Zhang Zhiping, Bae Bongmin, Cuddleston Winston H, Miura Pedro
Department of Biology, University of Nevada, Reno, Reno, NV, USA.
Department of Genetics and Genome Sciences, UConn Health, Farmington, CT, USA.
bioRxiv. 2023 Mar 23:2023.03.23.533999. doi: 10.1101/2023.03.23.533999.
Nervous system development is associated with extensive regulation of alternative splicing (AS) and alternative polyadenylation (APA). AS and APA have been extensively studied in isolation, but little is known about how these processes are coordinated. Here, the coordination of cassette exon (CE) splicing and APA in was investigated using a targeted long-read sequencing approach we call Pull-a-Long-Seq (PL-Seq). This cost-effective method uses cDNA pulldown and Nanopore sequencing combined with an analysis pipeline to resolve the connectivity of alternative exons to alternative 3' ends. Using PL-Seq, we identified genes that exhibit significant differences in CE splicing depending on connectivity to short versus long 3'UTRs. Genomic long 3'UTR deletion was found to alter upstream CE splicing in short 3'UTR isoforms and ELAV loss differentially affected CE splicing depending on connectivity to alternative 3'UTRs. This work highlights the importance of considering connectivity to alternative 3'UTRs when monitoring AS events.
神经系统发育与可变剪接（AS）和可变聚腺苷酸化（APA）的广泛调控相关。AS和APA已被分别广泛研究，但对于这些过程如何协调却知之甚少。在此，我们使用一种名为Pull-a-Long-Seq（PL-Seq）的靶向长读长测序方法，研究了盒式外显子（CE）剪接与APA在[具体内容缺失]中的协调情况。这种经济高效的方法利用cDNA下拉和纳米孔测序，并结合一个分析流程，以解析可变外显子与可变3'末端之间的连接关系。使用PL-Seq，我们鉴定出了一些基因，这些基因根据与短3'UTR或长3'UTR的连接情况，在CE剪接上表现出显著差异。发现基因组长3'UTR缺失会改变短3'UTR异构体中的上游CE剪接，并且ELAV缺失对CE剪接的影响因与可变3'UTR的连接情况而异。这项工作突出了在监测AS事件时考虑与可变3'UTR连接情况的重要性。
