Fig. 3. Muscle-specific sequences in human and murine archvillin. (A) Schematic representation of archvillin cDNA and protein domain structure showing the amino acid similarity of each domain in the predicted proteins (percentages), the amino acid sequences of the two muscle-specific coding inserts, the five predicted nuclear targeting sequences (blue bars), the predicted coiled-coil domain (green patterned box) and the location of a 23 bp insert sequence found in clones M03 and M08 (asterisk). Gold shading denotes the relative extents of the two N-terminal muscle-specific inserts and the highly conserved C-terminal villin/gelsolin homology regions. If stable protein can be produced from clones M03 and M08, the C-terminal sequence after Q-1948 in mouse archvillin would be altered to ALFSFLWKILEVLTSRPACSSSAAPPETSLRQSLCTPHKRPLPSAPCLSCRRTCTARRSQLSSLLTTITRCTSGKAGGPLKTR, resulting in a smaller, more basic protein (227 kDa, pI=8.16) without the villin-like headpiece. (B) Fluorescence localization in C2C12 myotubes expressing a chimera of EGFP and murine archvillin insert 1 sequence (MAV 257-629, left) or EGFP alone (right) is consistent with the prediction that muscle insert 1 contains functional nuclear targeting sequences. Bar, 5 µm. (C) The muscle-specific upstream exon M-3 contains regions of high sequence similarity. Regions of 254 nt and 34 nt in exon M-3 that exhibit 88% and 94% identity, respectively, between human and mouse archvillin cDNAs are shown (arrows). Intron locations are denoted schematically by thick vertical bars, and intron sizes are shown. Exons –2 and –1 are present in both muscle and non-muscle cDNAs. Exon M-3 is consistently observed in muscle-specific cDNAs and in ESTs from muscle-rich tissues. Exon nomenclature was based on the location of the initiator AUG because the large size of the first muscle-specific intron and the growing number of ESTs with homology to other upstream genomic sequences suggest the potential for many other exons encoding supervillin/archvillin 5'-UTR sequences. Upstream open reading frames (uORFs), two of which are conserved across species (thick bars) are present in human (HAV) and murine (MAV) archvillin 5'-UTRs.