Nucleic Acids Research Advance Access published online on September 22, 2006
Nucleic Acids Research, doi:10.1093/nar/gkl673
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© 2006 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Immunoprecipitation of spliceosomal RNAs by antisera to galectin-1 and galectin-3
Department of Microbiology and Molecular Genetics, Michigan State University East Lansing, MI 48824, USA 1 Department of Biochemistry and Molecular Biology, Michigan State University East Lansing, MI 48824, USA
*To whom correspondence should be addressed. Tel: +1 517 355 6463/1541; Fax: +1 517 353 8957; Email: patter13{at}msu.edu
Received August 29, 2006. Accepted August 30, 2006.
We have shown that galectin-1 and galectin-3 are functionally redundant splicing factors. Now we provide evidence that both galectins are directly associated with spliceosomes by analyzing RNAs and proteins of complexes immunoprecipitated by galectin-specific antisera. Both galectin antisera co-precipitated splicing substrate, splicing intermediates and products in active spliceosomes. Protein factors co-precipitated by the galectin antisera included the Sm core polypeptides of snRNPs, hnRNP C1/C2 and Slu7. Early spliceosomal complexes were also immunoprecipitated by these antisera. When splicing reactions were sequentially immunoprecipitated with galectin antisera, we found that galectin-1 containing spliceosomes did not contain galectin-3 and vice versa, providing an explanation for the functional redundancy of nuclear galectins in splicing. The association of galectins with spliceosomes was (i) not due to a direct interaction of galectins with the splicing substrate and (ii) easily disrupted by ionic conditions that had only a minimal effect on snRNP association. Finally, addition of excess amino terminal domain of galectin-3 inhibited incorporation of galectin-1 into splicing complexes, explaining the dominant-negative effect of the amino domain on splicing activity. We conclude that galectins are directly associated with splicing complexes throughout the splicing pathway in a mutually exclusive manner and they bind a common splicing partner through weak protein–protein interactions.
Present address: Jung W. Park, Department of Genetics and Developmental Biology, University of Connecticut Health Center, Farmington, CT 06030, USA
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors
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