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The U1 snRNP protein U1C recognizes the 5′ splice site in the absence of base pairing

Nature volume 419, pages 86–90 (2002)Cite this article

Abstract

Splicing of precursor messenger RNA takes place in the spliceosome, a large RNA/protein macromolecular machine1. Spliceosome assembly occurs in an ordered pathway in vitro and is conserved between yeast and mammalian systems. The earliest step is commitment complex formation in yeast or E complex formation in mammals, which engages the pre-mRNA in the splicing pathway and involves interactions between U1 small nuclear ribonucleoprotein (snRNP) and the pre-mRNA 5′ splice site2,3. Complex formation depends on highly conserved base pairing between the 5′ splice site and the 5′ end of U1 snRNA, both in vivo and in vitro4,5,6,7. U1 snRNP proteins also contribute to U1 snRNP activity8,9,10. Here we show that U1 snRNP lacking the 5′ end of its snRNA retains 5′-splice-site sequence specificity. We also show that recombinant yeast U1C protein, a U1 snRNP protein, selects a 5′-splice-site-like sequence in which the first four nucleotides, GUAU, are identical to the first four nucleotides of the yeast 5′-splice-site consensus sequence. We propose that a U1C 5′-splice-site interaction precedes pre-mRNA/U1 snRNA base pairing and is the earliest step in the splicing pathway.

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Figure 1: In vitro selection with U1 snRNP.
Figure 2: Site-specific crosslinking of commitment complex proteins to the 5′-splice-site region in the presence and absence of pre-mRNA/U1snRNA base pairing.
Figure 3: Characterization of complex formation in different extracts and at different temperatures.
Figure 4: Characterization of sequence specificity of recombinant yU1C.
Figure 5: yU1C binding to single-stranded (ss) RNA and duplex substrates.

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Acknowledgements

We thank current and ex-colleagues for discussions, and B. Séraphin and M. Moore for comments on the manuscript. H.D. was supported by a Charles A. King Trust Fellowship. The work was also supported by the National Institutes of Health.

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  1. Howard Hughes Medical Institute, Department of Biology, MS008, Brandeis University, 415 South Street, Waltham, Massachusetts, 02454, USA

    Hansen Du & Michael Rosbash

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Correspondence to Michael Rosbash.

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Du, H., Rosbash, M. The U1 snRNP protein U1C recognizes the 5′ splice site in the absence of base pairing. Nature 419, 86–90 (2002). https://doi.org/10.1038/nature00947

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