The gene fl(2)d is needed for the sex-specific splicing of transformer pre-mRNA but not for double-sex pre-mRNA in Drosophila melanogaster

 In Drosophila melanogaster, regulation of the sex determination genes throughout development occurs by sex-specific splicing of their products. The first gene is Sex-lethal(Sxl). The downstream target of Sxl is the gene transformer (tra): the Sxl protein controls the female-specific splicing of the Tra pre-mRNA. The downstream target of the gene tra is the gene double-sex (dsx): the Tra protein of females, controls the female-specific splicing of the Dsx pre-mRNA. We have identified a gene, female-lethal-2-d fl(2)d, whose function is required for the female-specific splicing of Sxl pre-mRNA. In this report we analyze whether the gene fl(2)d is also required for the sex-specific splicing of both Tra and Dsx pre-mRNAs. We found that the Sxl protein is not sufficient for the female-specific splicing of Tra pre-mRNA, the fl(2)d function also being necessary. This gene, however, is not required for the female-specific splicing of Dsx pre-mRNA. Received:23 May 1996 Accepted:3 July 1996     

The gene fl(2)d is required for various Sxl-controlled processes in Drosophila females

Summary In Drosophila melanogaster, the gene Sex-lethal (Sxl) controls the processes of sex determination, dosage compensation, oogenesis and sexual behaviour. The control of Sxl is by alternative splicing of its primary RNA. We have identified a gene, female-lethal-2-d (fl(2)d), which is needed for the female-specific splicing of Sxl RNA and which also has a vital function independent of Sxl. Here we analyse other aspects of the gene fl(2)d. Specifically, we have analysed the effect of the temperature-sensitive mutation fl(2)d ¹ on the viability of adult flies homozygous for this mutation. We have found that the viability of the mutant females is reduced, while that of the mutant males is not affected. In addition, the capacity of the mutant females to be inseminated is considerably reduced, whilst all the mutant males are able to inseminate females. These effects on females are suppressed by Sxl ^M1. However, the fat body cells of fl(2)d ¹ homozygous females are able to synthesize yolk proteins at the restrictive temperature. We have also carried out, in males, a clonal analysis of fl(2)d ², a mutation lethal in both sexes. We have found that the clones are fully viable. We conclude that the gene fl(2)d seems to be necessary during the adult life of females for the processes that require Sxl ⁺ activity. Moreover, the Sxl-independent vital function of fl(2)d seems to be required in both sexes only during larval development. Offprint requests to: L. Sánchez     

The mechanism of sex-specific splicing at the doublesex gene is different between Drosophila melanogaster and Bombyx mori

We have previously reported that Bmdsx, a homologue of the sex-determining gene, doublesex (dsx), was found to be sex-specifically expressed in various tissues at larval, pupal, and adult stages in the silkworm, Bombyx mori, and was alternatively spliced to yield male- and female-specific mRNAs. To reveal sex-specific differences in splicing patterns of Bmdsx pre-mRNA, the genomic sequence was determined and compared with male- and female-specific Bmdsx cDNA sequences. The open reading frame (ORF) consisted of five exons. Exons 3 and 4 were specifically incorporated into the female type of Bmdsx mRNA. On the other hand, exon 2 was spliced to exon 5 to produce the male type mRNA of Bmdsx. As in the case of Drosophila dsx, the OD2 domain was separated by a female-specific intron into sex-independent and sex-dependent regions. Sex-specific splicing occurred in equivalent positions in the Drosophila dsx gene. However, unlike Drosophila dsx, the female-specific introns showed no weak 3′ splice sites, and the TRA/TRA-2 binding site related sequences were not found in the female-specific exon, nor even in any other regions of the Bmdsx gene. Moreover, an in vitro splicing reaction consisting of HeLa cell nuclear extracts showed that the female-type of Bmdsx mRNA represented the default splicing. These findings suggest that the structural features of the sex-specific splicing patterns of Bmdsx pre-mRNA are similar to those of Drosophila dsx but the regulation of sex-specific alternative splicing of Bmdsx pre-mRNA is different.     

BCAS2 is essential for Drosophila viability and functions in pre-mRNA splicing

Here, we show that dBCAS2 (CG4980, human Breast Carcinoma Amplified Sequence 2 ortholog) is essential for the viability of Drosophila melanogaster. We find that ubiquitous or tissue-specific depletion of dBCAS2 leads to larval lethality, wing deformities, impaired splicing, and apoptosis. More importantly, overexpression of hBCAS2 rescues these defects. Furthermore, the C-terminal coiled-coil domain of hBCAS2 binds directly to CDC5L and recruits hPrp19/PLRG1 to form a core complex for splicing in mammalian cells and can partially restore wing damage induced by knocking down dBCAS2 in flies. In summary, Drosophila and human BCAS2 share a similar function in RNA splicing, which affects cell viability.     

Human splicing factor SF3a, but not SF1, is essential for pre-mRNA splicing in vivo

The three subunits of human splicing factor SF3a are essential for the formation of the functional 17S U2 snRNP and prespliceosome assembly in vitro. RNAi-mediated depletion indicates that each subunit is essential for viability of human cells. Knockdown of single subunits results in a general block in splicing strongly suggesting that SF3a is a constitutive splicing factor in vivo. In contrast, splicing of several endogenous and reporter pre-mRNAs is not affected after knockdown of SF1, which functions at the onset of spliceosome assembly in vitro and is essential for cell viability. Thus, SF1 may only be required for the splicing of a subset of pre-mRNAs. We also observe a reorganization of U2 snRNP components in SF3a-depleted cells, where U2 snRNA and U2-B' are significantly reduced in nuclear speckles and the nucleoplasm, but still present in Cajal bodies. Together with the observation that the 17S U2 snRNP cannot be detected in extracts from SF3a-depleted cells, our results provide further evidence for a function of Cajal bodies in U2 snRNP biogenesis.     

Polymorphism in the couch potato gene clines in eastern Australia but is not associated with ovarian dormancy in Drosophila melanogaster

Natural selection can generate parallel latitudinal clines in traits and gene frequencies across continents, but these have rarely been linked. An amino acid (isoleucine to lysine, or I462K) polymorphism of the couch potato (cpo) gene in Drosophila melanogaster is thought to control female reproductive diapause cline in North America (Schmidt et al. 2008, Proc Natl Acad Sci USA, 105, 16207-16211). Here, we show that under standard diapause-inducing conditions (12 °C and short photoperiod) (Saunders et al. 1989, Proc Natl Acad Sci USA, 86, 3748-3752), egg maturation in Australian flies is delayed, but not arrested at previtellogenic stages. At 12 °C, the phenotypic distribution in egg development was bimodal at stages 8 and 14 and showed a strong nonlinear pattern on the east coast of Australia, with incidence of egg maturation delay (ovarian dormancy) increasing both toward tropical and temperate climates. Furthermore, we found no evidence for an association between the cpo I462K polymorphism and ovarian dormancy at either 12 or 10 °C (when egg maturation was often delayed at stage 7). Owing to strong linkage disequilibrium, the latitudinal cline in cpo allele frequencies was no longer evident once variation in the In(3R)P inversion polymorphism was taken into account. Our results suggest that the standard diapause-inducing conditions (12 °C and short photoperiod) were not sufficient to cause the typical previtellogenic developmental arrest in Australian flies and that the cpo I462K polymorphism does not explain the observed delay in egg development. In conclusion, ovarian dormancy does not show a simple latitudinal cline, and the lack of cpo-dormancy association suggests a different genetic basis to reproductive dormancy in North America and Australia.     

Alternative splicing of the Drosophila Dscam pre-mRNA is both temporally and spatially regulated

The Drosophila melanogaster Down syndrome cell adhesion molecule (Dscam) gene encodes an axon guidance receptor that can express 38,016 different mRNAs by virtue of alternative splicing. The Dscam gene contains 95 alternative exons that are organized into four clusters of 12, 48, 33, and 2 exons each. Although numerous Dscam mRNA isoforms can be synthesized, it remains to be determined whether different Dscam isoforms are synthesized at different times in development or in different tissues. We have investigated the alternative splicing of the Dscam exon 4 cluster, which contains 12 mutually exclusive alternative exons, and found that Dscam exon 4 alternative splicing is developmentally regulated. The most highly regulated exon, 4.2, is infrequently used in early embryos but is the predominant exon 4 variant used in adults. Moreover, the developmental regulation of exon 4.2 alternative splicing is conserved in D. yakuba. In addition, different adult tissues express distinct collections of Dscam mRNA isoforms. Given the role of Dscam in neural development, these results suggest that the regulation of alternative splicing plays an important role in determining the specificity of neuronal wiring. In addition, this work provides a framework to determine the mechanisms by which complex alternative splicing events are regulated.     

The heat shock consensus sequence is not sufficient for hsp70 gene expression in Drosophila melanogaster.

A hybrid gene in which the expression of an Escherichia coli beta-galactosidase gene was placed under the control of a Drosophila melanogaster 70,000-dalton heat shock protein (hsp70) gene promoter was constructed. Mutant derivatives of this hybrid gene which contained promoter sequences of different lengths were prepared, and their heat-induced expression was examined in D. melanogaster and COS-1 (African green monkey kidney) cells. Mutants with 5' nontranscribed sequences of at least 90 and up to 1,140 base pairs were expressed strongly in both cell types. Mutants with shorter 5' extensions (of at least 63 base pairs) were transcribed and translated efficiently in COS-1 but not at all in D. melanogaster cells. Thus, in contrast to the situation in COS-1 cells, the previously defined heat shock consensus sequence which is located between nucleotides 62 and 48 of the hsp70 gene 5' nontranscribed DNA segment is not sufficient for the expression of the D. melanogaster gene in homologous cells. A second consensus-like element 69 to 85 nucleotides upstream from the cap site is postulated to be also involved in the heat-induced expression of the hsp70 gene in D. melanogaster cells.     

Aberrant pre-mRNA maturation is caused by LINE insertions into introns of the white gene of Drosophila melanogaster.

Insertional mutagenesis screens have provided thousands of mutant alleles for analysing genes of varied functions in Drosophila melanogaster. We here document mechanisms of insertional mutagenesis by a LINE element, the I factor, by determining the molecular structure of RNAs produced from two alleles of the white gene of D.melanogaster, wIR1 and wIR6. These alleles result from insertion of the I factor into introns of the gene. We show that sequences present within the element direct aberrant splicing and termination events. When the I factor is inserted within the white first intron it may lead to the use of a cryptic 3' splice site which does not contain the dinucleotide AG. This splicing gives rise to a chimeric messenger RNA whose synthesis is controlled differently in tissues where the mutated gene is expressed. When the I factor is inserted within the white last intron it induces synthesis of truncated mRNAs. These results provide, for the first time, mechanisms for I factor insertional mutagenesis. They are discussed in the more general context of RNA processing in Drosophila and the evolution of eukaryotic gene introns.     

Dual function for U2AF(35) in AG-dependent pre-mRNA splicing

The splicing factor U2AF is required for the recruitment of U2 small nuclear RNP to pre-mRNAs in higher eukaryotes. The 65-kDa subunit of U2AF (U2AF(65)) binds to the polypyrimidine (Py) tract preceding the 3' splice site, while the 35-kDa subunit (U2AF(35)) contacts the conserved AG dinucleotide at the 3' end of the intron. It has been shown that the interaction between U2AF(35) and the 3' splice site AG can stabilize U2AF(65) binding to weak Py tracts characteristic of so-called AG-dependent pre-mRNAs. U2AF(35) has also been implicated in arginine-serine (RS) domain-mediated bridging interactions with splicing factors of the SR protein family bound to exonic splicing enhancers (ESE), and these interactions can also stabilize U2AF(65) binding. Complementation of the splicing activity of nuclear extracts depleted of U2AF by chromatography in oligo(dT)-cellulose requires, for some pre-mRNAs, only the presence of U2AF(65). In contrast, splicing of a mouse immunoglobulin M (IgM) M1-M2 pre-mRNA requires both U2AF subunits. In this report we have investigated the sequence elements (e.g., Py tract strength, 3' splice site AG, ESE) responsible for the U2AF(35) dependence of IgM. The results indicate that (i) the IgM substrate is an AG-dependent pre-mRNA, (ii) U2AF(35) dependence correlates with AG dependence, and (iii) the identity of the first nucleotide of exon 2 is important for U2AF(35) function. In contrast, RS domain-mediated interactions with SR proteins bound to the ESE appear to be dispensable, because the purine-rich ESE present in exon M2 is not essential for U2AF(35) activity and because a truncation mutant of U2AF(35) consisting only of the pseudo-RNA recognition motif domain and lacking the RS domain is active in our complementation assays. While some of the effects of U2AF(35) can be explained in terms of enhanced U2AF(65) binding, other activities of U2AF(35) do not correlate with increased cross-linking of U2AF(65) to the Py tract. Collectively, the results argue that interaction of U2AF(35) with a consensus 3' splice site triggers events in spliceosome assembly in addition to stabilizing U2AF(65) binding, thus revealing a dual function for U2AF(35) in pre-mRNA splicing.