Intron retention in the alternatively spliced region of RON results from weak 3' splice site recognition
Intron retention in the alternatively spliced region of RON results from weak 3' splice site recognition
The RON gene encodes a tyrosine kinase receptor for macrophage-stimulating protein. A constitutively active isoform that arises by skipping of exon 11 is expressed in carcinomas and contributes to an invasive phenotype. However, a high proportion of the mRNA expressed from the endogenous gene, or from transfected minigenes, appears to retain introns 10 and 11. It is not known whether this represents specific repression or the presence of weak splicing signals. We have used chimeric pre-mRNAs spliced in vitro to investigate the reason for intron retention. A systematic test showed that, surprisingly, the exon sequences known to modulate exon 11 skipping were not limiting, but the 3' splice site regions adjacent to exons 11 and 12 were too weak to support splicing when inserted into a globin intron. UV-crosslinking experiments showed binding of hnRNP F/H just 5' of these regions, but the hnRNP F/H target sequences did not mediate inhibition. Instead, the failure of splicing is linked to weak binding of U2AF65, and spliceosome assembly stalls prior to formation of any of the ATP-dependent complexes. We discuss mechanisms by which U2AF65 binding is facilitated in vivo.
Alternative Splicing, Base Sequence, Consensus Sequence, Cross-Linking Reagents, HeLa Cells, Humans, Introns, Molecular Sequence Data, Protein Binding, RNA Splice Sites, RNA, Messenger, Receptor Protein-Tyrosine Kinases, beta-Globins, Journal Article, Research Support, Non-U.S. Gov't
1-12
Smith, Lindsay D.
1d44c2d0-d5af-411e-b6cd-9b5633f2eb1e
Lucas, Christian M.
4a0bbbd0-49b6-4da3-9d8c-588a6e8e238f
Eperon, Ian C.
ded0b4c2-da7f-4de2-814b-13a30d0202ee
2013
Smith, Lindsay D.
1d44c2d0-d5af-411e-b6cd-9b5633f2eb1e
Lucas, Christian M.
4a0bbbd0-49b6-4da3-9d8c-588a6e8e238f
Eperon, Ian C.
ded0b4c2-da7f-4de2-814b-13a30d0202ee
Smith, Lindsay D., Lucas, Christian M. and Eperon, Ian C.
(2013)
Intron retention in the alternatively spliced region of RON results from weak 3' splice site recognition.
PLoS ONE, 8 (10), , [e77208].
(doi:10.1371/journal.pone.0077208).
Abstract
The RON gene encodes a tyrosine kinase receptor for macrophage-stimulating protein. A constitutively active isoform that arises by skipping of exon 11 is expressed in carcinomas and contributes to an invasive phenotype. However, a high proportion of the mRNA expressed from the endogenous gene, or from transfected minigenes, appears to retain introns 10 and 11. It is not known whether this represents specific repression or the presence of weak splicing signals. We have used chimeric pre-mRNAs spliced in vitro to investigate the reason for intron retention. A systematic test showed that, surprisingly, the exon sequences known to modulate exon 11 skipping were not limiting, but the 3' splice site regions adjacent to exons 11 and 12 were too weak to support splicing when inserted into a globin intron. UV-crosslinking experiments showed binding of hnRNP F/H just 5' of these regions, but the hnRNP F/H target sequences did not mediate inhibition. Instead, the failure of splicing is linked to weak binding of U2AF65, and spliceosome assembly stalls prior to formation of any of the ATP-dependent complexes. We discuss mechanisms by which U2AF65 binding is facilitated in vivo.
Other
journal.pone.0077208
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e-pub ahead of print date: 14 October 2013
Published date: 2013
Keywords:
Alternative Splicing, Base Sequence, Consensus Sequence, Cross-Linking Reagents, HeLa Cells, Humans, Introns, Molecular Sequence Data, Protein Binding, RNA Splice Sites, RNA, Messenger, Receptor Protein-Tyrosine Kinases, beta-Globins, Journal Article, Research Support, Non-U.S. Gov't
Identifiers
Local EPrints ID: 420578
URI: http://eprints.soton.ac.uk/id/eprint/420578
ISSN: 1932-6203
PURE UUID: 973746ad-815a-4ed8-9e39-0b7fdc10f605
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Date deposited: 10 May 2018 16:30
Last modified: 15 Mar 2024 19:32
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Author:
Lindsay D. Smith
Author:
Christian M. Lucas
Author:
Ian C. Eperon
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