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An exonic splicing silencer represses spliceosome assembly after ATP-dependent exon recognition

Nature Structural & Molecular Biology volume 13, pages 937–944 (2006)Cite this article

Abstract

Precursor messenger RNA splicing is catalyzed by the spliceosome, a macromolecular complex that assembles in a stepwise process. The spliceosome's dynamic nature suggests the potential for regulation at numerous points along the assembly pathway; however, thus far, naturally occurring regulation of splicing has only been found to influence a small subset of spliceosomal intermediates. Here we report that the exonic splicing silencer (ESS1) that represses splicing of PTPRC (encoding CD45) exon 4 does not function by the typical mechanism of inhibiting binding of U1 or U2 small nuclear ribonucleoproteins (snRNPs) to the splice sites. Instead, a U1-, U2- and ATP-dependent complex forms across exon 4 that is required for inhibiting progression to the U4–U6–U5 tri-snRNP–containing B complex. Such inhibition represents a new mechanism for splicing regulation and suggests that regulation can probably occur at many of the transitions along the spliceosome assembly pathway.

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Figure 1: ESS1 represses splicing of CD45 exon 4 in vitro by stalling spliceosome assembly at an unusual step.
Figure 2: Stalled complex has the hallmarks of a canonical A complex.
Figure 3: Complex assigned as B complex has hallmarks of tri-snRNP association.
Figure 4: ESS1-dependent repression and formation of the stalled complex does not require either flanking splice site.
Figure 5: Stalled complex has the hallmarks of an A-like complex assembled across an exon.
Figure 6: Formation of an AEC is required for ESS1-dependent repression of splicing.
Figure 7: Proteins that cause ESS1-dependent exon skipping inhibit B complex formation.
Figure 8: Model for ESS1 function in hyperstabilizing an AEC.

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Acknowledgements

We thank T. Nilsen, D. Black, S. Sharma and B. Graveley for helpful advice and critical reading of the manuscript. This work was supported by US National Institutes of Health grant R01 GM067719 and Welch Foundation grant I-1634. A.E.H. is supported by the Division of Cell and Molecular Biology Training Program grant at University of Texas Southwestern Medical Center (T32 GM08203). K.W.L. is an E.E. and Greer Garson Fogelson Scholar in Biomedical Research.

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  1. Department of Biochemistry, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, 75390-9038, Texas, USA

    Amy E House & Kristen W Lynch

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  1. Amy E House
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  2. Kristen W Lynch
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A.E.H. performed all experiments. A.E.H. and K.W.L. designed research, analyzed data and wrote the paper.

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Correspondence to Kristen W Lynch.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

A nonspecific competitor RNA does not alleviate the stall in assembly of and ESS1-repressed substrate. (PDF 425 kb)

Supplementary Fig. 2

Selective inactivation of snRNAs by oligo-directed RNase H digestion. (PDF 642 kb)

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House, A., Lynch, K. An exonic splicing silencer represses spliceosome assembly after ATP-dependent exon recognition. Nat Struct Mol Biol 13, 937–944 (2006). https://doi.org/10.1038/nsmb1149

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