Abstract
MiRNAs impact on the control of cell fate by regulating gene expression at the post-transcriptional level. Here, using mammalian muscle differentiation as a model and a phenotypic loss-of-function screen, we explored the function of miRNAs at the genome-wide level. We found that the depletion of a high number of miRNAs (63) impacted on differentiation of human muscle precursors, underscoring the importance of this post-transcriptional mechanism of gene regulation. Interestingly, a comparison with miRNA expression profiles revealed that most of the hit miRNAs did not show any significant variations of expression during differentiation. These constitutively expressed miRNAs might be required for basic and/or essential cell function, or else might be regulated at the post-transcriptional level. MiRNA inhibition yielded a variety of phenotypes, reflecting the widespread miRNA involvement in differentiation. Using a functional screen (the STarS - Suppressor Target Screen – approach, i. e. concomitant knockdown of miRNAs and of candidate target proteins), we discovered miRNA protein targets that are previously uncharacterized controllers of muscle-cell terminal differentiation. Our results provide a strategy for functional annotation of the human miRnome.
Highlights
The microRNA machinery [1] has an essential function during development [2]
Muscle cell terminal differentiation involves the fusion of myoblastic precursor cells into large multinucleated post-mitotic cells that express muscle-specific markers such as muscle Myosin Heavy Chain (MHC) and Muscle Creatine Kinase (MCK)
The proportion of multinucleated MHCpositive myotubes (i. e., cells that are positive for MHC fluorescence and have at least 3 nuclei) was scored in each
Summary
MicroRNAs (miRNAs) [1] are encoded in intergenic or intronic sequences as long precursors that are sequentially processed by the endonucleases Drosha and Dicer into short double-stranded sequences [3]. They regulate gene expression at the post-transcriptional level: in the cytoplasm, they guide the RISC complex, an Argonaute-containing complex of proteins, toward a target messenger RNA. We implemented a genome-wide miRNA loss-of-function screen using LNA-modified synthetic antisense oligonucleotides [6] in LHCN, a human skeletal muscle precursor cell line. Using a phenotypic screen based on co-suppression of miRNAs and putative targets that we named the STarS assay, we identified important proteins whose role in controlling differentiation had not been previously identified
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