Cloning And Function Analysis Of N-RAP And MOB: Two Novel Genes On Chromosome 10q

Cloning and function analysis of a novel gene: human nebulin-related anchoring protein (N-RAP)N-RAP (nebulin related anchoring protein) is a 185 kDa actin-binding LIM protein recently discovered in skeletal and cardiac muscle tissues in murine. It is proposed to serve as a link between the terminal actin of the myofibril and the protein complexes at the cell membrane and thus as an organizing center in the initial phase of myofibril assembly. In addition, the upregulation of N-RAP expression in MLP (muscle LIM protein) knockout as well as in TOT mouse (tropomodulin overexpressing transgenic mouse) might serve as an early marker for the development of DCM (dilated cardiomyopathy).But in human, the sequence and function of N-RAP remain unknown. In our previous study, suppression subtractive hybridization (SSH) and cDNA microarray hybridization were combined to identify genes whose expression is altered in normal adult and fetal skeletal muscle. To our interest, a new expressed sequence tag (EST) (GenBank accession number: BQ091951) similar to murine N-RAP gene was obtained. In this study, the full length of human N-RAP cDNA, which contains a 5088bp ORF (open reading frame), encodes a protein of 1695 amino acid residues, was successfully cloned. Human N-RAP was mapped to the genomic region between HABP2 and CASP7 on chromosome 10q25~q26, consisting of 41 exons and 40 introns. Corresponding EST sequences were found in muscle, heart, spinal cord and prostate tissue. Homology searches with the deduced 1695 amino acid protein sequence revealed human N-RAP shares 88% similarity with murine N-RAP, 90% with rat N-RAP, 63% with human Nebulin, 58% with rat Nebulin, 60% with chick Nebulin and 59% with murine Nebulin. The predicted protein contains LIM domain, which binds two zinc ions, does not bind DNA, seems to act as interface for protein-protein interaction, and nebulin repeats, tandem arrays of which are known to bind actin. PCR revealed human N-RAP was expressed in high abundane in adult brain, adult heart, and adult skeletal muscle, and in low abundance in adult bone marrow and fetal skeletal muscle, which is consistent with the in silico expression pattern identified by biomformatics analysis. Homology searches and domain query indicate that human N-RAP is a novel member of nebulin family, and an ortholog of N-RAP in human, suggesting it is related to myofibrillogenesis. In addition, human N-RAP has the cytosolic localization, which is consistent with bioinformatics prediction and, significantly, with its function requirement for taking part in myofibrilassembly.In conclusion, human N-RAP is proposed to be crucial for myoflbnllogenesis and the development of skeletal muscle, cardiac muscle and DCM.