Abstract
Fractionation of HeLa cell nuclear extracts by glycerol gradient centrifugation separates endogenous uracil-rich small nuclear ribonucleoprotein complexes (U snRNP) into numerous particles sedimenting from 7S to greater than 60S. Complexes sedimenting at 10S contain a single U snRNP (U1 snRNP) and galectin-3. Addition of antibodies specific for galectin-3 to fractions containing these 10S complexes coprecipitates U1 snRNP, indicating that a fraction of the U1 snRNP is associated with this galectin. Galectin-3 has been shown by depletion-reconstitution studies to be an integral splicing component involved both in spliceosome assembly and splicing activity. The first step in initiation of spliceosome assembly is binding of U1 snRNP to the 5′ splice site of the premessenger RNA substrate. The finding that U1 snRNP and galectin-3 are associated in splicing extracts hints that this complex affords a potential entry point for galectin-3 into the splicing pathway. Addition of U1 snRNP-galectin-3 complexes immunoselected from the 10S region of glycerol gradients to a U1-depleted nuclear extract initiates splicing activity with the formation of splicing intermediates and mature mRNA. This chapter describes the materials and methods for these experiments that document galectin-3–U1 snRNP complexes initiate the splicing reaction in a U1-depleted nuclear extract.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Dignam JD, Lebovitz RM, Roeder RG (1983) Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res 11:1475–1489
Lerner M, Steitz JA (1981) Snurps and scyrps. Cell 25:298–300
Maniatis T, Reed R (1987) The role of small nuclear ribonucleoprotein particles in pre-mRNA splicing. Nature 325:673–678
Hoskins AA, Friedman LJ, Gallagher SS, Crawford DJ, Anderson EG, Wombacher R, Ramirez N, Cornish VW, Gelles J, Moore MJ (2011) Ordered and dynamic assembly of single spliceosomes. Science 331:1289–1295
Dagher SF, Wang JL, Patterson RJ (1995) Identification of galectin-3 as a factor in pre-mRNA splicing. Proc Natl Acad Sci U S A 92:1213–1217
Vyakarnam A, Dagher SF, Wang JL, Patterson RJ (1997) Evidence for a role for galectin-1 in pre-mRNA splicing. Mol Cell Biol 17:4730–4737
Wang W, Park JW, Wang JL, Patterson RJ (2006) Immunoprecipitation of spliceosomal RNAs by antisera to galectin-1 and galectin-3. Nucleic Acids Res 34:5166–5174
Haudek KC, Voss PG, Locascio LE, Wang JL, Patterson RJ (2009) A mechanism for incorporation of galectin-3 into the spliceosome through its association with U1 snRNP. Biochemistry 48:7705–7712
Conway GC, Krainer AR, Spector DL, Roberts RJ (1989) Multiple splicing factors are released from endogenous complexes during in vitro pre-mRNA splicing. Mol Cell Biol 9:5273–5280
Haudek KC, Voss PG, Wang JL, Patterson RJ (2016) A 10S galectin-3-U1 snRNP complex assembles into active spliceosomes. Nucleic Acids Res 44:6391–6397
Patterson RJ, Haudek KC, Voss PG, Wang JL (2015) Examination of the role of galectins in pre-mRNA splicing. Methods Mol Biol 1207:431–449
Agrwal N, Sun Q, Wang SY, Wang JL (1993) Carbohydrate-binding protein 35. I. Properties of the recombinant polypeptide and the individuality of the domains. J Biol Chem 268:14932–14939
Acknowledgments
The work has been supported by National Science Foundation Grant MCB-0092919 and Michigan State University Intramural Research Grant 09-CDFP-2001 (to R.J.P.) and by National Institutes of Health Grant GM-38740 and Michigan AgBioResearch Project MICL02455 (to J.L.W.).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2022 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Voss, P.G., Haudek, K.C., Patterson, R.J., Wang, J.L. (2022). Galectin-3–U1 snRNP Complexes Initiate Splicing Activity in U1-Depleted Nuclear Extracts. In: Stowell, S.R., Arthur, C.M., Cummings, R.D. (eds) Galectins. Methods in Molecular Biology, vol 2442. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2055-7_38
Download citation
DOI: https://doi.org/10.1007/978-1-0716-2055-7_38
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-2054-0
Online ISBN: 978-1-0716-2055-7
eBook Packages: Springer Protocols