A 10S galectin-3-U1 snRNP complex assembles into active spliceosomes.

In previous studies, we reported that fractionation of HeLa cell nuclear extracts on glycerol gradients revealed an endogenous similar to 10S particle that contained galectin-3 and U1 snRNP and this particle was sufficient to load the galectin polypeptide onto a pre-mRNA substrate. We now document that this interaction between the galectin-3-U1 snRNP particle and the pre-mRNA results in a productive spliceo-somal complex, leading to intermediates and products of the splicing reaction. Nuclear extracts were depleted of U1 snRNP with a concomitant loss of splicing activity. Splicing activity in the U1-depleted extract can be reconstituted by the galectin-3-U1 snRNP particle, isolated by immunoprecipitation of the 10S region (fractions3-5) of the glycerol gradient with anti-galectin-3 antibodies. In contrast, parallel anti-galectin-3 immunoprecipitation of free galectin-3 molecules not in a complex with U1 snRNP (fraction 1 of the same gradient), failed to restore splicing activity. These results indicate that the galectin-3-U1 snRNP-pre-mRNA ternary complex is a functional E complex and that U1 snRNP is required to assemble galectin-3 onto an active spliceosome.