High levels of intron-containing RNAs are associated with expression of the Drosophila DOPA decarboxylase gene.

Abstract

We have examined the structure and expression during embryonic development of the Drosophila DOPA decarboxylase gene, Ddc. The Ddc gene is transcribed to make at least five different size classes of RNA. These RNA species first appear late in embryogenesis, coincident with induction of Dde enzyme activity. The most abundant and smallest RNA appears to be Ddc mRNA. The sequences encoding this RNA are split by two intervening sequences. Each of the larger RNA species contains some or all of the intervening sequences. We have noted two unusual features of Ddc expression during embryogenesis. First, the intervening-sequencecontaining RNAs are present as 20% or more of the polyadenylated Dde RNA molecules, an exceptionally high proportion. Second, these RNAs do not disappear as rapidly as Ddc mRNA after Ddc enzyme activity reaches fully induced levels. These observations indicate slow rates of RNA processing relative to mRNA half-life and suggest that post-transcriptional steps participate in regulating Ddc expression. Although four of the five RNA species were detected at multiple developmental stages during which Dde is expressed, one was found uniquely during embryogenesis. This RNA differs from Ddc mRNA in length and in time of expression during embryogenesis but is transcribed in the same orientation and from the same genomic sequences as the Ddc primary transcript.

Sequence and structure of the dopa decarboxylase gene of Drosophila: evidence for novel RNA splicing variants.

“…The sequence and hybridization properties of the longest cDNA indicate that it corresponds to the 2.3-kb RNA. We conclude that the 2.3-kb RNA is not a precursor of the 2.0-kb species as has been proposed (Beall and Hirsh, 1984) but rather is an alternative processing variant of Ddc mRNA. These results suggest a detailed model for the structure of the various Ddc RNAs and surprisingly offer evidence that translation of the 2.3-kb and 2.0-kb species may lead to the production of two distinct DDC protein isoforms.…”

“…Of 24 positive plaques detected, five were shown to bear recombinant inserts three of which appeared to represent sizeable Ddc cDNA inserts. These observations suggest an RNA abundance of 1.2 x 10-3% which is considerably lower than the 0.1-0.01 % estimated by Hirsh and Davidson (1981) (Figure 1) hybridizes to the 2.3-but not the 2.0-kb Ddc RNA (Beall and Hirsh, 1984;Gietz and Hodgetts, 1985;Swiderski and O'Connor, personal com-munication; unpublished observations), we conclude that this cDNA represents the 2.3-kb RNA. Since all of the RNA species hybridize to the EcoRIlEcoRI fragment located between 387 and 1139 and shown in Figure 1 (Beall and Hirsh, 1984;Gietz and Hodgetts, 1985) while neither of the cDNAs hybridize to this fragment, we conclude that the small 5' exon is not included in the cDNAs.…”

“…Excision of the 77-base intron and the 1031-base intron generates a single ORF, the majority of which comprises the large 3' exon. Beall and Hirsh (1984) have proposed that the 2.3-kb RNA is a processing intermediate leading to a mature 2.0-kb message and have proposed a structure for the 2.0-kb message on the basis of RNA blotting and R-loop analysis. We disagree with this interpretation because both the 2.0-and 2.3-kb RNA species are found on polysomes in approximately equal proportions (Gietz and Hodgetts, 1985), suggesting that both are translated, and because the cDNA indicates that the 2.3-kb mRNA exhibits all the elements of an mRNA.…”

“…Mutant adults carrying these alleles show learning disabilities under restrictive conditions attesting to the functional requirement for DDC activity in the central nervous system. RNA blots probed for Ddc transcripts reveal RNA species of 4.0, 3.0, 2.7, 2.3 and 2.0 kb (Beall and Hirsh, 1984;Gietz and Hodgetts, 1985). Based on a proposed gene structure determined by R-looping and blotting experiments, the 2.3-kb, the 2.7-kb and the 3.0-kb RNA species have all been interpreted as processing intermediates leading from a single 4.0-kb primary transcript to a single mature mRNA of 2.0 kb (Beall and Hirsh, 1984;Gietz and Hodgetts, 1985).…”

“…RNA blots probed for Ddc transcripts reveal RNA species of 4.0, 3.0, 2.7, 2.3 and 2.0 kb (Beall and Hirsh, 1984;Gietz and Hodgetts, 1985). Based on a proposed gene structure determined by R-looping and blotting experiments, the 2.3-kb, the 2.7-kb and the 3.0-kb RNA species have all been interpreted as processing intermediates leading from a single 4.0-kb primary transcript to a single mature mRNA of 2.0 kb (Beall and Hirsh, 1984;Gietz and Hodgetts, 1985). Gene transfer experiments show that all the sequences necessary for proper developmental expression of Ddc function are contained within a 7.2-kb fragment of genomic DNA (Scholnick et al, 1983;Marsh et al, 1985).…”