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Galectins pp 431-449 | Cite as

Examination of the Role of Galectins in Pre-mRNA Splicing

  • Ronald J. Patterson
  • Kevin C. Haudek
  • Patricia G. Voss
  • John L. WangEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1207)

Abstract

Several lines of evidence have been accumulated to indicate that galectin-1 and galectin-3 are two of the many proteins involved in nuclear splicing of pre-mRNA. First, nuclear extracts, capable of carrying out splicing of pre-mRNA in a cell-free assay, contain both of the galectins. Second, depletion of the galectins from nuclear extracts, using either lactose affinity chromatography or immunoadsorption with antibodies, results in concomitant loss of splicing activity. Third, addition of either galectin-1 or galectin-3 to the galectin-depleted extract reconstitutes the splicing activity. Fourth, the addition of saccharides that bind to galectin-1 and galectin-3 with high affinity (e.g., lactose or thiodigalactoside) to nuclear extract results in inhibition of splicing whereas parallel addition of saccharides that do not bind to the galectins (e.g., cellobiose) fail to yield the same effect. Finally, when a splicing reaction is subjected to immunoprecipitation by antibodies directed against galectin-1, radiolabeled RNA species corresponding to the starting pre-mRNA substrate, the mature mRNA product, and intermediates of the splicing reaction are coprecipitated with the galectin. Similar results were also obtained with antibodies against galectin-3. This chapter describes two key assays used in our studies: one reports on the splicing activity by looking at product formation on a denaturing gel; the other reports on the intermediates of spliceosome assembly using non-denaturing or native gels.

Key words

RNA processing Pre-mRNA splicing Spliceosome Cell-free splicing assay Galectin 

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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Ronald J. Patterson
    • 1
  • Kevin C. Haudek
    • 2
  • Patricia G. Voss
    • 3
  • John L. Wang
    • 3
    Email author
  1. 1.Department of Microbiology and Molecular GeneticsMichigan State UniversityEast LansingUSA
  2. 2.Center for Engineering Education Research, College of EngineeringMichigan State UniversityEast LansingUSA
  3. 3.Department of Biochemistry and Molecular BiologyMichigan State UniversityEast LansingUSA

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