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.
Alternative Splicing
/ genetics
Animals
Cell Adhesion Molecules
/ genetics
Conserved Sequence
Down Syndrome
/ genetics
Drosophila Proteins
/ genetics
Drosophila melanogaster
/ genetics
Escherichia coli
/ genetics
Exons
/ genetics
Gene Expression
/ genetics
Gene Transfer Techniques
Humans
Introns
/ genetics
Neurons
/ metabolism
RNA Splicing
/ genetics
Transcription Factors
/ genetics
Journal
Nucleic acids research
ISSN: 1362-4962
Titre abrégé: Nucleic Acids Res
Pays: England
ID NLM: 0411011
Informations de publication
Date de publication:
20 02 2019
20 02 2019
Historique:
accepted:
03
12
2018
revised:
22
11
2018
received:
02
10
2018
pubmed:
13
12
2018
medline:
21
8
2019
entrez:
13
12
2018
Statut:
ppublish
Résumé
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.
Identifiants
pubmed: 30541104
pii: 5239037
doi: 10.1093/nar/gky1254
pmc: PMC6379703
doi:
Substances chimiques
Cell Adhesion Molecules
0
DSCAM protein, human
0
Drosophila Proteins
0
Dscam1 protein, Drosophila
0
GAL4 protein, Drosophila
0
Transcription Factors
0
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Pagination
1389-1403Subventions
Organisme : Biotechnology and Biological Sciences Research Council
ID : BB/F000855/1
Pays : United Kingdom
Informations de copyright
© The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research.
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