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.

N-RAP expression during mouse heart development

“…2A-C). We previously showed that N-RAP exon 12 is not expressed in adult hearts (Mohiddin et al, 2003). By using the exon 9 -14 primers, we observed a single PCR product from cardiac tissue between E10.5 and P21 that is consistent with omission of exon 12; the cardiac transcript in this region is 105 bases smaller than the predominant product from adult skeletal muscle, which includes exon 12 (Fig.…”

“…N-RAP is an actin-binding LIM protein that is concentrated at the ends of striated muscle cells (Luo et al, 1997;Zhang et al, 2001;Mohiddin et al, 2003). The N-RAP gene structure and coding sequence are highly conserved between mice and humans.…”

“…The N-RAP gene structure and coding sequence are highly conserved between mice and humans. As illustrated in Figure 2A, in both species, the protein consists of an N-terminal LIM domain followed by repetitive actin-binding modules that are homologous to those found in nebulin (Mohiddin et al, 2003), a giant protein that is thought to act as a length-regulating template for sarcomeric actin filaments in skeletal muscle (Kruger et al, 1991;Labeit et al, 1991;Labeit and Kolmerer, 1995;Wang et al, 1996). The N-RAP repeats are arranged as a series of five C-terminal super repeats, each composed of seven single repeats.…”

“…In between the LIM domain and super repeats are 10 or 11 single repeats. Alternative splicing of N-RAP has been reported for exon 12 (Mohiddin et al, 2003;Gehmlich et al, 2004) and exon 39 (Gehmlich et al, 2004). Each of these exons encodes a single 35-to 36-residue module.…”

“…Each of these exons encodes a single 35-to 36-residue module. In both humans and mice, exon 12 is not expressed in adult cardiac muscle but is included in ϳ90% of the N-RAP transcripts in adult skeletal muscle; therefore, the full-length N-RAP isoforms that include or exclude exon 12 have been termed N-RAP-s and N-RAP-c, for skeletal and cardiac muscle, respectively (Mohiddin et al, 2003).…”