Interaction of the human autoantigen p150 with splicing snRNPs

B. J. Blencowe, M. Carmo-Fonseca, S. E. Behrens, R. Luhrmann, A. I. Lamond (Lead / Corresponding author)

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Abstract

An important goal of studies on pre-mRNA splicing is to identify factors that mediate the snRNP-snRNP and snRNP-pre-mRNA interactions that take place in the spliceosome. The U4/U6 snRNP is one of the four snRNPs that are subunits of spliceosomes. A rare patient autoimmune serum (MaS serum) has recently been identified that specifically immunoprecipitates U4/U6 snRNP from HeLa cell extracts through recognition of a 150 kDa autoantigen (p150). Here we show that in addition to U4/U6 snRNP, p150 can also be detected associated with 20 S U5, U4/U6.U5 and 17 S U2 snRNPs, but not with U1 snRNP. In each particle p150 is present in sub-stoichiometric levels relative to the major snRNP proteins. We show that MaS serum selectively immunoprecipitates a sub-population of U4/U6 snRNPs in which the m3G-cap structure is masked and that p150 is preferentially associated with U6 snRNA in the U4/U6 particle. Anti-p150 antibodies show widespread nucleoplasmic staining, excluding nucleoli, with an elevated concentration in coiled bodies. This changes to a discrete punctate pattern when cells are treated with α-amanitin. Both the cytological and biochemical data indicate that the p150 autoantigen is a snRNP-associated factor in vivo. We also present biochemical evidence confirming that assembly of U4/U6 and U5 snRNPs into a U4/U6.U5 tri-snRNP particle is an integral step in the spliceosome assembly pathway. Addition of the purified U4/U6.U5 tri-snRNP restores splicing activity to inactivated HeLa nuclear extracts in which splicing had been inhibited by specific depletion of either the U4/U6 or U5 snRNPs.

Original languageEnglish
Pages (from-to)685-697
Number of pages13
JournalJournal of Cell Science
Volume105
Issue number3
Publication statusPublished - 1 Jul 1993

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Keywords

  • Autoantigen
  • snRNP
  • Splicing

Cite this

Blencowe, B. J., Carmo-Fonseca, M., Behrens, S. E., Luhrmann, R., & Lamond, A. I. (1993). Interaction of the human autoantigen p150 with splicing snRNPs. Journal of Cell Science, 105(3), 685-697.