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- 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.

Purification and Characterization of a Novel Isozyme of Chitinase fromBombyx mori

“…58) We also proposed an alternative splicing mechanism of B. mori chitinase, by which some chitinase isozymes can be produced at the mRNA level from one gene. 59) In addition to alternative splicing, we found post-translational processing of B. mori chitinase at the protein level in vitro during long storage: 88-kDa chitinase to 65-kDa chitnase, and 65-kDa chitinase to 54-kDa chitnase. 28) Therefore, it is possible that 75-kDa chitnase is produced by post-translational processing, probably from 88-kDa chitinase, in vivo.…”

In silicoIdentification of Novel Chitinase-Like Proteins in the Silkworm,Bombyx mori, Genome

“…The presence of this splicing mechanism in the B. mori chitinases also contributes to the interpretation of the variations reported in studies of the insect's cDNAs. All of the mRNA products are from a single gene and functionally active (Babiker et al 2002). Mikitani et al (2000) found a novel DNA type transposon in BmCht5 that shows similarity to the TC-like transposable element.…”

Transcriptome Analysis of the Silkworm (Bombyx mori) by High-Throughput RNA Sequencing

“…Only a few AS events have been reported in silkworm. The genes that are known to be alternatively spliced include those that encode antitrypsin [25], arginases [26], lipophorin receptor [27], transformer-2 [28], chitinase [29], annexin IX [30], and GATA-type transcription factors [31]. By aligning ESTs with the silkworm genomic sequence, Xia et al [32] detected 277 AS forms in 235 silkworm genes.…”