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The complex set of late transcripts from the Drosophila sex determination gene sex-lethal encodes multiple related polypeptides.

ABSTRACT: Sex-lethal (Sxl), a key sex determination gene in Drosophila melanogaster, is known to express a set of three early transcripts arising during early embryogenesis and a set of seven late transcripts occurring from midembryogenesis through adulthood. Among the late transcripts, male-specific mRNAs were distinguished from their female counterparts by the presence of an extra exon interrupting an otherwise long open reading frame (ORF). We have now analyzed the structures of the late Sxl transcripts by cDNA sequencing, Northern (RNA) blotting, primer extension, and RNase protection. The late transcripts appear to use a common 5' end but differ at their 3' ends by the use of alternative polyadenylation sites. Two of these sites lack canonical AATAAA sequences, and their use correlates in females with the presence of a functional germ line, suggesting possible tissue-specific polyadenylation. Besides the presence of the male-specific exon, no additional sex-specific splicing events were detected, although a number of non-sex-specific splicing variants were observed. In females, the various forms of late Sxl transcript potentially encode up to six slightly different polypeptides. All of the protein-coding differences occur outside the previously defined ribonucleoprotein motifs. One class of Sxl mRNAs also includes a second long ORF in the same frame as the first ORF but separated from it by a single ochre codon. The function of this second ORF is unknown. Significant amounts of apparently partially processed Sxl RNAs were observed, consistent with the hypothesis that the regulated Sxl splices occur relatively slowly.

SUBMITTER: Samuels ME

PROVIDER: S-EPMC361104 | biostudies-other | 1991 Jul

REPOSITORIES: biostudies-other

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The complex set of late transcripts from the Drosophila sex determination gene sex-lethal encodes multiple related polypeptides.

Samuels M E ME Schedl P P Cline T W TW

Molecular and cellular biology 19910701 7

Sex-lethal (Sxl), a key sex determination gene in Drosophila melanogaster, is known to express a set of three early transcripts arising during early embryogenesis and a set of seven late transcripts occurring from midembryogenesis through adulthood. Among the late transcripts, male-specific mRNAs were distinguished from their female counterparts by the presence of an extra exon interrupting an otherwise long open reading frame (ORF). We have now analyzed the structures of the late Sxl transcript ...[more]

PMID: 1710769

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Project description:BACKGROUND: Sex determining mechanisms are evolutionarily labile and related species often use different primary signals and gene regulatory networks. This is well illustrated by the sex determining cascade of Drosophila fruitflies, which have recruited Sex-lethal as the master switch and cellular memory of sexual identity, a role performed in other insects by the gene transformer. Here we investigate the evolutionary change in the coding sequences of sex determining genes associated with the recruitment of Sex-lethal. We analyze sequences of Sex-lethal itself, its Drosophila paralogue sister-or-Sex-lethal and downstream targets transformer and doublesex. RESULTS: We find that the recruitment of sister-or-Sex-lethal was associated with a number of adaptive amino acid substitutions, followed by a tightening of purifying selection within the Drosophila clade. Sequences of the paralogue sister-or-Sex-lethal, in contrast, show a signature of rampant positive selection and relaxation of purifying selection. The recruitment of Sex-lethal as top regulator and memory gene is associated with a significant release from purifying selection in transformer throughout the Drosophila clade. In addition, doublesex shows a signature of positive selection and relaxation of purifying selection in the Drosophila clade. A similar pattern is seen in sequences from the sister Tephritidae clade. CONCLUSIONS: The pattern of molecular evolution we observe for Sex-lethal and its paralogue sister-or-Sex-lethal is not characteristic of a duplication followed by neo-functionalization. Rather, evidence suggests a sub-functionalization scenario achieved through the evolution of sophisticated splicing. As expected, we find that transformer evolves under relaxed purifying selection after the recruitment of Sex-lethal in Drosophila. Finally, the observation of doublesex adaptation in both Drosophila and Tephritidae suggests that these changes are due to ongoing adaptation of downstream sex-specific regulation, rather than being associated the recruitment of Sex-lethal and the resulting change in the topology of the sex determining cascade.

Project description:N6-methyladenosine (m6A) has recently emerged as a widespread and conserved RNA modification that modulates messenger RNA (mRNA) processing and activity. As most m6A studies have been conducted in cultured cells, we established Drosophila as a model system that combines molecular and genomic analyses of m6A with investigations of organismal biology. We first apply miCLIP sequencing to map m6A modifications at single-nucleotide resolution during Drosophila embryogenesis. We next characterize Drosophila components of the m6A "writer" methyltransferase complex [MTC factors, METTL3/IME4, METTL14, FL(2)D and Nito], and characterize m6A-binding properties of the YTH domain-containing proteins, YT521-B and CG6422. We complement this by generating a collection of mutants in m6A writer and reader components. While Drosophila orthologs of mammalian MTC factors that have additional roles in splicing are lethal, our mutants in METTL and YTH factors are viable but present specific developmental and behavioral defects. We note that several mammalian m6A factors were originally identified as Drosophila regulators of Sxl splicing, which generates the master determinant of female identity. While our mutants in dedicated writer and reader machinery are not required to accumulate Sxl protein in the ovary, maternal loss of m6A writers and readers collaborates with reduction of Sxl to induce female lethality. Our data indicate Sxl is directly regulated by the m6A pathway, since miCLIP data show Sxl is a substantial target of intronic m6A in early embryos, in addition to bearing exonic m6A. Consistent with this, female-specific Sxl splicing is defective in m6A pathway mutants. YT521-B appears to be the major effector of m6A for Sxl regulation, as it shows much stronger genetic interactions with Sxl than CG6422 and YT521-B overexpression can induce female-specific Sxl splicing. Overall, our transcriptomic and genetic toolkit for studying the m6A pathway in Drosophila reveals a major biological utilization in establishing sex-specific splicing.

Dataset Information

The complex set of late transcripts from the Drosophila sex determination gene sex-lethal encodes multiple related polypeptides.

Publications

The complex set of late transcripts from the Drosophila sex determination gene sex-lethal encodes multiple related polypeptides.

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