PRJNA771049
3'UTR connected alternative splicing revealed by PL-Seq in Drosophila
This study is to investigate the 3'UTR linked cassatte exon (CE) alternative splicing in Drosophila melanogaster 16-18 hr embryos or adult heads. There are four genotypes used in the experiments: w1118 wild type control, a elav5 mutant, Khc-73 long 3'UTR mutant and Dscam1 long 3'UTR mutant. Totle RNAs were extracted from 16-18 hr embryos or adult heads; SMARTer cDNA synthesis kit was used to generate oligo dT primed full length cDNA; custom designed probes were used to pull down cDNA from genes of interest; sequencing libraries were prepared using Oxford Nanopore ligation sequencing kit LSK110 and then loaded on to flongle cell and sequenced using MinION sequencer. Reads were mapped onto the Drosophila melanogaster genome (dm6) and a series of filtering were performed to retain reads that meet speficit criterias for alternative splicing quantification and parse reads to short and long 3'UTR isoforms as well. We found that 23/28 genes we examined showed discrepancy of cassette exon(CE) splicing between short and long 3'UTR isoforms, and the 3'UTR dependent splicing discrepancy was de-regulated in elav mutant. Genomic long 3'UTR deletion affects the upstream CE splicing while the genomic deletion of the CE(exon 19 in Dscam1 and exon 15 in Khc-73) and its flanking introns didn't affect the 3' polyA site choice.