A mechanism for incorporation of galectin-3 into the spliceosome through its association with U1 snRNP

TitleA mechanism for incorporation of galectin-3 into the spliceosome through its association with U1 snRNP
Publication TypeJournal Article
Year of Publication2009
AuthorsHaudek, KC, Voss PG, Locascio LE, Wang JL, Patterson RJ
JournalBiochemistry
Volume48
Pagination7705-12
Date PublishedAug 18
ISBN Number1520-4995 (Electronic) 0006-2960 (Linking)
Accession Number19603745
KeywordsAnimals, Galectin 3/genetics/*metabolism, Hela Cells, Humans, Macromolecular Substances/metabolism, Ribonucleoprotein, U1 Small Nuclear/genetics/*metabolism, RNA Precursors/genetics/metabolism, RNA Splicing, Spliceosomes/genetics/*metabolism
AbstractPreviously, we showed that galectin-1 and galectin-3 are redundant pre-mRNA splicing factors associated with the spliceosome throughout the splicing pathway. Here we present evidence for the association of galectin-3 with snRNPs outside of the spliceosome (i.e., in the absence of pre-mRNA splicing substrate). Immunoprecipitation of HeLa nuclear extract with anti-galectin-3 resulted in the coprecipitation of the five spliceosomal snRNAs, core Sm polypeptides, and the U1-specific protein, U1 70K. When nuclear extract was fractionated on glycerol gradients, some galectin-3 molecules cosedimented with snRNP complexes. This cosedimentation represents bona fide galectin-3--snRNP complexes as (i) immunoprecipitation of gradient fractions with anti-galectin-3 yielded several complexes with varying ratios of snRNAs and associated proteins and (ii) the distribution of galectin-3--snRNP complexes was altered when the glycerol gradient was sedimented in the presence of lactose, a galectin ligand. A complex at approximately 10S showed an association of galectin-3 with U1 snRNP that was sensitive to treatment with ribonuclease A. We tested the ability of this U1 snRNP to recognize an exogenous pre-mRNA substrate. Under conditions that assemble early splicing complexes, we found this isolated galectin-3--U1 snRNP particle was sufficient to load galectin-3 onto a pre-mRNA substrate, but not onto a control RNA lacking splice sites. Pretreatment of the U1 snRNP with micrococcal nuclease abolished the assembly of galectin-3 onto this early complex. These data identify galectin-3 as a polypeptide associated with snRNPs in the absence of splicing substrate and describe a mechanism for the assembly of galectin-3 onto the forming spliceosome.
URLhttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19603745
DOI10.1021/bi900071b
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