Sex-specific expression of alternative transcripts in Drosophila

Abstract

Background: Many genes produce multiple transcripts due to alternative splicing or utilization of alternative transcription initiation/termination sites. This 'transcriptome expansion' is thought to increase phenotypic complexity by allowing a single locus to produce several functionally distinct proteins. However, sex, genetic and developmental variation in the representation of alternative transcripts has never been examined systematically. Here, we describe a genome-wide analysis of sex-specific expression of alternative transcripts in Drosophila melanogaster.

Results: We compared transcript profiles in males and females from eight Drosophila lines (OregonR and 2b, and 6 RIL) using a newly designed 60-mer oligonucleotide microarray that allows us to distinguish a large proportion of alternative transcripts. The new microarray incorporates 7,207 oligonucleotides, satisfying stringent binding and specificity criteria that target both the common and the unique regions of 2,768 multi-transcript genes, as well as 12,912 oligonucleotides that target genes with a single known transcript. We estimate that up to 22% of genes that produce multiple transcripts show a sex-specific bias in the representation of alternative transcripts. Sexual dimorphism in overall transcript abundance was evident for 53% of genes. The X chromosome contains a significantly higher proportion of genes with female-biased transcription than the autosomes. However, genes on the X chromosome are no more likely to have a sexual bias in alternative transcript representation than autosomal genes.

Conclusion: Widespread sex-specific expression of alternative transcripts in Drosophila suggests that a new level of sexual dimorphism at the molecular level exists.

Figure 1

Experimental approach used to detect sex-specific splicing. Probes designed based on sequence clustering may target either constitutive or alternatively transcribed exons. Each panel shows a different example of probe distribution among constitutive and alternatively transcribed regions. For instance, '2+1+1' indicates that the corresponding gene has two probes targeting a common region and one probe targeting each of two alternatively transcribed regions, '3+1' indicates that the gene has three common probes and one probe that targets an alternatively transcribed region, and so on. For each probe, the figure shows its designating number, location in the transcript, and the ratio of the normalized and log-transformed (natural log) values between females (numerator) and males (denominator). Note that different probes that target the same subset of transcripts have similar values for the normalized log transformed male/female expression ratios, even if they are located in different exons. In contrast, probes that target alternatively spliced regions have different values for the normalized log transformed male/female expression ratios.

Figure 2

Sex-specific amplification of alternative transcripts from nine genes that showed significant sex by probe interaction in the microarray data (unc-13, mud, jupiter, r, aret, CG4662, CG10899, garz, Akap200; see Table 3). The graph shows the average CTs for each exon junction in males and females of the OregonR line. CT values were calculated by performing qPCR with SYBR^® Green I dye chemistry on three bioreplicates consisting of four virgin males and females, and correspond to the number of cycles when the fluorescence intensity was significantly above background during the exponential phase of amplification; dark blue, male transcript 1; light blue, male transcript 2; green, male transcript 3; red, female transcript 1; pink, female transcript 2; orange, female transcript 3.

Figure 3

Examples of transcript clustering. Transcripts were clustered by BLAT and then aligned in ClustalW. Some of the more common clustering patterns are depicted. (a) Two transcripts, each with a unique region of at least 80 bases and a common region of at least 80 bases; (b) two transcripts, each with a unique region of at least 80 bases, and a common region between 40 and 79 bases; (c) two transcripts with a common region of at least 80 bases, a unique region of at least 80 bases and a unique region of at least 50 bases; (d) two sequences with a gapped alignment.