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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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Time course analysis of spliceosomal RNAs precipitated by anti-gal-1 or anti-gal-3 antiserum. 32P-MINX was incubated with ATP in NE at 30°C. At the times indicated, the reactions were subjected to galectin affinity adsorption and the bound fraction separated into two parts. RNA was extracted from one portion and analyzed by denaturing gel electrophoresis. MINX RNA in splicing reactions at 0, 5, 10, 20, 40 and 60 min (input) and MINX RNA precipitated by anti-gal-1 or anti-gal-3 antiserum. Arrows at left indicate degraded RNA species in the input material.
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fig2: Time course analysis of spliceosomal RNAs precipitated by anti-gal-1 or anti-gal-3 antiserum. 32P-MINX was incubated with ATP in NE at 30°C. At the times indicated, the reactions were subjected to galectin affinity adsorption and the bound fraction separated into two parts. RNA was extracted from one portion and analyzed by denaturing gel electrophoresis. MINX RNA in splicing reactions at 0, 5, 10, 20, 40 and 60 min (input) and MINX RNA precipitated by anti-gal-1 or anti-gal-3 antiserum. Arrows at left indicate degraded RNA species in the input material.

Mentions: To characterize in greater detail galectin-containing splicing complexes, splicing reactions were subjected to antiserum selection during a time course experiment and RNAs (Figures 2 and 3) and some of the proteins (Figure 4) in the immunoprecipitates were characterized. Each galectin antisera co-immunoprecipitated the splicing substrate throughout the time course of the splicing reaction. At the earliest times sampled, both antisera precipitated MINX pre-mRNA, most probably in the form of H/E complexes (see below). Splicing intermediates and ligated exons were precipitated as they appeared during the kinetic analysis (Figure 2, beginning at 20 min for the anti-gal-1 time course and at 40 min for the anti-gal-3 time course). Less spliceosomal RNAs were precipitated in the gal-3 time course compared to the gal-1 time course in this experiment due to use of a lower quantity of anti-gal-3 serum for precipitation. An internal control for the specificity of RNA precipitation is apparent in these analyses. Degraded RNAs of the gal-1 time course observed at 0, 5 and 10 min (highlighted by arrows to the left of the input lanes in Figure 2) are not detected in the immunoprecipitated complexes at these times. Both galectin antisera appeared to immunoprecipitate the excised lariat RNA species preferentially (compare the ratios of lariat to pre-mRNA species in the precipitates and in the input at the 40 and 60 min time points in Figure 2). Both observations (i.e. no precipitation of degraded RNAs and preferential precipitation of free lariat) argue against non-specific adsorption of radioactive RNA species to beads since the precipitated RNAs do not reflect the same relative amounts of the different RNA species in the sample used for immunoselection (input).


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

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

Time course analysis of spliceosomal RNAs precipitated by anti-gal-1 or anti-gal-3 antiserum. 32P-MINX was incubated with ATP in NE at 30°C. At the times indicated, the reactions were subjected to galectin affinity adsorption and the bound fraction separated into two parts. RNA was extracted from one portion and analyzed by denaturing gel electrophoresis. MINX RNA in splicing reactions at 0, 5, 10, 20, 40 and 60 min (input) and MINX RNA precipitated by anti-gal-1 or anti-gal-3 antiserum. Arrows at left indicate degraded RNA species in the input material.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: Time course analysis of spliceosomal RNAs precipitated by anti-gal-1 or anti-gal-3 antiserum. 32P-MINX was incubated with ATP in NE at 30°C. At the times indicated, the reactions were subjected to galectin affinity adsorption and the bound fraction separated into two parts. RNA was extracted from one portion and analyzed by denaturing gel electrophoresis. MINX RNA in splicing reactions at 0, 5, 10, 20, 40 and 60 min (input) and MINX RNA precipitated by anti-gal-1 or anti-gal-3 antiserum. Arrows at left indicate degraded RNA species in the input material.
Mentions: To characterize in greater detail galectin-containing splicing complexes, splicing reactions were subjected to antiserum selection during a time course experiment and RNAs (Figures 2 and 3) and some of the proteins (Figure 4) in the immunoprecipitates were characterized. Each galectin antisera co-immunoprecipitated the splicing substrate throughout the time course of the splicing reaction. At the earliest times sampled, both antisera precipitated MINX pre-mRNA, most probably in the form of H/E complexes (see below). Splicing intermediates and ligated exons were precipitated as they appeared during the kinetic analysis (Figure 2, beginning at 20 min for the anti-gal-1 time course and at 40 min for the anti-gal-3 time course). Less spliceosomal RNAs were precipitated in the gal-3 time course compared to the gal-1 time course in this experiment due to use of a lower quantity of anti-gal-3 serum for precipitation. An internal control for the specificity of RNA precipitation is apparent in these analyses. Degraded RNAs of the gal-1 time course observed at 0, 5 and 10 min (highlighted by arrows to the left of the input lanes in Figure 2) are not detected in the immunoprecipitated complexes at these times. Both galectin antisera appeared to immunoprecipitate the excised lariat RNA species preferentially (compare the ratios of lariat to pre-mRNA species in the precipitates and in the input at the 40 and 60 min time points in Figure 2). Both observations (i.e. no precipitation of degraded RNAs and preferential precipitation of free lariat) argue against non-specific adsorption of radioactive RNA species to beads since the precipitated RNAs do not reflect the same relative amounts of the different RNA species in the sample used for immunoselection (input).

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