Abstract
| Original language | English |
|---|---|
| Pages (from-to) | 1389–1403 |
| Number of pages | 15 |
| Journal | Nucleic Acids Research |
| Volume | 47 |
| Issue number | 3 |
| Early online date | 12 Dec 2018 |
| DOIs | |
| Publication status | Published - 20 Feb 2019 |
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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 journal › Article
}
TY - JOUR
T1 - 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
AU - Haussmann, Irmgard
AU - Ustaoglu, Pinar
AU - Brauer, Ulrike
AU - Hemani, Yash
AU - Dix, Thomas C
AU - Soller, Matthias
N1 - 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.
PY - 2019/2/20
Y1 - 2019/2/20
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=85065535248&partnerID=8YFLogxK
U2 - 10.1093/nar/gky1254
DO - 10.1093/nar/gky1254
M3 - Article
VL - 47
SP - 1389
EP - 1403
JO - Nucleic Acids Research
JF - Nucleic Acids Research
SN - 0305-1048
IS - 3
ER -