Alternative RNA splicing that is spatially regulated: generation of transcripts from the Antennapedia gene of Drosophila melanogaster with different protein-coding regions.

Abstract

We have shown previously that transcription of the Drosophila homeotic gene Antennapedia results in four major RNA species which differ in long 5'-and 3'-untranslated sequences. The protein-coding portion of these transcripts, however, is located in exons common to all. Using RNase protection assays and further cDNA clone isolation, we have now detected two alternative splicing events between exons of this region. These result in four RNA variations which, if translated, would encode a family of Antennapedia proteins. By analyzing transcripts from various developmental stages and isolated tissues, we show that alternative splicing is under strict temporal and spatial regulation. For example, while similar patterns of splicing were found for all wild-type thoracic imaginal disks examined, these differed distinctly from the patterns observed in neural tissues. Our results suggest that individual RNAs may be associated with different biological roles, and provide molecular evidence that the Antennapedia gene is involved in multiple functions.The Antennapedia (Antp) gene of Drosophila inelanogaster is a homeotic locus required for the proper development of thoracic segments. Mutants lacking this gene die as embryos, and cells of the thorax are transformed in an anterior direction (9,32,45,55). Antp also functions later in development in the imaginal disks (the presumptive adult tissues), promoting thoracic development while repressing the development of head structures (1,46,54). These roles are further exemplified by dominant mutations of this locus which cause abnormal activation of Antp expression in head imaginal disks. This ectopic transcription results in the transformation of head structures into thoracic tissues (12,22).Structurally, the Antp gene represents a complex transcriptional unit (22,27,48,53). It is composed of two functionally independent promoters, two polyadenylation sites, and eight exons spanning more than 100 kilobases (kb) of genomic DNA sequence (Fig. 1A). Four major classes of Antp transcripts are produced, two 5.0 and two 3.5 kb in size (Fig. 1B). These RNAs differ by extensive 5'-and 3'-untranslated sequences but share a common open reading frame (ORF) region involving exons 5 through 8.The localization of the Antp RNAs and proteins provides molecular evidence for its involvement in thoracic development. These products are found in a complex distribution pattern which undergoes dynamic changes as development proceeds. During early embryogenesis, both Antp RNAs and proteins are detected throughout the thoracic and abdominal regions, whereas by the end of this period and throughout the larval instar stages, these products are much more restricted to the thoracic segments (7,22,29,32,53,58). Within these domains of expression, Antp is active in both the ectoderm (epidermal and neural tissues) and the mesoderm. However, its products are usually detected in only a subset of the cells within a given region. (26,42). An example with consequences for Drosophila development is the finding that t...

Functional synthetic Antennapedia genes and the dual roles of YPWM motif and linker size in transcriptional activation and repression

“…The longlinker variant is expressed predominantly in embryonic stages, and equal amounts of both variants are detected in larval, pupal, and adult stages (17). Thus, different Antp transcripts might play distinct, albeit similar, roles in development.…”

“…When the YPWM motif is replaced by WRPW, a domain interacting with the generic transcriptional corepressor Groucho (Gro) (16), Antp repressive activity is retained in vivo. Finally, we evaluate the effect of either a long or a short linker between the YPWM motif and the HD, comprising eight or four amino acids, respectively, which occur naturally in different Antp isoforms (17), and find that they play an important role in transcriptional potency and/or specificity in vivo.…”

The roles of Drosophila cyclins A and B in mitotic control

“…Northern blots were probed with RNA probes synthesized with T7 polymerase from a linearized Bluescript plasmid containing the cyclin B cDNA as described by Stroeher et al (1988). Nylon filters were hybridized for 14 hr at 58°C in 50% formamide, 5× SSC, 50 mM NaPO 4 (pH 7.0), 5× Denhardt's solution, 0.1% SDS, 200 (μg/ml salmon sperm DNA.…”

Tissue-specific alternative splicing of the Drosophila dopa decarboxylase gene is affected by heat shock.

“…Examples of these genes include the vertebrate genes encoding c-Src (4,5,22,29), N-CAM (28,34), calcitonin/CGRP (32), and substance P/K (26). There exist similar alternatively spliced genes in the fruit fly, including neuroglian (12), antennapedia (41), Ultrabithorax (17), and dopa decarboxylase (25).…”