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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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Analysis of spliceosomes precipitated by various antisera at increasing salt concentrations. HeLa NE was incubated with 32P-MINX for 60 min in the presence of ATP. Immobilized pre-immune serum (lanes 2, 6 and 10); anti-gal-1 (lanes 3, 7 and 11); anti-gal-3 (lanes 4, 8 and 12) or anti-Sm (lanes 5, 9 and 13) was incubated with the reactions in 60 mM KCl-buffer for 60 min. Beads were then washed extensively with 60 mM KCl-buffer (lanes 2–5); 130 mM KCl-buffer (lanes 6–9); or 250 mM KCl-buffer (lanes 10–13). The bound fractions were eluted and RNA analyzed by denaturing gel electrophoresis and quantitated by phosphorimage analysis (Molecular Dynamics) following autoradiography. Lane 1 indicates the RNA subjected to immunoprecipitation.
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fig9: Analysis of spliceosomes precipitated by various antisera at increasing salt concentrations. HeLa NE was incubated with 32P-MINX for 60 min in the presence of ATP. Immobilized pre-immune serum (lanes 2, 6 and 10); anti-gal-1 (lanes 3, 7 and 11); anti-gal-3 (lanes 4, 8 and 12) or anti-Sm (lanes 5, 9 and 13) was incubated with the reactions in 60 mM KCl-buffer for 60 min. Beads were then washed extensively with 60 mM KCl-buffer (lanes 2–5); 130 mM KCl-buffer (lanes 6–9); or 250 mM KCl-buffer (lanes 10–13). The bound fractions were eluted and RNA analyzed by denaturing gel electrophoresis and quantitated by phosphorimage analysis (Molecular Dynamics) following autoradiography. Lane 1 indicates the RNA subjected to immunoprecipitation.

Mentions: The strength of the association of galectins with spliceosomes was evaluated in relation to the stable association of the snRNPs with splicing complexes. Splicing complexes formed after a 60 min. splicing reaction (Figure 9, lane 1) were incubated with each galectin antiserum or pre-immune serum in 60 mM KCl buffer (the buffer used for optimal splicing efficiency). Aliquots of the antibody-bound spliceosomes were then washed with 60 mM (lanes 2–5), 130 mM (lanes 6–9) or 250 mM (lanes 10–13) KCl buffers. The bound fractions were eluted and analyzed for radiolabeled RNA. Salt concentrations of 130 or 250 mM released most of the splicing substrate from the galectin-selected columns (>90% of the spliceosomes were released as determined by quantitation from phosphorimage analysis) whereas 130 mM KCl had no effect on spliceosomes selected by anti-Sm antiserum. Even when the salt was increased to 250 mM KCl, ∼20% of the snRNPs remained stably associated with spliceosomes. The loss of spliceosomal RNAs from the antibody columns was due to dissociation of the complexes from each galectin and not due to release of the galectins from their respective antibody. At 130 mM KCl, virtually no gal-1 or gal-3 was released from the immobilized antibodies compared to the galectins bound at 60 mM KCl. At 250 mM KCl, more than 70% of the galectins remained bound to the antibodies (data not shown). We conclude that the association of galectins with the splicing machinery is sensitive to perturbation of ionic strength.


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

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

Analysis of spliceosomes precipitated by various antisera at increasing salt concentrations. HeLa NE was incubated with 32P-MINX for 60 min in the presence of ATP. Immobilized pre-immune serum (lanes 2, 6 and 10); anti-gal-1 (lanes 3, 7 and 11); anti-gal-3 (lanes 4, 8 and 12) or anti-Sm (lanes 5, 9 and 13) was incubated with the reactions in 60 mM KCl-buffer for 60 min. Beads were then washed extensively with 60 mM KCl-buffer (lanes 2–5); 130 mM KCl-buffer (lanes 6–9); or 250 mM KCl-buffer (lanes 10–13). The bound fractions were eluted and RNA analyzed by denaturing gel electrophoresis and quantitated by phosphorimage analysis (Molecular Dynamics) following autoradiography. Lane 1 indicates the RNA subjected to immunoprecipitation.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC1636441&req=5

fig9: Analysis of spliceosomes precipitated by various antisera at increasing salt concentrations. HeLa NE was incubated with 32P-MINX for 60 min in the presence of ATP. Immobilized pre-immune serum (lanes 2, 6 and 10); anti-gal-1 (lanes 3, 7 and 11); anti-gal-3 (lanes 4, 8 and 12) or anti-Sm (lanes 5, 9 and 13) was incubated with the reactions in 60 mM KCl-buffer for 60 min. Beads were then washed extensively with 60 mM KCl-buffer (lanes 2–5); 130 mM KCl-buffer (lanes 6–9); or 250 mM KCl-buffer (lanes 10–13). The bound fractions were eluted and RNA analyzed by denaturing gel electrophoresis and quantitated by phosphorimage analysis (Molecular Dynamics) following autoradiography. Lane 1 indicates the RNA subjected to immunoprecipitation.
Mentions: The strength of the association of galectins with spliceosomes was evaluated in relation to the stable association of the snRNPs with splicing complexes. Splicing complexes formed after a 60 min. splicing reaction (Figure 9, lane 1) were incubated with each galectin antiserum or pre-immune serum in 60 mM KCl buffer (the buffer used for optimal splicing efficiency). Aliquots of the antibody-bound spliceosomes were then washed with 60 mM (lanes 2–5), 130 mM (lanes 6–9) or 250 mM (lanes 10–13) KCl buffers. The bound fractions were eluted and analyzed for radiolabeled RNA. Salt concentrations of 130 or 250 mM released most of the splicing substrate from the galectin-selected columns (>90% of the spliceosomes were released as determined by quantitation from phosphorimage analysis) whereas 130 mM KCl had no effect on spliceosomes selected by anti-Sm antiserum. Even when the salt was increased to 250 mM KCl, ∼20% of the snRNPs remained stably associated with spliceosomes. The loss of spliceosomal RNAs from the antibody columns was due to dissociation of the complexes from each galectin and not due to release of the galectins from their respective antibody. At 130 mM KCl, virtually no gal-1 or gal-3 was released from the immobilized antibodies compared to the galectins bound at 60 mM KCl. At 250 mM KCl, more than 70% of the galectins remained bound to the antibodies (data not shown). We conclude that the association of galectins with the splicing machinery is sensitive to perturbation of ionic strength.

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