Alternative splicing of the primary transcript generates heterogeneity within the products of the gene for Bombyx mori chitinase

Abstract

The gene of chitinase in the silkworm, Bombyx mori, generates four mRNA products by alternative splicing. Nucleotide sequences of the entire gene for chitinase and respective cDNAs demonstrate that the pre-mRNA undergoes alternative splicing at both the 5' and 3' regions. At the 5' region, the pre-mRNA experienced differential splicing through two alternative 5'-intron consensus splicing sites. These products differ in the last amino acid of the signal peptide and the first amino acid of the mature N-terminal sequences: one with Cys(20)-Ala(21) and the other with Ser(20)-Asp(21). The product with Cys(20)-Ala(21) residues is one amino acid larger than the other with Ser(20)-Asp(21). At the 3' region the pre-mRNA of the chitinase gene undergoes alternative splicing in three different fashions. It is spliced either through retaining or excluding the upstream 121-bp direct repeat found at the 3' region of the coding sequences or through retaining or excluding of an insertion of 9 bp in a combinatorial manner. Retention or exclusion of the upstream 121-bp direct repeat results in a protein with a deduced amino acid sequence similar in size to the one retaining both direct repeats. However, exclusion of the insert of the 9 bp from the mRNA results in a protein with 22 extra amino acids. All of the mRNA products appear to be generated from a single gene as demonstrated by testing the 3' region of the genomic DNA and variant chitinase mRNA products. B. mori chitinase expression in the fifth instar larvae epidermal tissues appears to be developmentally regulated, but the phenomenon of alternative splicing of the pre-mRNA is not stage-dependent. Furthermore, the four mRNA products showed chitinase activity when expressed in Escherichia coli, which demonstrates the role of the alternative splicing process in generating multiple isoforms of the silkworm's chitinase.