Immunoprecipitation of spliceosomal RNAs by antisera to galectin-1 and galectin-3.
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.
Wang
Weizhong
W
Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824, USA.
Park
Jung W
JW
Wang
John L
JL
Patterson
Ronald J
RJ
D037483:Galectin 1 / Q000032:analysis* / Q000276:immunology / Q000302:isolation & purification; D037502:Galectin 3 / Q000032:analysis* / Q000276:immunology / Q000302:isolation & purification; D006367:HeLa Cells; D006801:Humans; D007106:Immune Sera; D047468:Immunoprecipitation; D011189:Potassium Chloride / Q000494:pharmacology; D017434:Protein Structure, Tertiary; D012322:RNA Precursors / Q000378:metabolism; D012326:RNA Splicing; D012333:RNA, Messenger / Q000032:analysis* / Q000302:isolation & purification; D017381:Spliceosomes / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism
D037483:Galectin 1; D037502:Galectin 3; D007106:Immune Sera; C445499:LGALS1 protein, human; D012322:RNA Precursors; D012333:RNA, Messenger; D011189:Potassium Chloride