Association of galectin-1 and galectin-3 with Gemin4 in complexes containing the SMN protein

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 macromolecu...

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Veröffentlicht in:Nucleic acids research 2001-09, Vol.27 (17), p.3595
Hauptverfasser: Park, Jung W, Voss, Patricia G, Grabski, Sharon, Wang, John L, Patterson, Ronald J
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Voss, Patricia G
Grabski, Sharon
Wang, John L
Patterson, Ronald J
description 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.
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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. 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title Association of galectin-1 and galectin-3 with Gemin4 in complexes containing the SMN protein
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