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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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Sequential immunoprecipitation of splicing complexes by gal-1 and gal-3 antisera. (A) protocol for sequential immunoprecipitation. Splicing reactions were incubated for 60 min with 32P-MINX and divided into equal aliquots. The aliquots were incubated with the indicated antiserum as described in Materials and Methods. Part of the unbound fraction was then incubated with the other galectin antiserum. After washing, the bound material from the first and second immunoprecipitations was eluted and analyzed for MINX RNA (B). Lanes 1, 5, 7 and 9 represent aliquots of the bound fraction used for immunoprecipitation (input). Lanes 2–4, represent the MINX RNA bound to the indicated antiserum in the first immunoprecipitate and lanes 6, 8 and 10 represent MINX RNA bound to the indicated antiserum in the second immunoprecipitate. (C) western blotting analysis of gal-1 and gal-3 in the bound (lanes 2–4) and unbound (lanes 5–7) fractions of the first immunoprecipitation.
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fig6: Sequential immunoprecipitation of splicing complexes by gal-1 and gal-3 antisera. (A) protocol for sequential immunoprecipitation. Splicing reactions were incubated for 60 min with 32P-MINX and divided into equal aliquots. The aliquots were incubated with the indicated antiserum as described in Materials and Methods. Part of the unbound fraction was then incubated with the other galectin antiserum. After washing, the bound material from the first and second immunoprecipitations was eluted and analyzed for MINX RNA (B). Lanes 1, 5, 7 and 9 represent aliquots of the bound fraction used for immunoprecipitation (input). Lanes 2–4, represent the MINX RNA bound to the indicated antiserum in the first immunoprecipitate and lanes 6, 8 and 10 represent MINX RNA bound to the indicated antiserum in the second immunoprecipitate. (C) western blotting analysis of gal-1 and gal-3 in the bound (lanes 2–4) and unbound (lanes 5–7) fractions of the first immunoprecipitation.

Mentions: To distinguish between these two formal possibilities (spliceosomes precipitated by one galectin antibody contain one or both nuclear galectins), we performed sequential immunoprecipitations as outlined in Figure 6A. Standard splicing reactions were incubated for 60 min. and divided into two equal portions. One aliquot was immunoprecipitated with anti-gal-1 and the other with anti-gal-3. The unbound fractions were then subjected to a second immunoprecipitation using the other galectin antiserum. Radiolabeled RNA in the bound fractions from each immunoprecipitation was analyzed (Figure 6B). Roughly the same quantity of spliceosomes was precipitated by the anti-gal-1 antiserum in the two sequential selections (compare lanes 3 and 10). Similar results were obtained following the two anti-gal-3 immunoprecipitations (compares lanes 4 and 8). In order to interpret these results, the efficiency of each galectin antiserum to quantitatively immunoprecipitate its cognate antigen was determined. We analyzed the bound and unbound fractions from the first immunoprecipitation for gal-1 and gal-3 (Figure 6C). The bound fraction from the first anti-gal-1 precipitate showed only gal-1 (Figure 6C, lane 3) with no detectable gal-3. Further, the unbound fraction of this precipitation showed nearly quantitative depletion of gal-1 (lane 6; the amount of gal-1 in this fraction represents <10% of the total gal-1 in the reaction used for immunoprecipitation). Similar results were obtained with gal-3. Analysis of the bound fraction of the first anti-gal-3 precipitation showed only gal-3 (lane 4) and nearly all of gal-3 was removed by this immunoprecipitation (lane 5; <15% of the total gal-3 in the reaction remained in the unbound fraction of the first precipitation). We interpret these data to indicate that gal-1 and gal-3 were quantitatively removed during the initial immunoselection and that the two galectins reside on different splicing complexes. Finally, spliceosomal RNAs could be immunoprecipitated by anti-Sm serum from the material remaining after the two sequential galectin adsorptions (data not shown), indicating that some spliceosomal complexes contained neither gal-1 nor gal-3.


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

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

Sequential immunoprecipitation of splicing complexes by gal-1 and gal-3 antisera. (A) protocol for sequential immunoprecipitation. Splicing reactions were incubated for 60 min with 32P-MINX and divided into equal aliquots. The aliquots were incubated with the indicated antiserum as described in Materials and Methods. Part of the unbound fraction was then incubated with the other galectin antiserum. After washing, the bound material from the first and second immunoprecipitations was eluted and analyzed for MINX RNA (B). Lanes 1, 5, 7 and 9 represent aliquots of the bound fraction used for immunoprecipitation (input). Lanes 2–4, represent the MINX RNA bound to the indicated antiserum in the first immunoprecipitate and lanes 6, 8 and 10 represent MINX RNA bound to the indicated antiserum in the second immunoprecipitate. (C) western blotting analysis of gal-1 and gal-3 in the bound (lanes 2–4) and unbound (lanes 5–7) fractions of the first immunoprecipitation.
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fig6: Sequential immunoprecipitation of splicing complexes by gal-1 and gal-3 antisera. (A) protocol for sequential immunoprecipitation. Splicing reactions were incubated for 60 min with 32P-MINX and divided into equal aliquots. The aliquots were incubated with the indicated antiserum as described in Materials and Methods. Part of the unbound fraction was then incubated with the other galectin antiserum. After washing, the bound material from the first and second immunoprecipitations was eluted and analyzed for MINX RNA (B). Lanes 1, 5, 7 and 9 represent aliquots of the bound fraction used for immunoprecipitation (input). Lanes 2–4, represent the MINX RNA bound to the indicated antiserum in the first immunoprecipitate and lanes 6, 8 and 10 represent MINX RNA bound to the indicated antiserum in the second immunoprecipitate. (C) western blotting analysis of gal-1 and gal-3 in the bound (lanes 2–4) and unbound (lanes 5–7) fractions of the first immunoprecipitation.
Mentions: To distinguish between these two formal possibilities (spliceosomes precipitated by one galectin antibody contain one or both nuclear galectins), we performed sequential immunoprecipitations as outlined in Figure 6A. Standard splicing reactions were incubated for 60 min. and divided into two equal portions. One aliquot was immunoprecipitated with anti-gal-1 and the other with anti-gal-3. The unbound fractions were then subjected to a second immunoprecipitation using the other galectin antiserum. Radiolabeled RNA in the bound fractions from each immunoprecipitation was analyzed (Figure 6B). Roughly the same quantity of spliceosomes was precipitated by the anti-gal-1 antiserum in the two sequential selections (compare lanes 3 and 10). Similar results were obtained following the two anti-gal-3 immunoprecipitations (compares lanes 4 and 8). In order to interpret these results, the efficiency of each galectin antiserum to quantitatively immunoprecipitate its cognate antigen was determined. We analyzed the bound and unbound fractions from the first immunoprecipitation for gal-1 and gal-3 (Figure 6C). The bound fraction from the first anti-gal-1 precipitate showed only gal-1 (Figure 6C, lane 3) with no detectable gal-3. Further, the unbound fraction of this precipitation showed nearly quantitative depletion of gal-1 (lane 6; the amount of gal-1 in this fraction represents <10% of the total gal-1 in the reaction used for immunoprecipitation). Similar results were obtained with gal-3. Analysis of the bound fraction of the first anti-gal-3 precipitation showed only gal-3 (lane 4) and nearly all of gal-3 was removed by this immunoprecipitation (lane 5; <15% of the total gal-3 in the reaction remained in the unbound fraction of the first precipitation). We interpret these data to indicate that gal-1 and gal-3 were quantitatively removed during the initial immunoselection and that the two galectins reside on different splicing complexes. Finally, spliceosomal RNAs could be immunoprecipitated by anti-Sm serum from the material remaining after the two sequential galectin adsorptions (data not shown), indicating that some spliceosomal complexes contained neither gal-1 nor gal-3.

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