title: Genomic organization, alternative splicing, and expression of human and mouse N-RAP, a nebulin-related LIM protein of striated muscle.
abstract: Linkage analysis identifies 10q24-26 as a disease locus for dilated cardiomyopathy (DCM), a region including the N-RAP gene. N-RAP is a nebulin-like LIM protein that may mediate force transmission and myofibril assembly in cardiomyocytes. We describe the sequence, genomic structure, and expression of human N-RAP, as well as an initial screen to determine whether N-RAP mutations cause cardiomyopathy. Human expressed sequence tag databases were searched with the published 3,528-bp mouse N-RAP open reading frame (ORF). Putative cDNA sequences were interrogated by direct sequencing from cardiac and skeletal muscle RNA. We identified two human N-RAP isoforms with ORFs of 5,085 bp (isoform C) and 5,190 bp (isoform S), encoding products of 193-197 kDa. Genomic database searches localize N-RAP to human chromosome 10q25.3 and match isoforms C and S to 41 and 42 exons. Only isoform C is detected in human cardiac RNA; in skeletal muscle, approximately 10% is isoform C and approximately 90% is isoform S. We investigated apparent differences between human N-RAP cDNA and mouse sequences. Two mouse N-RAP isoforms with ORFs of 5,079 and 5,184 bp were identified with approximately 85% similarity to human isoforms; published mouse sequences include cloning artifacts truncating the ORF. Murine and human isoforms have similar gene structure, tissue specificity, and size. N-RAP is especially conserved within its nebulin-like and LIM domains. We expressed both N-RAP isoforms and the previously described truncated N-RAP in embryonic chick cardiomyocytes. All constructs targeted to myofibril precursors and the cell periphery, and inhibited myofibril assembly. Several human N-RAP polymorphisms were detected, but none were unique to cardiomyopathy patients. N-RAP is highly conserved and exclusively expressed in cardiac and skeletal muscle. Genetic abnormalities remain excellent candidate causes for cardiac and skeletal myopathies.
authors.affiliation: National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892, USA. mohiddis@nhlbi.nih.gov
authors.forename: Saidi A
authors.initials: SA
authors.lastname: Mohiddin
authors.forename: Shajia
authors.initials: S
authors.lastname: Lu
authors.forename: John-Paul
authors.initials: JP
authors.lastname: Cardoso
authors.forename: Stefanie
authors.initials: S
authors.lastname: Carroll
authors.forename: Sanjaya
authors.initials: S
authors.lastname: Jha
authors.forename: Robert
authors.initials: R
authors.lastname: Horowits
authors.forename: Lameh
authors.initials: L
authors.lastname: Fananapazir
mesh_terms: D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D002642:Chick Embryo; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000235:genetics* / Q000378:metabolism; D018482:Muscle, Skeletal / Q000378:metabolism*; D009206:Myocardium / Q000378:metabolism*; D020033:Protein Isoforms; D017422:Sequence Analysis, DNA
chemical_list: D009124:Muscle Proteins; C108533:NRAP protein, human; C491567:Nrap protein, mouse; D020033:Protein Isoforms