Physiological apoptosis of polar cells during Drosophila oogenesis is mediated by Hid-dependent regulation of Diap1.

Although much has been learned in recent years about the apoptotic machinery, the mechanisms underlying survival and death choices during development of metazoans remain less clearly understood. During early oogenesis in Drosophila, a small excess in the number of specialized somatic cells, called polar cells (PCs), produced at follicle extremities is reduced to exactly two cells through apoptosis by mid-oogenesis. We have found that PCs destined to die first lose their apical contacts and then round up and shrink progressively until they disappear. Caspases are activated only once the cells have begun to shrink, suggesting that they are implicated in this part of the process, but not in the initial loss of cell polarity. Loss-of-function analyses based on mutant, clonal and RNAi approaches show that among the RHG family of pro-apoptotic factors, Hid is specifically necessary for PC apoptosis, as well as the initiator caspase Dronc and its adaptor Dark/Apaf-1, and likely several effector caspases, in particular Drice. In addition, we show that Hid protein and transcripts accumulate specifically in PCs destined to die, while the anti-apoptotic factor Diap1 is downregulated in these cells in a hid-dependent manner. Therefore, our results implicate the Hid-Diap1 module as an important regulatory point in a developmental case of apoptosis.

Regulated splicing of an alternative exon of beta-tropomyosin pre-mRNAs in myogenic cells depends on the strength of pyrimidine-rich intronic enhancer elements.

Alternative splicing of chicken beta-tropomyosin (beta-TM) pre-mRNAs ensures that in nonmuscle cells, only exon 6A is expressed, whereas in skeletal muscle, exon 6B is utilized preferentially. We have previously shown that efficient splicing of the nonmuscle exon 6A requires two pyrimidine-rich splicing enhancers (S4 and I5Y) that are present in the introns flanking exon 6A. Here, we examined the function of the S4 and I5Y elements by replacing them within beta-TM minigenes by other pyrimidine- and purine-rich sequence elements and analyzing splicing in transfected quail nonmuscle and muscle cells. Several features of these splicing regulatory elements were revealed by this study. First, a wide variety of pyrimidine-rich sequences can replace the intronic S4 splicing enhancer, indicating that pyrimidine composition, rather than sequence specificity, determines activity for this element. Second, one type of purine-rich sequence (GARn), normally found within exons, can also replace the S4 splicing enhancer. Third, the diverse elements tested exhibit differential activation of the splice sites flanking exon 6A and different positional constraints. Fourth, the strength of the S4 splicing enhancer is appropriately set to obtain proper regulation of the transition from exon 6A splicing in myoblasts to exon 6B splicing in myotubes, but this splicing regulatory element is not the target for cell-type-specific splicing factors.

Sequence divergence associated with species-specific splicing of the nonmuscle beta-tropomyosin alternative exon.

Alternative splicing of vertebrate beta-tropomyosin transcripts ensures mutually exclusive expression of internal exons 6A and 6B in nonmuscle and skeletal muscle cells, respectively. Recently, we reported that this splicing regulation requires species-specific elements, since the splicing profile for the chicken, rat, and Xenopus beta-tropomyosin alternative exons is not reproduced in transfection experiments when heterologous myogenic cells are used. By analyzing the splicing pattern of hybrid chicken/rat beta-TM constructions transfected into both quail and mouse cell lines, we demonstrate that chicken beta-tropomyosin exon 6A is flanked by stronger splicing signals than rat exon 6A, thus leading to the misregulation of splicing in heterologous cells. We have characterized three splicing signals that contribute to this difference: 1) nonconsensus nucleotide differences at positions +4 and +6 in the donor site downstream of exon 6A, 2) differences in the pyrimidine composition between the branch site and acceptor site upstream of exon 6A, and 3) a pyrimidine-rich intronic exon 6A splicing enhancer present upstream of exon 6A only in the chicken beta-TM gene. The functional divergence between splicing signals in two homologous vertebrate genes reveals species-specific strategies for proper modulation of splicing of alternative exons.

Splicing of the alternative exons of the chicken, rat, and Xenopus beta tropomyosin transcripts requires class-specific elements.

The diversity of protein isoforms is often generated from single genes by alternative splicing of the primary transcript. Using transfection of beta tropomyosin minigene constructs into homologous and heterologous cell systems, we show that there are differences, among higher vertebrates, in the components of the splicing machinery which control the conserved regulated splicing pattern of two mutually exclusive exons (6A and 6B) present in this gene. These experiments demonstrate that genes which give rise to alternative transcripts may require an appropriate combination of splicing factors which are species-specific, or at least restricted to the same taxonomic subgroup (class). An important practical implication is that the splicing of these genes may be deregulated in heterologous systems in vitro and in vivo, i.e. in transgenic animals.

Splicing of retrotransposon insertions from transcripts of the Drosophila melanogaster vermilion gene in a revertant.

A mutation of the Drosophila melanogaster vermilion (v) gene known as v1 is caused by the insertion of a 412 retrotransposon into the 5' untranslated region of the first exon. Mutants carrying this insertion accumulate a low level of mRNA from which most of the transposon sequences have been eliminated by splicing at cryptic sites within transposon sequences. Here, we demonstrate that a revertant of the v1 allele called v+37 is caused by the insertion of a second retrotransposon, the B104/roo element, into a site near one end of the 412 element. The revertant strain accumulates a higher level of mRNA from which most of both transposons have been removed by splicing at new donor sites introduced by the B104/roo insertion and the same acceptor site within 412. Mutations at suppressor of sable [su(s)], which increase the accumulation of v1 transcripts, slightly elevate the level of v+37 RNA. In addition, we show that the first v intron downstream of the 412 insertion is not efficiently removed in the v1 mutant, and suppressor and reversion mutations increase the proportion of transcripts that are properly spliced at that downstream intron. Thus, it appears that both the suppressor and reversion mutations exert an effect at the level of pre-mRNA splicing.

A retrotransposon 412 insertion within an exon of the Drosophila melanogaster vermilion gene is spliced from the precursor RNA.

Three alleles of the Drosophila melanogaster vermilion (v) gene are suppressed by recessive mutations at the suppressor of sable [su(s)], gene. Previous work has established that these alleles have identical insertions of the 412 retrotransposon in the 5'-untranslated region of the gene. Despite the transposon insertion in an exon, v mutants accumulate trace amounts of apparently wild-type-sized transcripts in a su(s)+ background, and the level of v transcript accumulation is increased by su(s) mutations. Here, we have characterized transcripts from a suppressible v mutant in both su(s)+ and su(s)- backgrounds by S1 nuclease protection experiments and sequence analysis of polymerase chain reaction (PCR) generated cDNA clones. We find that transposon sequences are imprecisely eliminated from v mutant transcripts by splicing at donor and acceptor sites located near the ends of the 412 retrotransposon. Four different 5' donor sites are alternatively spliced to a single 3' acceptor site. The implications of this finding are discussed in relation to possible functions of the su(s)+ gene product.

Structure and transcription of the Drosophila melanogaster vermilion gene and several mutant alleles.

The nucleotide sequence and intron-exon structure of the Drosophila melanogaster vermilion (v) gene have been determined. In addition, the sites of several mutations and the effects of these mutations on transcription have been examined. The major v mRNA is generated upon splicing six exons of lengths (5' to 3') 83, 161, 134, 607, 94, and 227 nucleotides (nt). A minor species of v mRNA is initiated at an upstream site and has a 5' exon of at least 152 nt which overlaps the region included in the 83-nt exon of the major v RNA. The three v mutations, v1, v2, and vk, which can be suppressed by mutations at suppressor of sable, su(s), are insertions of transposon 412 at the same position in exon 1, 36 nt downstream of the major transcription initiation site. Despite the 7.5-kilobase insertion in these v alleles, a reduced level of wild-type-sized mRNA accumulates in suppressed mutant strains. The structure and transcription of several unsuppressible v alleles have also been examined. The v36f mutation is a B104/roo insertion in intron 4 near the splice donor site. A mutant carrying this alteration accumulates a very low level of mRNA that is apparently polyadenylated at a site within the B104/roo transposon. The v48a mutation, which deletes approximately 200 nt of DNA, fuses portions of exons 3 and 4 without disruption of the translational reading frame. A smaller transcript accumulates at a wild-type level, and thus an altered, nonfunctional polypeptide is likely to be synthesized in strains carrying this mutation. The v(H2a) mutants has a P element insertion in exon 6 within the coding region.