Characterization And Functional Analysis Of Drosophila Melanogaster Myosin Chaperone Unc 45 Dunc 45 Gene PDF Download

Drosophila melanogaster has a single myosin alkali light chain gene which encodes for two protein isoforms by developmentally regulated alternative splicing of the primary transcript. All six of the exons in the gene are present in the mRNA of larval muscles and the tubular and abdominal muscles of the adults. A novel mRNA species present exclusively in the adult and pupal Indirect Flight Muscle (IFM) lacks the fifth exon, thus encoding a MLC-ALK isoform with a variant carboxyl terminus. All introns of the transcript contain the established concensus splicing signals with the exception of intron 4. In this intron, a non-canonical polypurine stretch replaces the concensus polypyrimidine, rendering it a likely regulatory site. Because the transcripts are colinear with the gene throughout development the alternative splicing pattern in the IFM appears to be regulated at the level of splice site choice. The goal of this research is to identify the cis-regulatory sequences that control the choice between alternative larval and IFM-specific splicing pathways. I have developed a transient expression system for Drosophila Schneider 2 cultured cells utilizing the Drosophila metallothionein promoter to direct transcription of transfected MLC-ALK minigenes. This analysis demonstrated that the larval-specific splicing pathway represents the default splicing of the MLC-ALK transcripts. Analysis of mutant minigene transcripts revealed that splicing in the IFM-specific pathway is not the result of blockage or incapacitation of either splice acceptor or/and donor sequences flanking exon 5. The structures of the mutant mRNAs suggest that utilization of the IFM-specific pathway requires trans-acting factors which are absent in the cultured cells. Furthermore, analysis of mutant and hybrid minigene transcripts identified a unique cis-regulatory sequence proximal to the splice donor of intron 4, required for efficient utilization of the larval-specific splicing pathway. Mutations in intron 4 inhibit removal of the downstream intron 5 suggesting that an ordered pathway of intron removal is employed for larval-specific splicing. On the basis of these results a model of the mechanism of tissue and temporal regulation of alternative splicing of the MLC-ALK transcripts is presented.