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Immunoprecipitation of spliceosomal RNAs by antisera to galectin-1 and galectin-3.

Wang W, Park JW, Wang JL, Patterson RJ - Nucleic Acids Res. (2006)

Bottom Line: Now we provide evidence that both galectins are directly associated with spliceosomes by analyzing RNAs and proteins of complexes immunoprecipitated by galectin-specific antisera.Early spliceosomal complexes were also immunoprecipitated by these antisera.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824, USA.

ABSTRACT
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.

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Immunoprecipitation of spliceosomes by anti-Sm antiserum. Splicing reactions were incubated with ATP for 60 min. and then were immunoprecipitated with human anti-Sm antiserum or human IgG (control). Bound fractions were eluted and RNA analyzed by gel electrophoresis and autoradiography (A) and gal-1 detected by western blotting (B).
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fig5: Immunoprecipitation of spliceosomes by anti-Sm antiserum. Splicing reactions were incubated with ATP for 60 min. and then were immunoprecipitated with human anti-Sm antiserum or human IgG (control). Bound fractions were eluted and RNA analyzed by gel electrophoresis and autoradiography (A) and gal-1 detected by western blotting (B).

Mentions: Galectins should be detected in spliceosomes isolated by precipitation with antiserum directed against another splicing factor. A reciprocal co-immunoprecipitation experiment was performed using anti-Sm antisera (Figure 5). As expected, spliceosomes selected by the Sm antiserum contained the splicing substrate, intermediates and products. Immunoprecipitation with human IgG revealed only background levels of splicing RNAs. Most importantly, gal-1 was detected in the Sm selected complexes, but not detected in the material precipitated by the control human IgG. These data strongly support our contention that galectins are splicing factors associated with the splicing machinery.


Immunoprecipitation of spliceosomal RNAs by antisera to galectin-1 and galectin-3.

Wang W, Park JW, Wang JL, Patterson RJ - Nucleic Acids Res. (2006)

Immunoprecipitation of spliceosomes by anti-Sm antiserum. Splicing reactions were incubated with ATP for 60 min. and then were immunoprecipitated with human anti-Sm antiserum or human IgG (control). Bound fractions were eluted and RNA analyzed by gel electrophoresis and autoradiography (A) and gal-1 detected by western blotting (B).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1636441&req=5

fig5: Immunoprecipitation of spliceosomes by anti-Sm antiserum. Splicing reactions were incubated with ATP for 60 min. and then were immunoprecipitated with human anti-Sm antiserum or human IgG (control). Bound fractions were eluted and RNA analyzed by gel electrophoresis and autoradiography (A) and gal-1 detected by western blotting (B).
Mentions: Galectins should be detected in spliceosomes isolated by precipitation with antiserum directed against another splicing factor. A reciprocal co-immunoprecipitation experiment was performed using anti-Sm antisera (Figure 5). As expected, spliceosomes selected by the Sm antiserum contained the splicing substrate, intermediates and products. Immunoprecipitation with human IgG revealed only background levels of splicing RNAs. Most importantly, gal-1 was detected in the Sm selected complexes, but not detected in the material precipitated by the control human IgG. These data strongly support our contention that galectins are splicing factors associated with the splicing machinery.

Bottom Line: Now we provide evidence that both galectins are directly associated with spliceosomes by analyzing RNAs and proteins of complexes immunoprecipitated by galectin-specific antisera.Early spliceosomal complexes were also immunoprecipitated by these antisera.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824, USA.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus