10 research outputs found

    Loss of peroxiredoxin-2 exacerbates eccentric contraction-induced force loss in dystrophin-deficient muscle

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    Force loss in skeletal muscle exposed to eccentric contraction is often attributed to injury. We show that EDL muscles from dystrophin-deficient mdx mice recover 65% of lost force within 120 min of eccentric contraction and exhibit minimal force loss when the interval between contractions is increased from 3 to 30 min. A proteomic screen of mdx muscle identified an 80% reduction in the antioxidant peroxiredoxin-2, likely due to proteolytic degradation following hyperoxidation by NADPH Oxidase 2. Eccentric contraction-induced force loss in mdx muscle was exacerbated by peroxiredoxin-2 ablation, and improved by peroxiredoxin-2 overexpression or myoglobin knockout. Finally, overexpression of γcyto- or βcyto-actin protects mdx muscle from eccentric contraction-induced force loss by blocking NADPH Oxidase 2 through a mechanism dependent on cysteine 272 unique to cytoplasmic actins. Our data suggest that eccentric contraction-induced force loss may function as an adaptive circuit breaker that protects mdx muscle from injurious contractions

    RNA targeting with CRISPR–Cas13

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    RNA has important and diverse roles in biology, but molecular tools to manipulate and measure it are limited. For example, RNA interference1-3 can efficiently knockdown RNAs, but it is prone to off-target effects4, and visualizing RNAs typically relies on the introduction of exogenous tags5. Here we demonstrate that the class 2 type VI6,7 RNA-guided RNA-targeting CRISPR-Cas effector Cas13a8(previously known as C2c2) can be engineered for mammalian cell RNA knockdown and binding. After initial screening of 15 orthologues, we identified Cas13a from Leptotrichia wadei (LwaCas13a) as the most effective in an interference assay in Escherichia coli. LwaCas13a can be heterologously expressed in mammalian and plant cells for targeted knockdown of either reporter or endogenous transcripts with comparable levels of knockdown as RNA interference and improved specificity. Catalytically inactive LwaCas13a maintains targeted RNA binding activity, which we leveraged for programmable tracking of transcripts in live cells. Our results establish CRISPR-Cas13a as a flexible platform for studying RNA in mammalian cells and therapeutic development.National Institute of Mental Health (U.S.) (Grant 5DP1-MH100706)National Institute of Mental Health (U.S.) (Grant 1R01-MH110049

    Disease-proportional proteasomal degradation of missense dystrophins

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