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 ∼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.
Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824, USA.
Kevin C
KC
Haudek
Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824, USA.
Patricia G
PG
Voss
Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824, USA.
John L
JL
Wang
Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824, USA patter13@msu.edu.
Ronald J
RJ
Patterson
D001798:Blood Proteins; D002457:Cell Extracts / Q000235:genetics; D002467:Cell Nucleus / Q000235:genetics; D002474:Cell-Free System; D037502:Galectin 3 / Q000235:genetics* / Q000378:metabolism; D037161:Galectins; D006367:HeLa Cells; D006801:Humans; D012322:RNA Precursors / Q000235:genetics* / Q000378:metabolism; D012326:RNA Splicing / Q000235:genetics; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000235:genetics* / Q000378:metabolism; D017381:Spliceosomes / Q000235:genetics* / Q000378:metabolism
D001798:Blood Proteins; D002457:Cell Extracts; D037502:Galectin 3; D037161:Galectins; C428445:LGALS3 protein, human; D012322:RNA Precursors; D017412:Ribonucleoprotein, U1 Small Nuclear