Brain and muscle express a unique alternative transcript of alpha II spectrin

Alternative splicing of pre-mRNA transcripts of alpha and beta spectrin has emerged as an important generator of diversity in this gene family, yet th e functional consequences and extent of this diversity remains unknown. We have cloned and characterized full-length alpha II spectrin cDNA from human fetal brain (GenBank U83867 and U26396). On the basis of the predicted ami no acid sequence, 11 amino acid substitutions, presumably representing poly morphisms, have been identified that distinguish this alpha II spectrin fro m human lung fibroblast aII spectrin. In addition, human fetal brain spectr in displays a novel five amino acid insertion in repeat 15 that arises from alternative mRNA splicing and that distinguishes this spectrin from lung f ibroblast aII spectrin. This discovery, together with two previously identi fied regions of alternative mRNA splicing in (alpha II spectrin suggest tha t as many as eight different splice forms of the mature protein might exist if all combinations (at inserts I, 2, and 3) of alternative mRNA splicing are utilized. To assess this possibility, the tissue distribution of altern ative exon usage was investigated by semiquantitative PCR with intron-jumpi ng primer sets. Tissues examined were from mouse and included heart, kidney , lung, liver, thymus, spleen, brain, ovary, testis, and skeletal muscle, a s well as mouse embryonic tissue. Transcripts both with and without insert 1, representing a 60 bp insertion within (alpha II spectrin repeat 10, were identified in all tissues. In contrast, transcripts with insert 2, the nov el 15 bp insertion reported here, were only expressed in brain, heart, skel etal muscle, and embryonic tissue. In all tissues examined only transcripts positive for insert 3, an 18 bp insertion in repeat 21, were amplified, ev en under conditions in which a 30% level of insert 3 negative transcript co uld be easily detected in artificially prepared control samples. All combin ations of insert 1 and insert 2 were identified together in individual tran scripts, verifying at least four distinct isoforms of alpha II spectrin. Th ese have been named alpha II Sigma 1 through alpha II Sigma 4, in accord wi th current spectrin naming conventions. Dynamic molecular modeling of the 1 5th repeat unit incorporating insert 2 predicts that the spliced sequence f orms a loop between helices A and B, and suggests that this insert might co nstitute a novel protein interaction site. The presence of this sequence in alpha II Sigma 3 and alpha II Sigma 4 spectrin suggests a specialized and heretofore unanticipated function for the 15th repeat of this molecule.