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

Biochemistry. 2009 Aug 18;48(32):7705-12. doi: 10.1021/bi900071b.

Abstract

Previously, 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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Galectin 3 / genetics
  • Galectin 3 / metabolism*
  • HeLa Cells
  • Humans
  • Macromolecular Substances / metabolism
  • RNA Precursors / genetics
  • RNA Precursors / metabolism
  • RNA Splicing
  • Ribonucleoprotein, U1 Small Nuclear / genetics
  • Ribonucleoprotein, U1 Small Nuclear / metabolism*
  • Spliceosomes / genetics
  • Spliceosomes / metabolism*

Substances

  • Galectin 3
  • Macromolecular Substances
  • RNA Precursors
  • Ribonucleoprotein, U1 Small Nuclear