Abstract
Igfn1 is a complex locus that codes for multiple splicing variants of Immunoglobulin- and Fibronectin-like domain containing proteins predominantly expressed in skeletal muscle. To reveal possible roles for Igfn1, we applied non-selective knock-down by shRNAs as well as specific targeting of Igfn1 exon 13 by CRISPR/Cas9 mutagenesis in C2C12 cells. Decreased expression of Igfn1 variants via shRNAs against the common 3’-UTR region caused a total blunting of myoblast fusion, but did not prevent expression of differentiation markers. Targeting of N-terminal domains by elimination of exon 13 via CRISPR/Cas9 mediated homologous recombination, also resulted in fusion defects as well as large multinucleated cells. Expression of IGFN1_v1 partially rescued fusion and myotube morphology in the Igfn1 exon 13 knock-out cell line, indicating a role for this variant in myoblast fusion and differentiation. However, in vivo overexpression of IGFN1_v1 or the Igfn1 Exon 13 CRISPR/Cas9 targeting vector did not result in significant size changes in transfected fibres.
Highlights
Igfn1 was identified as a protein fragment in a Yeast-two-hybrid assay using the KY protein as bait [1]
C2C12 proliferating myoblasts were transduced with sh-1-IGFN1 viruses using a scrambled short hairpin RNA (shRNA) as control
To determine whether differentiation was inhibited in the knock-down cell line we looked at markers of adhesion and differentiation and, for reference, these markers were profiled throughout C2C12 differentiation (Fig 2A)
Summary
Igfn was identified as a protein fragment in a Yeast-two-hybrid assay using the KY protein as bait [1]. The largest IGFN1 protein isoform (referred to as IGFN1) contains a series of 11 fibronectin and immunoglobulin-like domains distributed in three N- and eight C-terminal domains separated by a large disordered segment. The domain composition of IGFN1 is reminiscent of other sarcomeric proteins associated with the actin cytoskeleton (e.g., myosin binding protein C, filamin C, myotilin, myopalladin or titin, see [6] for a review). These proteins are proposed to act as crosslinkers and bear an inherent flexible structure that allows them to maintain protein interactions through cycles of contraction and relaxation [7]
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