This project proposes to continue our studies on Carbohydrate Binding Protein 35 (CBP35), a predominantly intracellular lectin that binds to galactose-containing glycoconjugates. The polypeptide (M/T about 35,000) consists of two domains: a proline- and glycine-rich domain at the NH2- terminal half and a carbohydrate recognition domain at the COOH-terminal half, with extensive homology to other S-type lectins. The addition of serum growth factors to quiescent cultures of mouse 3T3 fibroblasts increased the expression of CBP35, in terms of elevated transcription rate of the gene, increased accumulation of mRNA, and increased amount of the protein that is translocated to the nucleus. Analysis of CBP35 in the cytoplasm and nucleoplasm suggests that the lectin is associated with a ribonucleoprotein complex, as indicated by its position of sedimentation on sucrose and cesium sulfate gradients. Using a cell-free assay for the splicing of intervening sequences from pre-mRNA, we found that saccharides and antibodies that bind to CBP35 perturb the splicing reaction, whereas control reagents failed to yield the same effort. On the basis of these results, the specific aim of the proposed research includes: (1) to determine the effect on cell-free splicing activity when a nuclear extract is depleted of CBP35 and to attempt to reconstitute the system by readdition of CBP35; (2) to transfect cells with CBP35 cDNA in the sense and antisense orientations and to monitor the growth and the status of mRNA in these cells; (3) to identify the RNA and protein components of the CBP35- ribonucleoprotein complex; and (4) to search for a homolog of CBP35 in yeasts and to study the in vivo consequences of a gene knockout via integrative disruption.
| Haudek, Kevin C; Voss, Patricia G; Wang, John L et al. (2016) A 10S galectin-3-U1 snRNP complex assembles into active spliceosomes. Nucleic Acids Res 44:6391-7 |
| Haudek, Kevin C; Spronk, Kimberly J; Voss, Patricia G et al. (2010) Dynamics of galectin-3 in the nucleus and cytoplasm. Biochim Biophys Acta 1800:181-9 |
| Haudek, Kevin C; Voss, Patricia G; Locascio, Lauren E et al. (2009) A mechanism for incorporation of galectin-3 into the spliceosome through its association with U1 snRNP. Biochemistry 48:7705-12 |
| Gray, Richard M; Davis, Michael J; Ruby, Katherine M et al. (2008) Distinct effects on splicing of two monoclonal antibodies directed against the amino-terminal domain of galectin-3. Arch Biochem Biophys 475:100-8 |
