Journal Article

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

Kevin C. Haudek, Patricia G. Voss, John L. Wang and Ronald J. Patterson

in Nucleic Acids Research

Volume 44, issue 13, pages 6391-6397
Published in print July 2016 | ISSN: 0305-1048
Published online April 2016 | e-ISSN: 1362-4962 | DOI: http://dx.doi.org/10.1093/nar/gkw303

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In previous studies, we reported that fractionation of HeLa cell nuclear extracts on glycerol gradients revealed an endogenous ∼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 spliceosomal 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 (fractions 3–5) 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.

Journal Article.  4810 words.  Illustrated.

Subjects: Chemistry ; Biochemistry ; Bioinformatics and Computational Biology ; Genetics and Genomics ; Molecular and Cell Biology

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