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© The Rockefeller University Press, 0021-9525/1999//1355 $5.00
The Journal of Cell Biology, Volume 145, Number 7, , 1999 1355-1368

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Combined Biochemical and Electron Microscopic Analyses Reveal the Architecture of the Mammalian U2 snRNP



Angela Krämer*, Patric Grüter*, Karsten Gröning*, and Berthold Kastner{ddagger}

* Département de Biologie Cellulaire, Université de Genève, CH-1211 Genève 4, Switzerland; and {ddagger} Institut für Molekularbiologie und Tumorforschung, Philipps-Universität, D-35037 Marburg, Germany

The 17S U2 small nuclear ribonucleoprotein particle (snRNP) represents the active form of U2 snRNP that binds to the pre-mRNA during spliceosome assembly. This particle forms by sequential interactions of splicing factors SF3b and SF3a with the 12S U2 snRNP. We have purified SF3b and the 15S U2 snRNP, an intermediate in the assembly pathway, from HeLa cell nuclear extracts and show that SF3b consists of four subunits of 49, 130, 145, and 155 kD. Biochemical analysis indicates that both SF3b and the 12S U2 snRNP are required for the incorporation of SF3a into the 17S U2 snRNP. Nuclease protection studies demonstrate interactions of SF3b with the 5' half of U2 small nuclear RNA, whereas SF3a associates with the 3' portion of the U2 snRNP and possibly also interacts with SF3b. Electron microscopy of the 15S U2 snRNP shows that it consists of two domains in which the characteristic features of isolated SF3b and the 12S U2 snRNP are conserved. Comparison to the two-domain structure of the 17S U2 snRNP corroborates the biochemical results in that binding of SF3a contributes to an increase in size of the 12S U2 domain and possibly induces a structural change in the SF3b domain.

Key Words: electron microscopy • pre-mRNA splicing • spliceosome • splicing factor • U2 small nuclear ribonucleoprotein particle

Abbreviations used in this paper: PAS, protein A–Sepharose; SAP, spliceosome-associated protein; SF, splicing factor; snRNA, small nuclear RNA; snRNP, small nuclear ribonucleoprotein particle.

Patric Grüter's present address is 5 rue Cherbuliez, CH-1207 Genève, Switzerland. Karsten Gröning's present address is Nikolaus-Otto-Str. 8, D-59557 Lippstadt, Germany.


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