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Mammalian Mss51 is a Skeletal Muscle-Specific Gene Modulating Cellular Metabolism

View Article: PubMed Central - PubMed

ABSTRACT

Background:: The transforming growth factor β (TGF-β) signaling pathways modulate skeletal muscle growth, regeneration, and cellular metabolism. Several recent gene expression studies have shown that inhibition of myostatin and TGF-β1 signaling consistently leads to a significant reduction in expression of Mss51, also named Zmynd17. The function of mammalian Mss51 is unknown although a putative homolog in yeast is a mitochondrial translational activator.

Objective:: The objective of this work was to characterize mammalian MSS51.

Methods:: Quantitative RT-PCR and immunoblot of subcellular fractionation were used to determine expression patterns and localization of Mss51. The CRISPR/Cas9 system was used to reduce expression of Mss51 in C2C12 myoblasts and the function of Mss51 was evaluated in assays of proliferation, differentiation and cellular metabolism.

Results:: Mss51 was predominantly expressed in skeletal muscle and in those muscles dominated by fast-twitch fibers. In vitro, its expression was upregulated upon differentiation of C2C12 myoblasts into myotubes. Expression of Mss51 was modulated in response to altered TGF-β family signaling. In human muscle, MSS51 localized to the mitochondria. Its genetic disruption resulted in increased levels of cellular ATP, β-oxidation, glycolysis, and oxidative phosphorylation.

Conclusions:: Mss51 is a novel, skeletal muscle-specific gene and a key target of myostatin and TGF-β1 signaling. Unlike myostatin, TGF-β1 and IGF-1, Mss51 does not regulate myoblast proliferation or differentiation. Rather, Mss51 appears to be one of the effectors of these growth factors on metabolic processes including fatty acid oxidation, glycolysis and oxidative phosphorylation.

No MeSH data available.


Related in: MedlinePlus

CRISPR/Cas9-disruption of Mss51 locus in C2C12 myoblasts. (A) Schematic of theMss51 genomic locus with the CRISPR guide RNA (gRNA) target sequence enlarged, predicted cut site marked in red, exons marked in black, UTRs in white. At the cut site, a double-strand break occurred and was re-sealed by non-homologous end joining. (B) Production of Mss51-disrupted cells was achieved by co-transfection of a dual-expression plasmid encoding the CRISPR gRNA (red) and the Cas9 endonuclease (blue), and a reporter plasmid encoding a disrupted GFP with two homology arms (dark green) flanking the CRISPR target site (red), which was cleaved by Cas9, re-sealed by homology-directed repair, and expressed functional GFP. Two populations of GFP-positive cells were collected by FACS sorting and expanded for further analysis – Mss51-disrupted cells were collected as shown and in parallel, control cells were transfected with a GFP expression plasmid and subjected to the same FACS process. (C) Mss51 expression measured by qRT-PCR in control and Mss51-disrupted populations over 6 days of differentiation, normalized to expression in proliferating control cells (Day 0) using reference genes Tfam and TBP (n = 3). **p <  0.01,***p <  0.001.
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jnd-2-4-jnd150119-g004: CRISPR/Cas9-disruption of Mss51 locus in C2C12 myoblasts. (A) Schematic of theMss51 genomic locus with the CRISPR guide RNA (gRNA) target sequence enlarged, predicted cut site marked in red, exons marked in black, UTRs in white. At the cut site, a double-strand break occurred and was re-sealed by non-homologous end joining. (B) Production of Mss51-disrupted cells was achieved by co-transfection of a dual-expression plasmid encoding the CRISPR gRNA (red) and the Cas9 endonuclease (blue), and a reporter plasmid encoding a disrupted GFP with two homology arms (dark green) flanking the CRISPR target site (red), which was cleaved by Cas9, re-sealed by homology-directed repair, and expressed functional GFP. Two populations of GFP-positive cells were collected by FACS sorting and expanded for further analysis – Mss51-disrupted cells were collected as shown and in parallel, control cells were transfected with a GFP expression plasmid and subjected to the same FACS process. (C) Mss51 expression measured by qRT-PCR in control and Mss51-disrupted populations over 6 days of differentiation, normalized to expression in proliferating control cells (Day 0) using reference genes Tfam and TBP (n = 3). **p <  0.01,***p <  0.001.

Mentions: In order to decrease Mss51 expression, the Mss51 genomic locus was disrupted in C2C12 cells using the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system for genome engineering [27]. Guide RNA (gRNA) target sequences and plasmids were designed and synthesized through the University of Massachusetts Medical School’s Mutagenesis Core, which provided a plasmid expressing both the CRISPR gRNA and the RNA-guided DNA endonuclease Cas9 under independent promoters, as well as a reporter plasmid containing a non-functional GFP ORF, disrupted by homology arms flanking DNA sequence containing the Mss51-specific CRISPR target sequence [28]. Proliferating C2C12 cells were transfected with the CRISPR/Cas9 plasmid and disrupted GFP reporter plasmid using an Amaxa Nucleofector II and Cell Line Nucleofector Kit V (Lonza, Walkersville, MD, USA) according to manufacturer’s instructions. The target sequence within Mss51, depicted in Fig. 4A, was cleaved by the CRISPR/Cas9 complex and re-sealed by non-homologous end joining. Simultaneously, the CRISPR/Cas9 complex also cleaved the disrupted GFP reporter plasmid, which was re-sealed by homology-directed repair using the homology arms flanking the cut site, allowing for functional expression of GFP only when the CRISPR/Cas9 complex was present. After 24 hours, transfected cells were isolated by fluorescence-activated cell sorting using a FACSAria Ilu (BD Biosciences, Franklin Lakes, NJ, USA), shown schematically in Fig. 4B. In parallel, control cells were transfected with a plasmid expressing functional GFP and subjected to the same cell sorting process as the Mss51-disrupted cells. Pooled populations of Mss51-disrupted and control cells were collected and expanded for comparative analysis. Cells were allowed to differentiate into myotubes for six days before analysis unless otherwise stated.


Mammalian Mss51 is a Skeletal Muscle-Specific Gene Modulating Cellular Metabolism
CRISPR/Cas9-disruption of Mss51 locus in C2C12 myoblasts. (A) Schematic of theMss51 genomic locus with the CRISPR guide RNA (gRNA) target sequence enlarged, predicted cut site marked in red, exons marked in black, UTRs in white. At the cut site, a double-strand break occurred and was re-sealed by non-homologous end joining. (B) Production of Mss51-disrupted cells was achieved by co-transfection of a dual-expression plasmid encoding the CRISPR gRNA (red) and the Cas9 endonuclease (blue), and a reporter plasmid encoding a disrupted GFP with two homology arms (dark green) flanking the CRISPR target site (red), which was cleaved by Cas9, re-sealed by homology-directed repair, and expressed functional GFP. Two populations of GFP-positive cells were collected by FACS sorting and expanded for further analysis – Mss51-disrupted cells were collected as shown and in parallel, control cells were transfected with a GFP expression plasmid and subjected to the same FACS process. (C) Mss51 expression measured by qRT-PCR in control and Mss51-disrupted populations over 6 days of differentiation, normalized to expression in proliferating control cells (Day 0) using reference genes Tfam and TBP (n = 3). **p <  0.01,***p <  0.001.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4664537&req=5

jnd-2-4-jnd150119-g004: CRISPR/Cas9-disruption of Mss51 locus in C2C12 myoblasts. (A) Schematic of theMss51 genomic locus with the CRISPR guide RNA (gRNA) target sequence enlarged, predicted cut site marked in red, exons marked in black, UTRs in white. At the cut site, a double-strand break occurred and was re-sealed by non-homologous end joining. (B) Production of Mss51-disrupted cells was achieved by co-transfection of a dual-expression plasmid encoding the CRISPR gRNA (red) and the Cas9 endonuclease (blue), and a reporter plasmid encoding a disrupted GFP with two homology arms (dark green) flanking the CRISPR target site (red), which was cleaved by Cas9, re-sealed by homology-directed repair, and expressed functional GFP. Two populations of GFP-positive cells were collected by FACS sorting and expanded for further analysis – Mss51-disrupted cells were collected as shown and in parallel, control cells were transfected with a GFP expression plasmid and subjected to the same FACS process. (C) Mss51 expression measured by qRT-PCR in control and Mss51-disrupted populations over 6 days of differentiation, normalized to expression in proliferating control cells (Day 0) using reference genes Tfam and TBP (n = 3). **p <  0.01,***p <  0.001.
Mentions: In order to decrease Mss51 expression, the Mss51 genomic locus was disrupted in C2C12 cells using the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system for genome engineering [27]. Guide RNA (gRNA) target sequences and plasmids were designed and synthesized through the University of Massachusetts Medical School’s Mutagenesis Core, which provided a plasmid expressing both the CRISPR gRNA and the RNA-guided DNA endonuclease Cas9 under independent promoters, as well as a reporter plasmid containing a non-functional GFP ORF, disrupted by homology arms flanking DNA sequence containing the Mss51-specific CRISPR target sequence [28]. Proliferating C2C12 cells were transfected with the CRISPR/Cas9 plasmid and disrupted GFP reporter plasmid using an Amaxa Nucleofector II and Cell Line Nucleofector Kit V (Lonza, Walkersville, MD, USA) according to manufacturer’s instructions. The target sequence within Mss51, depicted in Fig. 4A, was cleaved by the CRISPR/Cas9 complex and re-sealed by non-homologous end joining. Simultaneously, the CRISPR/Cas9 complex also cleaved the disrupted GFP reporter plasmid, which was re-sealed by homology-directed repair using the homology arms flanking the cut site, allowing for functional expression of GFP only when the CRISPR/Cas9 complex was present. After 24 hours, transfected cells were isolated by fluorescence-activated cell sorting using a FACSAria Ilu (BD Biosciences, Franklin Lakes, NJ, USA), shown schematically in Fig. 4B. In parallel, control cells were transfected with a plasmid expressing functional GFP and subjected to the same cell sorting process as the Mss51-disrupted cells. Pooled populations of Mss51-disrupted and control cells were collected and expanded for comparative analysis. Cells were allowed to differentiate into myotubes for six days before analysis unless otherwise stated.

View Article: PubMed Central - PubMed

ABSTRACT

Background:: The transforming growth factor &beta; (TGF-&beta;) signaling pathways modulate skeletal muscle growth, regeneration, and cellular metabolism. Several recent gene expression studies have shown that inhibition of myostatin and TGF-&beta;1 signaling consistently leads to a significant reduction in expression of Mss51, also named Zmynd17. The function of mammalian Mss51 is unknown although a putative homolog in yeast is a mitochondrial translational activator.

Objective:: The objective of this work was to characterize mammalian MSS51.

Methods:: Quantitative RT-PCR and immunoblot of subcellular fractionation were used to determine expression patterns and localization of Mss51. The CRISPR/Cas9 system was used to reduce expression of Mss51 in C2C12 myoblasts and the function of Mss51 was evaluated in assays of proliferation, differentiation and cellular metabolism.

Results:: Mss51 was predominantly expressed in skeletal muscle and in those muscles dominated by fast-twitch fibers. In vitro, its expression was upregulated upon differentiation of C2C12 myoblasts into myotubes. Expression of Mss51 was modulated in response to altered TGF-&beta; family signaling. In human muscle, MSS51 localized to the mitochondria. Its genetic disruption resulted in increased levels of cellular ATP, &beta;-oxidation, glycolysis, and oxidative phosphorylation.

Conclusions:: Mss51 is a novel, skeletal muscle-specific gene and a key target of myostatin and TGF-&beta;1 signaling. Unlike myostatin, TGF-&beta;1 and IGF-1, Mss51 does not regulate myoblast proliferation or differentiation. Rather, Mss51 appears to be one of the effectors of these growth factors on metabolic processes including fatty acid oxidation, glycolysis and oxidative phosphorylation.

No MeSH data available.


Related in: MedlinePlus