IRES-mediated translation of utrophin A is enhanced by glucocorticoid treatment in skeletal muscle cells.
Glucocorticoids are currently the only drug treatment recognized to benefit Duchenne muscular dystrophy (DMD) patients. The nature of the mechanisms underlying the beneficial effects remains incompletely understood but may involve an increase in the expression of utrophin. Here, we show that treatment of myotubes with 6alpha-methylprednisolone-21 sodium succinate (PDN) results in enhanced expression of utrophin A without concomitant increases in mRNA levels thereby suggesting that translational regulation contributes to the increase. In agreement with this, we show that PDN treatment of cells transfected with monocistronic reporter constructs harbouring the utrophin A 5'UTR, causes an increase in reporter protein expression while leaving levels of reporter mRNAs unchanged. Using bicistronic reporter assays, we further demonstrate that PDN enhances activity of an Internal Ribosome Entry Site (IRES) located within the utrophin A 5'UTR. Analysis of polysomes demonstrate that PDN causes an overall reduction in polysome-associated mRNAs indicating that global translation rates are depressed under these conditions. Importantly, PDN causes an increase in the polysome association of endogenous utrophin A mRNAs and reporter mRNAs harbouring the utrophin A 5'UTR. Additional experiments identified a distinct region within the utrophin A 5'UTR that contains the inducible IRES activity. Together, these studies demonstrate that a translational regulatory mechanism involving increased IRES activation mediates, at least partially, the enhanced expression of utrophin A in muscle cells treated with glucocorticoids. Targeting the utrophin A IRES may thus offer an important and novel therapeutic avenue for developing drugs appropriate for DMD patients.
Miura
Pedro
P
Department of Cellular and Molecular Medicine, Centre for Neuromuscular Disease, University of Ottawa, Ottawa, Ontario, Canada.
Andrews
Meghan
M
Holcik
Martin
M
Jasmin
Bernard J
BJ
D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D008776:Methylprednisolone Hemisuccinate / Q000494:pharmacology*; D051379:Mice; D018482:Muscle, Skeletal / Q000187:drug effects* / Q000378:metabolism; D014176:Protein Biosynthesis / Q000187:drug effects*; D012333:RNA, Messenger / Q000235:genetics; D012270:Ribosomes / Q000378:metabolism*; D049411:Utrophin / Q000235:genetics*
D017931:DNA Primers; D012333:RNA, Messenger; C483600:Utrn protein, mouse; D049411:Utrophin; D008776:Methylprednisolone Hemisuccinate