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Abstract
Muscle stem cells (MuSCs) have the ability to carry out the specialized function of cell polarization, which is required for the production of one repopulating cell and one myogenic progenitor cell with muscle regeneration capabilities. The mechanisms which regulate proteins involved in establishing MuSC polarity such as Dmd and Itga7 are currently not well understood. Herein, we define the RNA-binding protein Quaking (QKI) as a major regulator alternative splicing of key MuSC polarity factors including Dmd, Itga7, Mark2, and Numb. We generate a conditional QKI knockout mouse, and for the first time it is shown in vivo that deficiency of QKI in MuSCs results in reduced asymmetric cell divisions, leading to a loss of the myogenic progenitor cell population and striking muscle regeneration defects. Transcriptomic analysis of QKI-deficient MuSCs identifies QKI as a regulator of the splicing events which give rise to the mutually exclusive Itga7-X1 and -X2 isoforms. We observe increased X1 expression in QKI-deficient MuSCs and recapitulate this splicing event using antisense oligonucleotide directed against a quaking binding site within the Itga7 mRNA. Interestingly, recreating this single splicing event is detrimental to the polarization of Itga7 and Dmd proteins, and leads to a drastic reduction of the myogenic progenitor population, highlighting the significance of QKI-mediated alternative splicing of Itga7 in maintaining MuSC polarity. Altogether, these findings define a novel role for QKI as a post-transcriptional regulator of MuSC polarity.
Highlights
Muscle regeneration is a complex process which begins with the activation of tissue-resident quiescent muscle stem cells (MuSCs) (Montarras et al, 2013)
We report that major polaritydetermining factors including Integrin Alpha-7 (Itga7) and Dmd undergo defective alternative splicing (AS) in QKI-deficient primary MuSCs
Treatment of wild type MuSCs with ASOs directed against the QKI response element (QRE) in Itga7 intron 4 led to exon 5 inclusion and expression of the Itga7-X1 isoform known to express extracellular linker domain and interfere with polarization of both Dmd and Itga7
Summary
Muscle regeneration is a complex process which begins with the activation of tissue-resident quiescent muscle stem cells (MuSCs) (Montarras et al, 2013). Dystrophin (Dmd)-deficient MuSCs were observed to have asymmetric division defects as a result of faulty localization of polarity-determining proteins Mark and Pard, leading to a loss of myogenic progenitor cells (Dumont et al, 2015). The Numb protein segregates to one daughter cell during mitosis, along with an unequal distribution of template DNA to provide different identities to the resulting daughter cells (Shinin et al, 2006). These findings lay important groundwork towards a better understanding of the complexities of asymmetric MuSC division. The role of post-transcriptional regulatory networks mediated by RNAbinding proteins (RBPs) in cell polarity and asymmetric MuSC division is not understood
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