An ultraconserved pseudo 5' splice site fine-tunes development by regulating alternative splicing within TOR-related pathways

Introns have expanded dramatically during evolution, in which ultraconserved RNA motifs may play essential regulatory roles. Sharing the sequence with the actual 5' splice sites (5'SSs), pseudo-5'SSs are widespread in introns but never spliced. We searched homologous introns and identified eight ultraconserved pseudo-5'SSs (UCP-5'SSs). The most conserved one resides in the animal ENOX1/Enox genes that are involved in plasma electron transport and cell enlargement. In vivo deletion of this 9-nt UCP-5'SS in Drosophila results in a significantly enlarged ovary and increased fecundity. We demonstrate that this UCP-5'SS is a silencer for alternative splicing (AS) regulation of an upstream ultraconserved essential exon through interaction with the U1 snRNP-core proteins. The AS changes are observed in all the tested Drosophila mutants from the dTOR and Insulin-like pathways. Remarkably, loss of this UCP-5'SS significantly mitigates the changes. Conservatively, multiple-source human cells treated with the mTOR/Insulin pathway inhibitors also change the AS and specifically increase the translation of U1-70K. This study reveals an ultraconserved regulatory network in which a short intronic RNA motif functions as a sensor of TOR-related pathways during ovarian development. Overall design: To investigate the function of conserved motifs in the enox gene of fruit flies, we established strains with deletions in the enox motif and the enox gene, followed by RNA-seq analysis.