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A multiprotein complex mediates the ATP-dependent assembly of spliceosomal U snRNPs

Nature Cell Biology volume 3, pages 945–949 (2001)Cite this article

Abstract

The spliceosomal snRNPs U1, U2, U4 and U5 contain a common RNP structure termed the Sm core that is formed by the binding of Sm proteins onto the U snRNA. Although isolated Sm proteins assemble spontaneously onto U snRNAs in vitro, there is increasing evidence that SMN and its interactor Gemin2 are involved in this process in vivo. Here, we describe a cell-free assay system for the assembly of U snRNPs that closely reproduces in vivo conditions. Using this system, we show that assembly of U1 snRNP depends on ATP. Immunodepletion of SMN–Gemin2 from the extract abolished assembly even though the extract contained high levels of Sm proteins. An affinity-purified macromolecular SMN complex consisting of 16 components including all Sm proteins restored assembly in the immunodepleted extract. These data provide the first direct evidence that a complex containing SMN and Gemin2 mediates the active assembly of spliceosomal U snRNPs.

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Figure 1: In vitro assembly of U1 snRNP in Xenopus laevis egg extract.
Figure 2: ATP-dependent assembly of U1 snRNP in egg extract.
Figure 3: Immunodepletion of SMN and Gemin2 from egg extract abolishes U1 snRNP assembly.
Figure 4: Molecular characterization of a nuclear SMN complex.
Figure 5: In vitro reconstitution of U1 snRNP assembly with purified SMN complexes.

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Acknowledgements

We are indebted to B. Laggerbauer and R. Terns for critically reading the manuscript, R. Lührmann and D. Schümperli for continuous discussion, O. Kelm and C. Kambach for providing reagents, and G. Sowa for technical support. This work was supported by the Boehringer Ingelheim Fonds (to D.B.), the Swiss National Science Foundation (no. 31-52619.97) (to R P.) and grants of the DFG (Fi-573/2-2).

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  1. Max-Planck Institute of Biochemistry, Am Klopferspitz 18a, Martinsried, D-82152, Germany

    Gunter Meister, Dirk Bühler, Friedrich Lottspeich & Utz Fischer

  2. University of Bern, Baltzerstr. 4, Bern, CH-3012, Switzerland

    Ramesh Pillai

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  2. Dirk Bühler
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Correspondence to Utz Fischer.

Supplementary information

Supplementary figures and table

Figure S1 The amino acid sequence of p175. (PDF 777 kb)

Figure S2 No de novo methylation of Sm proteins during assembly of U1 snRNP in vitro.

Figure S3 UV crosslink of SmG to U1 snRNA.

Table 1 Identification of three novel components of SMN complexes NSCII and CSC.

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Meister, G., Bühler, D., Pillai, R. et al. A multiprotein complex mediates the ATP-dependent assembly of spliceosomal U snRNPs. Nat Cell Biol 3, 945–949 (2001). https://doi.org/10.1038/ncb1101-945

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