Sex-lethal regulates back-splicing and generation of the sex-differentially expressed circular RNAs.

Conversely to canonical splicing, back-splicing connects the upstream 3' splice site (SS) with a downstream 5'SS and generates exonic circular RNAs (circRNAs) that are widely identified and have regulatory functions in eukaryotic gene expression. However, sex-specific back-splicing in Drosophila has not been investigated and its regulation remains unclear. Here, we performed multiple RNA analyses of a variety sex-specific Drosophila samples and identified over ten thousand circular RNAs, in which hundreds are sex-differentially and -specifically back-spliced. Intriguingly, we found that expression of SXL, an RNA-binding protein encoded by Sex-lethal (Sxl), the master Drosophila sex-determination gene that is only spliced into functional proteins in females, promoted back-splicing of many female-differential circRNAs in the male S2 cells, whereas expression of a SXL mutant (SXLRRM) did not promote those events. Using a monoclonal antibody, we further obtained the transcriptome-wide RNA-binding sites of SXL through PAR-CLIP. After splicing assay of mini-genes with mutations in the SXL-binding sites, we revealed that SXL-binding on flanking exons and introns of pre-mRNAs facilitates back-splicing, whereas SXL-binding on the circRNA exons inhibits back-splicing. This study provides strong evidence that SXL has a regulatory role in back-splicing to generate sex-specific and -differential circRNAs, as well as in the initiation of sex-determination cascade through canonical forward-splicing.

Roles of minor spliceosome in intron recognition and the convergence with the better understood major spliceosome.

Catalyzed by spliceosomes in the nucleus, RNA splicing removes intronic sequences from precursor RNAs in eukaryotes to generate mature RNA, which also significantly increases proteome complexity and fine-tunes gene expression. Most metazoans have two coexisting spliceosomes; the major spliceosome, which removes >99.5% of introns, and the minor spliceosome, which removes far fewer introns (only 770 at present have been predicted in the human genome). Both spliceosomes are large and dynamic machineries, each consisting of five small nuclear RNAs (snRNAs) and more than 100 proteins. However, the dynamic assembly, catalysis, and protein composition of the minor spliceosome are still poorly understood. With different splicing signals, minor introns are rare and usually distributed alone and flanked by major introns in genes, raising questions of how they are recognized by the minor spliceosome and how their processing deals with the splicing of neighboring major introns. Due to large numbers of introns and close similarities between the two machinery, cooperative, and competitive recognition by the two spliceosomes has been investigated. Functionally, many minor-intron-containing genes are evolutionarily conserved and essential. Mutations in the minor spliceosome exhibit a variety of developmental defects in plants and animals and are linked to numerous human diseases. Here, we review recent progress in the understanding of minor splicing, compare currently known components of the two spliceosomes, survey minor introns in a wide range of organisms, discuss cooperation and competition of the two spliceosomes in splicing of minor-intron-containing genes, and contributions of minor splicing mutations in development and diseases. This article is categorized under: RNA Processing > Processing of Small RNAs RNA Processing > Splicing Mechanisms RNA Structure and Dynamics > RNA Structure, Dynamics and Chemistry.