Plasmid-based gap-repair recombineered transgenes reveal a central role for introns in mutually exclusive alternative splicing in Down Syndrome Cell Adhesion Molecule exon 4

Irmgard Haussmann, Pinar Ustaoglu, Ulrike Brauer, Yash Hemani, Thomas C Dix, Matthias Soller

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Abstract

Alternative splicing is a key feature of human genes, yet studying its regulation is often complicated by large introns. The Down Syndrome Cell Adhesion Molecule (Dscam) gene from Drosophila is one of the most complex genes generating vast molecular diversity by mutually exclusive alternative splicing. To resolve how alternative splicing in Dscam is regulated, we first developed plasmid-based UAS reporter genes for the Dscam variable exon 4 cluster and show that its alternative splicing is recapitulated by GAL4-mediated expression in neurons. We then developed gap-repair recombineering to very efficiently manipulate these large reporter plasmids in Escherichia coli using restriction enzymes or sgRNA/Cas9 DNA scission to capitalize on the many benefits of plasmids in phiC31 integrase-mediated transgenesis. Using these novel tools, we show that inclusion of Dscam exon 4 variables differs little in development and individual flies, and is robustly determined by sequences harbored in variable exons. We further show that introns drive selection of both proximal and distal variable exons. Since exon 4 cluster introns lack conserved sequences that could mediate robust long-range base-pairing to bring exons into proximity for splicing, our data argue for a central role of introns in mutually exclusive alternative splicing of Dscam exon 4 cluster.
Original languageEnglish
Pages (from-to)1389–1403
Number of pages15
JournalNucleic Acids Research
Volume47
Issue number3
Early online date12 Dec 2018
DOIs
Publication statusPublished - 20 Feb 2019

Bibliographical note

Copyright The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which
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Funder

Biotechnology and Biological Science Research Council (BBSRC BB/K006827/1 and BB/F000855/1) (to I.U.H., U.B., M.S.); Darwin Trust (to Y.H.); Sukran Sinan Fund (to P.U.). Funding for open access charge: BBSRC institutional funding.

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