title: Association of galectin-1 and galectin-3 with Gemin4 in complexes containing the SMN protein.
abstract: In previous studies we showed that galectin-1 and galectin-3 are factors required for the splicing of pre-mRNA, as assayed in a cell-free system. Using a yeast two-hybrid screen with galectin-1 as bait, Gemin4 was identified as a putative interacting protein. Gemin4 is one component of a macromolecular complex containing approximately 15 polypeptides, including SMN (survival of motor neuron) protein. Rabbit anti-galectin-1 co-immunoprecipitated from HeLa cell nuclear extracts, along with galectin-1, polypeptides identified to be in this complex: SMN, Gemin2 and the Sm polypeptides of snRNPs. Direct interaction between Gemin4 and galectin-1 was demonstrated in glutathione S-transferase (GST) pull-down assays. We also found that galectin-3 interacted with Gemin4 and that it constituted one component of the complex co-immunoprecipitated with galectin-1. Indeed, fragments of either Gemin4 or galectin-3 exhibited a dominant negative effect when added to a cell-free splicing assay. For example, a dose-dependent inhibition of splicing was observed in the presence of exogenously added N-terminal domain of galectin-3 polypeptide. In contrast, parallel addition of either the intact galectin-3 polypeptide or the C-terminal domain failed to yield the same effect. Using native gel electrophoresis to detect complexes formed by the splicing extract, we found that with addition of the N-terminal domain the predominant portion of the radiolabeled pre-mRNA was arrested at a position corresponding to the H-complex. Inasmuch as SMN-containing complexes have been implicated in the delivery of snRNPs to the H-complex, these results provide strong evidence that galectin-1 and galectin-3, by interacting with Gemin4, play a role in spliceosome assembly in vivo.
authors.affiliation: Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824, USA.
authors.forename: J W
authors.initials: JW
authors.lastname: Park
authors.forename: P G
authors.initials: PG
authors.lastname: Voss
authors.forename: S
authors.initials: S
authors.lastname: Grabski
authors.forename: J L
authors.initials: JL
authors.lastname: Wang
authors.forename: R J
authors.initials: RJ
authors.lastname: Patterson
mesh_terms: D017398:Alternative Splicing / Q000187:drug effects; D000943:Antigens, Differentiation / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D037483:Galectin 1; D037502:Galectin 3; D005982:Glutathione Transferase / Q000008:administration & dosage / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006388:Hemagglutinins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D046911:Macromolecular Substances; D015778:Minor Histocompatibility Antigens; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D011233:Precipitin Tests; D011485:Protein Binding; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000008:administration & dosage / Q000235:genetics / Q000378:metabolism; D017411:Ribonucleoproteins, Small Nuclear; D012441:Saccharomyces cerevisiae / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid; D017381:Spliceosomes; D020798:Two-Hybrid System Techniques
chemical_list: D000943:Antigens, Differentiation; C406815:GEMIN4 protein, human; D037483:Galectin 1; D037502:Galectin 3; D006388:Hemagglutinins; D046911:Macromolecular Substances; D015778:Minor Histocompatibility Antigens; D009687:Nuclear Proteins; D012322:RNA Precursors; D011993:Recombinant Fusion Proteins; D017411:Ribonucleoproteins, Small Nuclear; D005982:Glutathione Transferase