Isolation, expression analysis and characterization of NEFA-interacting nuclear protein 30 and RING finger and SPRY domain containing 1 in skeletal muscle.
Muscle atrophy results from a range of physiological conditions, including immobilization, spinal cord damage, inflammation and aging. In this study we describe two genes, NEFA-interacting nuclear protein 30 (Nip30) and RING Finger and SPRY domain containing 1 (Rspry1), which have not previously been characterized or shown to be expressed in skeletal muscle. Furthermore, Nip30 and Rspry1 were transcriptionally induced in response to neurogenic muscle wasting in mice and were also found to be expressed endogenously at the RNA and protein level in C2C12 mouse muscle cells. Interestingly, during analysis of Nip30 and Rspry1 it was observed that these genes share a 230 base pair common regulatory region that contains several putative transcription regulatory elements. In order to assess the transcriptional activity of the Nip30 and Rspry1 regulatory regions, a fragment of the promoter of each gene was cloned, fused to a reporter gene, and transfected into cells. The Nip30 and Rspry1 reporters were both found to have significant transcriptional activity in cultured cells. Furthermore, the Nip30-Rspry1 common regulatory region contains a conserved E-box enhancer, which is an element bound by myogenic regulatory factors that function in the regulation of muscle-specific gene expression. Therefore, in order to determine if the predicted E-box was functional, Nip30 and Rspry1 reporters were transfected into cells ectopically expressing the myogenic regulatory factor, MyoD1, resulting in significant induction of both reporter genes. In addition, mutation of the conserved E-box element eliminated MyoD1 activation of the Nip30 and Rspry1 reporters. Finally, GFP-tagged Nip30 was found to localize to the nucleus, while GFP-tagged Rspry1 was found to localize to the cytoplasm of muscle cells.
University of North Florida, Department of Biology, 1 UNF Drive, Jacksonville, Florida, 32224. Electronic address: d.s.waddell@unf.edu.
David S
DS
Waddell
University of North Florida, Department of Biology, 1 UNF Drive, Jacksonville, Florida, 32224.
Paige J
PJ
Duffin
University of North Florida, Department of Biology, 1 UNF Drive, Jacksonville, Florida, 32224.
Ashley N
AN
Haddock
University of North Florida, Department of Biology, 1 UNF Drive, Jacksonville, Florida, 32224.
Virginia E
VE
Triplett
University of North Florida, Department of Biology, 1 UNF Drive, Jacksonville, Florida, 32224.
Jason J
JJ
Saredy
University of North Florida, Department of Biology, 1 UNF Drive, Jacksonville, Florida, 32224.
Karina M
KM
Kakareka
University of North Florida, Department of Biology, 1 UNF Drive, Jacksonville, Florida, 32224.
John T
JT
Eldredge
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D017124:Conserved Sequence; D003593:Cytoplasm / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D024721:E-Box Elements; D005786:Gene Expression Regulation; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000166:cytology / Q000502:physiology*; D009133:Muscular Atrophy / Q000235:genetics / Q000503:physiopathology; D017570:MyoD Protein / Q000235:genetics / Q000378:metabolism; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism; D017434:Protein Structure, Tertiary
D004268:DNA-Binding Proteins; D017570:MyoD Protein; C057363:MyoD1 myogenic differentiation protein; D009687:Nuclear Proteins; C000606810:Rspry1 protein, mouse; C000606809:nip30 protein, mouse
Atrophy; Gene expression; Gene regulation; Skeletal muscle