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Recognition of atypical 5′ splice sites by shifted base-pairing to U1 snRNA

Abstract

Accurate pre-mRNA splicing is crucial for gene expression. The 5′ splice site (5′ ss)—the highly diverse element at the 5′ end of introns—is initially recognized via base-pairing to the 5′ end of the U1 small nuclear RNA (snRNA). However, many natural 5′ ss have a poor match to the consensus sequence, and are predicted to be weak. Using genetic suppression experiments in human cells, we demonstrate that some atypical 5′ ss are actually efficiently recognized by U1, in an alternative base-pairing register that is shifted by one nucleotide. These atypical 5′ ss are phylogenetically widespread, and many of them are conserved. Moreover, shifted base-pairing provides an explanation for the effect of a 5′ ss mutation associated with pontocerebellar hypoplasia. The unexpected flexibility in 5′ ss–U1 base-pairing challenges an established paradigm and has broad implications for splice-site prediction algorithms and gene-annotation efforts in genome projects.

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Figure 1: Shifted base-pairing between atypical 5′ ss and the 5′ end of U1 snRNA.
Figure 2: Suppressor U1 snRNAs in the shifted register can rescue splicing.
Figure 3: Compensatory U1 mutations that restore shifted but not canonical base-pairing rescue splicing at atypical 5′ ss.
Figure 4: U1 but not U1A7 snRNA decoys reduce splicing via the atypical 5′ ss.
Figure 5: U6 snRNA does not base-pair to the atypical 5′ ss in a shifted register.

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Acknowledgements

We thank M. Hastings and D. Horowitz for insightful comments on the manuscript, R. Sachidanandam for helpful suggestions, and Y. Hua and Z. Zhang for technical advice. X.R. and A.R.K. acknowledge support from the US National Institutes of Health grant GM42699.

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X.R. performed the experiments and the in silico analyses; X.R. and A.R.K. contributed to the design of the study and to the preparation of the manuscript.

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Correspondence to Adrian R Krainer.

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Supplementary Figures 1–7 and Supplementary Tables 1 and 2 (PDF 1179 kb)

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Roca, X., Krainer, A. Recognition of atypical 5′ splice sites by shifted base-pairing to U1 snRNA. Nat Struct Mol Biol 16, 176–182 (2009). https://doi.org/10.1038/nsmb.1546

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