A mechanism for incorporation of galectin-3 into the spliceosome through its association with U1 snRNP.
Previously, we showed that galectin-1 and galectin-3 are redundant pre-mRNA splicing factors associated with the spliceosome throughout the splicing pathway. Here we present evidence for the association of galectin-3 with snRNPs outside of the spliceosome (i.e., in the absence of pre-mRNA splicing substrate). Immunoprecipitation of HeLa nuclear extract with anti-galectin-3 resulted in the coprecipitation of the five spliceosomal snRNAs, core Sm polypeptides, and the U1-specific protein, U1 70K. When nuclear extract was fractionated on glycerol gradients, some galectin-3 molecules cosedimented with snRNP complexes. This cosedimentation represents bona fide galectin-3--snRNP complexes as (i) immunoprecipitation of gradient fractions with anti-galectin-3 yielded several complexes with varying ratios of snRNAs and associated proteins and (ii) the distribution of galectin-3--snRNP complexes was altered when the glycerol gradient was sedimented in the presence of lactose, a galectin ligand. A complex at approximately 10S showed an association of galectin-3 with U1 snRNP that was sensitive to treatment with ribonuclease A. We tested the ability of this U1 snRNP to recognize an exogenous pre-mRNA substrate. Under conditions that assemble early splicing complexes, we found this isolated galectin-3--U1 snRNP particle was sufficient to load galectin-3 onto a pre-mRNA substrate, but not onto a control RNA lacking splice sites. Pretreatment of the U1 snRNP with micrococcal nuclease abolished the assembly of galectin-3 onto this early complex. These data identify galectin-3 as a polypeptide associated with snRNPs in the absence of splicing substrate and describe a mechanism for the assembly of galectin-3 onto the forming spliceosome.
Haudek
Kevin C
KC
Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824, USA.
Voss
Patricia G
PG
Locascio
Lauren E
LE
Wang
John L
JL
Patterson
Ronald J
RJ
D000818:Animals; D037502:Galectin 3 / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D046911:Macromolecular Substances / Q000378:metabolism; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism; D012326:RNA Splicing; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000235:genetics / Q000378:metabolism*; D017381:Spliceosomes / Q000235:genetics / Q000378:metabolism*
D037502:Galectin 3; D046911:Macromolecular Substances; D012322:RNA Precursors; D017412:Ribonucleoprotein, U1 Small Nuclear