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

Research output: Contribution to journalArticle

2 Citations (Scopus)
8 Downloads (Pure)

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

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Alternative Splicing
Cell Adhesion Molecules
Down Syndrome
Transgenes
Introns
Exons
Plasmids
Genes
Integrases
Gene Transfer Techniques
Conserved Sequence
Reporter Genes
Base Pairing
Diptera
Drosophila
Escherichia coli
Neurons
DNA
Enzymes

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
permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

Copyright © and Moral Rights are retained by the author(s) and/ or other copyright owners. A copy can be downloaded for personal non-commercial research or study, without prior permission or charge. This item cannot be reproduced or quoted extensively from without first obtaining permission in writing from the copyright holder(s). The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the copyright holders.

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.

Cite this

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

In: Nucleic Acids Research, Vol. 47, No. 3, 20.02.2019, p. 1389–1403.

Research output: Contribution to journalArticle

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