mRNA targets of miR-206 in C2C12 cells. We identified over one hundred mRNAs as putative miR-206 targets. Functional experiments on six of these targets indicate that Adam19, Bgn, Cbx5, Smarce1, and Spg20 are direct miR-206 targets in C2C12 cells. Our data show a unique and important role for miR-206 in myogenesis.

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Methods Mentioned

BETA
affinity purification
Illumina sequencing
xOP-seq
crosslinking immunoprecipitation
HITS-CLIP
transfection
FACS
Assay
PCR

Software Mentioned

Bedtools
DAVID Bioinformatics Resources
UCSC Genome Browser
HOMER
Bowtie2
HOMER ( Hypergeometric Optimization of Motif EnRichment )

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