Pseudouridine synthase 1 deficient mice, a model for Mitochondrial Myopathy with Sideroblastic Anemia, exhibit muscle morphology and physiology alterations

Abstract

Mitochondrial myopathy with lactic acidosis and sideroblastic anemia (MLASA) is an oxidative phosphorylation disorder, with primary clinical manifestations of myopathic exercise intolerance and a macrocytic sideroblastic anemia. One cause of MLASA is recessive mutations in PUS1, which encodes pseudouridine (Ψ) synthase 1 (Pus1p). Here we describe a mouse model of MLASA due to mutations in PUS1. As expected, certain Ψ modifications were missing in cytoplasmic and mitochondrial tRNAs from Pus1−/− animals. Pus1−/− mice were born at the expected Mendelian frequency and were non-dysmorphic. At 14 weeks the mutants displayed reduced exercise capacity. Examination of tibialis anterior (TA) muscle morphology and histochemistry demonstrated an increase in the cross sectional area and proportion of myosin heavy chain (MHC) IIB and low succinate dehydrogenase (SDH) expressing myofibers, without a change in the size of MHC IIA positive or high SDH myofibers. Cytochrome c oxidase activity was significantly reduced in extracts from red gastrocnemius muscle from Pus1−/− mice. Transmission electron microscopy on red gastrocnemius muscle demonstrated that Pus1−/− mice also had lower intermyofibrillar mitochondrial density and smaller mitochondria. Collectively, these results suggest that alterations in muscle metabolism related to mitochondrial content and oxidative capacity may account for the reduced exercise capacity in Pus1−/− mice.

Dual nature of pseudouridylation in U2 snRNA: Pus1p-dependent and Pus1p-independent activities in yeasts and higher eukaryotes

“…Pus1 knockout mice have been recently generated. In these mice, Pus1p-dependent modification of tRNAs was shown to Dual mechanism of positioning Ψ43 in U2 snRNA www.rnajournal.org 1061 be missing, but U2 snRNA was not tested (Mangum et al 2016). We analyzed U2 snRNA pseudouridylation in the Pus1 knockout mice and their wild-type siblings using RNA samples kindly provided by Jeffrey Patton and Diego Altomare (University of South Carolina).…”

The molecular genetics of sideroblastic anemia

“…65 Most PUS1 mutations are missense alleles, but 1 homozygous null sibling pair has been described, 66 indicating that PUS1 is not essential in mammals: a point supported by the mild phenotype and viability of Pus1 2/2 mice, which, like nearly all animal models of SA, do not develop bone marrow ring sideroblasts. 67 SIFD is a recently recognized entity caused by incomplete lossof-function mutations in the template-independent RNA polymerase TRNT1, also known as the CCA-adding enzyme, which catalyzes the sequential addition of cytosine, cytosine, and adenine ribonucleotides onto the 39 terminus of all cytosolic and mitochondrial tRNAs. 51,52 The tRNA 39 adenine resulting from TRNT1 activity is the substrate for aminoacyl-tRNAs synthetases such as YARS2, and tRNAs so charged are substrates for protein synthesis.…”

Role of gp130 in basal and exercise-trained skeletal muscle mitochondrial quality control

“…Serial transverse muscle sections (10 m) were cut from the midbelly of the tibialis anterior on a cryostat at Ϫ20°C and stored at Ϫ80°C until further analysis. Succinate dehydrogenase (SDH) enzyme activity was performed as previously described to determine myofiber oxidative capacity (36). Frozen sections were air dried at room temperature for 10 min, followed by incubation in a solution containing 0.2 M phosphate buffer (pH 7.4), 0.1 M MgCl 2, 0.2 M succinic acid, and 2.4 mM nitroblue tetrazolium at 37°C for 45 min.…”

“…Sections were then washed in distilled water for 3 min, dehydrated in 50% ethanol for 2 min, and mounted for viewing with mounting media. Digital images were acquired as previously described (36). The percentage of SDH-positive fibers was determined at ϫ20.…”