MicroRNA-486 is an epigenetic modulator of Duchenne muscular dystrophy pathologies [eCLIP-seq]

Duchenne muscular dystrophy (DMD) is an X-linked progressive muscle disorder resulting in muscle weakness and cardiomyopathy. MicroRNAs have shown to play a significant role in muscle development, metabolism, and disease pathologies. We demonstrated that miR-486 expression is reduced in DMD muscles and its expression levels correlate with dystrophic disease severity. miR-486 knockout mice developed disrupted myofiber architecture, decreased myofiber size, decreased locomotor activity, increased fibrosis, and metabolic defects that were exacerbated on the dystrophic mdx5cv background. We integrated RNA-sequencing and chimeric eCLIP-sequencing data to identify direct in vivo targets of miR-486 and associated dysregulated gene signatures in skeletal muscle. Together, our studies identify miR-486 as a driver of muscle remodeling in DMD, a useful biomarker for dystrophic disease progression, and highlight chimeric eCLIP-sequencing as a useful tool to identify direct in vivo microRNA target transcripts. Overall design: Whole TA muscles were isolated from male 6-month-old wild type and miR-486 KO mice (n = 3 total samples per genotype; performed in duplicate) and snap frozen in liquid nitrogen. The samples were processed for chimeric eCLIP-sequencing by Eclipse Bioinnovations Inc. (San Diego, CA). Approximately 80 mg of mouse muscles were homogenized by cryogenic pulverization. Samples were then were UV (254 nm) cross-linked twice at 400 mJ/cm2 using Stratalinker 2400 (Stratagene; San Diego, CA), on a bed of ice. Following cross-linking, the samples were sonicated (QSonica Q800R2; QSonica LLC, Newtown, CT) to shear the genomic DNA into smaller fragments. Ago2 immunoprecipitation using an Ago2 antibody (EclipseBioinnovations Inc.) was pre-coupled to anti-mouse Dynabeads (M-280 Sheep Anti-Mouse IgG Dynabeads, ThermoFisher Scientific 11201D), added to the homogenized lysate, and incubated overnight at 40C with gentile rocking. After immunoprecipitation, 2% of the sample was taken as the paired input sample. For chimeric eCLIP experiments, a standardized eCLIP protocol2 was modified to enable chimeric ligation of the miRNA and mRNA (Van Nostrand, E & Yeo, G, personal communication). miRNA-specific chimeric-eCLIP was performed by amplifying cDNA using a mouse miR-486a primer and a sequencing adapter-specific primer, following amplification with indexed primers. Chimeric eCLIP-sequencing Library Preparation: Whole TA muscles were isolated from male 6-month-old wild type and miR-486 KO mice (n = 5 total samples per genotype; performed in duplicate) and snap frozen in liquid nitrogen. The samples were processed for chimeric eCLIP-sequencing by Eclipse Bioinnovations Inc. (San Diego, CA). Approximately 80 mg of mouse muscles were homogenized by cryogenic pulverization. Samples were then UV (254 nm) cross-linked twice at 400 mJ/cm2 using Stratalinker 2400 (Stratagene; San Diego, CA), on a bed of ice. Following cross-linking, the samples were sonicated (QSonica Q800R2; QSonica LLC, Newtown, CT) to shear the genomic DNA into smaller fragments. Ago2 immunoprecipitation using an Ago2 antibody (EclipseBioinnovations Inc.) was pre-coupled to anti-mouse Dynabeads (M-280 Sheep Anti-Mouse IgG Dynabeads, ThermoFisher Scientific 11201D), added to the homogenized lysate, and incubated overnight at 40C with gentile rocking. After immunoprecipitation, 2% of the sample was taken as the paired input sample. For chimeric eCLIP experiments, a standardized eCLIP protocol was modified to enable chimeric ligation of the miRNA and mRNA (Van Nostrand, E & Yeo, G, personal communication). miRNA-specific chimeric-eCLIP was performed by amplifying cDNA using a mouse miR-486a primer and a sequencing adapter-specific primer, following amplification with indexed primers.