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The Drosophila suppressor-of-white-apricot [su(wa)] protein regulates/modulates at least two somatic RNA processing events. It is a potent regulator of its own expression. We report here new studies of this autoregulatory circuit. Among other things, our studies show the following. First, new evidence that su(wa) expression is autoregulated at the level of pre-mRNA splicing is reported. su(wa) protein represses accumulation of the fully spliced su(wa) mRNA encoding it and promotes accumulation of high levels of incompletely spliced su(wa) pre-mRNA. Second, the fully spliced su(wa) mRNA is sufficient for all known su(wa) genetic functions indicating that it encodes the sole su(wa) protein. Third, the incompletely spliced su(wa) pre-mRNAs resulting from autoregulation are not translated (probably as a result of nuclear retention) and apparently represent nonfunctional by-products. Fourth, the special circumstances of su(wa) expression during oogenesis allows maternal deposition exclusively of fully spliced su(wa) mRNA. Fifth, su(wa) protein immunolocalizes to nuclei consistent with its being a direct regulator of pre-mRNA processing. We discuss the implications of our results for mechanisms of splicing regulation and for developmental control of su(wa) expression.

Regulators responsible for the pervasive, nonsex-specific alternative pre-mRNA splicing characteristic of metazoans are almost entirely unknown or uncertain. We describe here a novel family of splicing regulators present throughout metazoans. Specifically, we analyze two nematode (Caenorhabditis elegans) genes. One, CeSWAP, is a cognate of the suppressor-of-white-apricot (DmSWAP) splicing regulator from the arthropod Drosophila. Our results define the ancient, conserved SWAP protein family whose members share a colinearly arrayed series of novel sequence motifs. Further, we describe evidence that the CeSWAP protein autoregulates its levels by feedback control of splicing of its own pre-mRNA analogously to the DmSWAP protein and as expected of a splicing regulator. The second nematode gene, Ceprp21, encodes an abundant nuclear cognate of the constitutive yeast splicing protein, prp21, on the basis of several lines of evidence. Our analysis defines prp21 as a second novel, ancient protein family. One of the motifs conserved in prp21 proteins--designated surp--is shared with SWAP proteins. Several lines of evidence indicate that both new families of surp-containing proteins act at the same (or very similar) step in early prespliceosome assembly. We discuss implications of our results for regulated metazoan pre-mRNA splicing.

We characterize spliceosome assembly in Drosophila embryonic nuclear extracts. Further, we show that these extracts contain high levels of a 5' to 3' exoribonuclease activity allowing rapid, convenient protection mapping of 5' splice site and branchpoint sequences. We use this assay to show, for the first time, that a regulated arthropod intron uses a remote branchpoint strikingly similar in structure to those observed previously in regulated vertebrate introns. These results provide new evidence that both regulated and constitutive splicing are similar in detail in vertebrates and arthropods indicating that the powerful genetic systems for analysis of splicing regulation in Drosophila are likely to be directly informative for regulated splicing throughout metazoa. In addition, we report formation of a novel class of intron-dependent complexes. Behavior of these complexes indicates that they represent a mutually exclusive, kinetically competing pathway with spliceosome assembly. We propose that this competition represents the basis for a kinetic proofreading mechanism enhancing fidelity of intron recognition. We also discuss possible implications of this model for regulated splicing.

The copia insertion responsible for the wamutation is 3' to the white promotor and in the same transcriptional orientation as white. First, we have analyzed the effects of the wa copia insertion on levels of polyadenylated white transcripts and find large, developmentally programmed effects. Second, we have isolated and sequenced an LTR-excision event involving the copia insertion at wa. This represents the first documented case of an LTR-excision event in Drosophila. This single copia LTR has developmentally programmed effects on white transcript levels qualitatively similar to the intact copia element. Third, we have characterized the structures of white transcripts from wa. We find polyadenylated white transcripts apparently having 3' termini in or near the 3' LTR of the wa copia insertion, as has been reported in limited studies of wa transcription in adults by others. These earlier studies also revealed wa transcripts apparently corresponding to polyadenylated terminus formation in the 5' LTR of the copia transposon; however, our more detailed studies reveal that these transcripts probably have other origins and that little, if any, polyadenylated terminus formation for white transcripts occurs in the 5' LTR of the wa copia insertion. Moreover, we find no polyadenylated terminus formation for white transcripts occurring in the single LTR of the wa LTR-excision product. Fourth, we find that each of three mutant alleles at su(wa) produces elevated levels of several classes of RNAs apparently corresponding to transcriptional readthrough of the wa copia transposon. Elevated levels of one presumptive readthrough transcript were observed previously in one su(wa)1 mutant strain. Fifth, we have confirmed the existence of a transcript initiated in the 3' LTR of the wa copia insertion and find the levels of this transcript to be strongly influenced by developmental stage and genetic background. Lastly, we have analyzed white transcripts produced by the whd 81b11 allele, which carries an insertion of copia in the opposite transcriptional orientation and in a different position than the wa copia insertion. In contrast to the wa copia insertion allele, the whd 81b11 allele produces polyadenylated white transcript levels very similar to the w + case at the stages examined. Moreover, the whd 81b11 copia element apparently produced polyadenylated terminus formation in white transcripts and we observe no effect of the allelic state of su(wa) on apparent readthrough of this stop site. We discuss some possible implications of our results for the properties of retrotransposons as developmentally complex insertional mutagens and for the functional organization of the copia transcription unit.

A new mutant allele (wDZL)at the white locus of Drosophila melanogaster is dominant to the wild-type allele, but apparently only when the two alleles are synapsed. When chromosomal rearrangements prevent somatic pairing between the two white alleles, wDZL is rendered recessive to wild type. This observation suggests that the dominance of wDZL is sensitive to a synapsis (transvection) effect. On the basis of this and other properties, it is proposed that wDZL causes the repression of transcription of a synapsed w+ allele, but not of a w+ allele elsewhere in the same nucleus. One model to account for this supposes that wDzL produces a repressor of white-locus transcription. This repressor is presumed to be so unstable that other white genes, removed from wDZL but in the same nucleus, are not detectably repressed. These properties may be simply understood if it is assumed that the repressor produced by the wDZL allele is an RNA molecule.