Analysis of Galectins as Splicing Factors

Michigan State University, East Lansing MI

Investigators

Abstract

This research will focus on galectins-1 and -3 as pre-mRNA splicing factors. Galectins are a family of saccharide-binding proteins that (i) has a specific affinity for galactose/lactose and (ii) shares a characteristic amino acid sequence in the carbohydrate recognition domain. In terms of cell biology, several galectins share another intriguing feature - dual localization. Some galectins are found in both the extracellular and intracellular compartments. This laboratory previously demonstrated that galectin-3 was a new and novel splicing factor and documented that another family member, galectin-1, exhibited nuclear localization and functioned as a splicing factor. Thus, the splicing activities of galectins-1 and -3 appear functionally redundant. The requirement of galectin-1 or -3 in pre-mRNA splicing indicates a second biologic role for these proteins in addition to their carbohydrate-binding activity. Are the two activities connected or autonomous? A galectin-1 mutant incapable of binding lactose is splicing competent in an assay carried out in the presence of lactose to inhibit endogenous galectins. These preliminary results hint that the splicing and carbohydrate-binding activities of galectin-1 are independent. Recently, using a yeast two-hybrid screen, this laboratory identified Gemin4 as an interacting partner for galectin-1 (and, by inference, galectin-3). Using an in vitro assay, the laboratory determined that both galectins directly bind to Gemin4. Gemin4 is a component of the SMN (survivor of motor neurons) complex - a large macromolecular machine containing at least 12 polypeptides found in the nucleus and the cytoplasm. The nuclear SMN complex localizes to subnuclear structures termed gems and functions to supply snRNP particles to the splicing apparatus. Preliminary immunoprecipitation experiments using anti-SMN and anti-galectin-1 antisera showed that both SMN and galectin-1 proteins are in a nuclear complex which also contains galectin-3, Gemin2, hnRNP C and Sm B/B' and D polypeptides of the snRNPs. These data provide in vivo evidence that galectins interact with a macromolecular complex involved in pre-mRNA splicing. An emerging theme in cell biology proposes that biosynthetic processes are orchestrated by complex macromolecular machines that are dynamically changing and interconnected. The convergence of the SMN complex and galectins-1/-3 on the spliceosomal H-complex makes it essential to characterize the various nuclear machines/complexes in detail. Thus, the specific objectives of this project include: (1) delineate and characterize components and interactions of nuclear complexes in which Gemin4 and galectins-1 and -3 have been found, (2) define the step(s) in which galectins-1 and -3 participate in splicing and, (3) define the role of the carbohydrate-binding site of galectins-1 and -3 in pre-mRNA splicing activity. An emerging concept in cell biology is that biosynthetic processes are orchestrated by complex, dynamically changing "machines" composed of many individual molecules, at least some of which can associate with and dissociate from the machine in response to changing cellular needs. This research promises to advance the understanding of this important process.