Conserved RNA cis-elements regulate alternative splicing of Lepidopteran doublesex.
Doublesex (dsx) is a downstream key regulator in insect sex determination pathway. In Drosophila, alternative splicing of Dm-dsx gene is sex-specifically regulated by transformer (tra), in which the functional TRA promotes female-specific Dm-dsx. However, the sex determination pathway in Lepidoptera is not well understood; here we focused on alternative splicing of doublesex (dsx) in two agricultural pests, Asian corn borer (Ostrinia furnacalis) and cotton bollworm (Helicoverpa armigera), as well as the silkworm (Bombyx mori). More than a dozen new alternative splicing isoforms of dsx were found in the Lepidopteran females, which exist in all tested developmental stages and differentiated tissues. Alignment of mRNA and protein sequences of doublesex revealed high conservation of this gene in Lepidoptera. Strength analysis of splice sites revealed a weak 5' splice site at intron 3 in Lepidopteran dsx, which was experimentally confirmed. Furthermore, we identified highly conserved RNA sequences in the Lepidopteran dsx, including RNA elements I (14 nt), II (11 nt), III (26 nt), IV (17 nt), 3E-1 (8 nt) and 3E-2 (8 nt). The RNA elements III and IV were previously found in exon 4 of B. mori dsx and bound with Bm-PSI, which suppressed the inclusion of exons 3 & 4 into the male-specific Bm-dsx. Then we identified and analyzed the homologous genes of Bm-psi in the two Lepidopteran pests, which expressed at similar levels and exhibited a unique isoform in the males and females from each Lepidoptera. Importantly, mutagenesis of Bm-dsx mini-genes and their expression in BmN cell line demonstrated that three RNA elements are involved in the female-specific alternative splicing of Bm-dsx. Mutations in the RNA cis-elements 3E-1 and 3E-2 resulted in decreased inclusion of exon 3 into the female-specific dsx mRNA, suggesting that these two elements would be exonic splicing enhancers that facilitate the recognition of the weak 5' splice site at intron 3 of Lepidopteran dsx. We propose that the 5' splice sites at intron 3 are weak, resulting in multiple alternative splicing events in intron 3 of female Lepidoptera dsx. Activation of the 5' splice site requires regulatory cis-elements in exons 3 for female-specific splicing of Lepidoptera dsx.
Key Laboratory of Insect Developmental and Evolutionary Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China.
Xiu-Ye
XY
Wang
Key Laboratory of Insect Developmental and Evolutionary Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China.
Zeng-Zhang
ZZ
Zheng
College of Life Sciences, Shanghai University, Shanghai 200444, China.
Hong-Sheng
HS
Song
Key Laboratory of Insect Developmental and Evolutionary Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China. Electronic address: yzxu@sibs.ac.cn.
Yong-Zhen
YZ
Xu
D017398:Alternative Splicing; D000818:Animals; D001483:Base Sequence; D012831:Bombyx / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017124:Conserved Sequence; D005091:Exons; D005260:Female; D005786:Gene Expression Regulation; D019476:Insect Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D007438:Introns; D008297:Male; D008969:Molecular Sequence Data; D009036:Moths / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D022821:RNA Splice Sites; D012045:Regulatory Sequences, Nucleic Acid; D013045:Species Specificity
D019476:Insect Proteins; D022821:RNA Splice Sites
AS; Alternative splicing; Bm; Bombyx mori; Doublesex; Ha; Helicoverpa armigera; Lepidoptera; Of; Ostrinia furnacalis; SS; alternative splicing; splice site