Stage-specific selection of alternative transcriptional initiation sites from the 5C actin gene of Drosophila melanogaster

Abstract

The transcription unit of the 5C actin gene exhibits a complex organization that is unique among the six actin genes of Drosophila melanogaster. Three different mRNA size classes showing distinct patterns of accumulation throughout development are detected on Northern blots. We have determined the structure of the various 5C actin transcripts by exon mapping using strand-specific RNA probes, primer extension analysis, and DNA sequences analysis of both cDNA and genomic clones. All the transcripts share a single protein-coding nucleotide sequence but are heterogeneous in the 5' and 3' untranslated regions. The 5' untranslated region of each transcript consists of either one of two small exons (exon 1 and exon 2) which are alternatively spliced to a single acceptor site 8 bp upstream from the translation initiation codon in exon 3. Results from primer extension analysis suggest that transcription can initiate from either exon 1 or exon 2, and also from a third site within exon 2. We detect an increase in the relative abundance of exon 1-containing transcripts at larval and pupal stages, as well as a change in the proportion of transcripts that initiate at either of the two exon 2 sites. Five polyadenylation sites have been found within three termination/processing regions that define the three size classes of polyadenylated transcripts. The results of our experiments indicate the existence in vivo of all possible combinations of 5' exon with 3' polyadenylation site. However, particular combinations of 5' initiation site and 3' polyadenylation site are preferred at certain developmental stages.