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Regulated tissue-specific expression of antagonistic pre-mRNA splicing factors

Published online by Cambridge University Press:  01 April 1998

AKEMI HANAMURA
Affiliation:
Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724-2208, USA
JAVIER F. CÁCERES
Affiliation:
Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724-2208, USA Present address: MRC Human Genetics Unit, Western General Hospital, Crewe Road, Edinburgh, Scotland, United Kingdom.
AKILA MAYEDA
Affiliation:
Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724-2208, USA
B. ROBERT FRANZA Jr.
Affiliation:
Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724-2208, USA
ADRIAN R. KRAINER
Affiliation:
Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724-2208, USA
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Abstract

The SR proteins are essential metazoan pre-mRNA splicing factors that can also influence the selection of alternative 5′ splice sites in a concentration-dependent manner. Their activity in alternative splicing in vitro is antagonized by members of the hnRNP A/B family of proteins. The opposite effects of members of these two families of antagonistic splicing factors in vitro and upon overexpression in vivo suggest that changes in their relative levels may be a natural mechanism for the regulation of alternative splicing in vivo. One prediction of this model is that the ratios of these antagonists should vary in different cell types and in other situations in which cellular or viral transcripts are differentially spliced. We raised monoclonal antibodies specific for SF2/ASF and used them to measure the abundance of SF2/ASF protein and its isoforms, its phosphorylation state in vivo and during splicing in vitro, and its association with the spliceosome. SF2/ASF exists predominantly or exclusively in a highly phosphorylated state in vivo in all cell types examined, and unphosphorylated protein was not detectable. Unphosphorylated recombinant SF2/ASF becomes rapidly phosphorylated under splicing conditions in HeLa cell extracts and associates stably with one or more exons of β-globin pre-mRNA. This interaction appears to persist through the splicing reaction and SF2/ASF remains bound to spliced mRNA. We compared the distribution of SF2/ASF to that of its antagonist, hnRNP A1, in different rat tissues and in immortal and transformed cell lines. We found that the protein levels of these antagonistic splicing factors vary naturally over a very wide range, supporting the notion that changes in the ratio of these proteins can affect alternative splicing of a variety of pre-mRNAs in vivo.

Type
Research Article
Information
RNA , Volume 4 , Issue 4 , April 1998 , pp. 430 - 444
Copyright
© 1998 RNA Society

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