Alternative splicing of calretinin mRNA leads to different forms of calretinin

cDNA clones for calretinin, a member of the troponin-C family of calcium-binding proteins, were isolated from a cDNA library of the human colon carcinoma cell line WiDr. Sequence analysis revealed two forms of alternatively spliced calretinin mRNAs encoding C-terminally truncated proteins. Exon 7 was either spliced to exon 9 (delta 8) or to exon 10 (delta 8,9); both resulted in a frame shift and a translational stop at the second codon of exon 9 (delta 8), or at codon 15 of exon 10 (delta 8,9), respectively. The presence of delta 8 and delta 8,9 calretinin mRNA in WiDr cells was confirmed using reverse-transcriptase PCR and sequence analysis of the amplicon, as well as by a ribonuclease protection assay. Co115/3 and three other human colon carcinoma cell lines were found, by reverse-transcriptase PCR to also contain delta 8,9 calretinin mRNA. The truncated proteins were able to bind calcium, as evidenced by a calcium blot of the delta 8 form (calretinin-20k) and delta 8,9 form (calretinin-22k) expressed in Escherichia coli. Immunohistochemical staining using an antiserum specific for the novel C-terminus of calretinin-22k confirmed its presence in WiDr, Co115/3 and three additional colon carcinoma cell lines. The fact that alternative splicing of calretinin was found in five different cell lines suggests that alternatively spliced calretinins fulfill a physiological function.