Regulation of an antisense RNA with the transition of neonatal to IIb myosin heavy chain during postnatal development and hypothyroidism in rat skeletal muscle. Am J Physiol Regul Integr Comp Physiol 302: R854 -R867, 2012. First published January 18, 2012 doi:10.1152/ajpregu.00591.2011.-Postnatal development of fast skeletal muscle is characterized by a transition in expression of myosin heavy chain (MHC) isoforms, from primarily neonatal MHC at birth to primarily IIb MHC in adults, in a tightly coordinated manner. These isoforms are encoded by distinct genes, which are separated by ϳ17 kb on rat chromosome 10. The neonatal-to-IIb MHC transition is inhibited by a hypothyroid state. We examined RNA products [mRNA, pre-mRNA, and natural antisense transcript (NAT)] of developmental and adult-expressed MHC genes (embryonic, neonatal, I, IIa, IIx, and IIb) at 2, 10, 20, and 40 days after birth in normal and thyroid-deficient rat neonates treated with propylthiouracil. We found that a long noncoding antisense-oriented RNA transcript, termed bII NAT, is transcribed from a site within the IIb-Neo intergenic region and across most of the IIb MHC gene. NATs have previously been shown to mediate transcriptional repression of sense-oriented counterparts. The bII NAT is transcriptionally regulated during postnatal development and in response to hypothyroidism. Evidence for a regulatory mechanism is suggested by an inverse relationship between IIb MHC and bII NAT in normal and hypothyroid-treated muscle. Neonatal MHC transcription is coordinately expressed with bII NAT. A comparative phylogenetic analysis also suggests that bII NAT-mediated regulation has been a conserved trait of placental mammals for most of the eutherian evolutionary history. The evidence in support of the regulatory model implicates long noncoding antisense RNA as a mechanism to coordinate the transition between neonatal and IIb MHC during postnatal development. long noncoding antisense RNA; thyroid hormone; gene transcription; phylogenetic; RT-PCR SIX MYOSIN HEAVY CHAIN (MHC) genes, each encoding a specific motor protein impacting contractile velocity and hence muscle performance, are expressed across the spectrum of skeletal muscle of mammals. In the rat the genes of the three fast MHC types, IIa, IIx, and IIb MHC, are clustered on chromosome 10 in a tandem alignment (IIa-IIx-IIb). These MHCs, along with the slow type I MHC, are the primary adult-specific isoforms. Also located on this genomic locus are the embryonic (Emb), situated 5= to IIa, and the neonatal (Neo), located 3= to IIb. The Emb and Neo are the predominant MHC isoforms expressed during fetal and early postnatal development (63). Postnatal development is characterized by replacement of these developmental isoforms with the adult isoforms of MHC, in a highly regulated and precisely coordinated manner, such that individual muscles attain various proportions of each MHC isoform to confer optimal function for a given usage pattern. Thyroid hormone, which is essential to normal development an...