Functional recognition of the 3′ splice site AG by the splicing factor U2AF35

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Abstract

In metazoans, spliceosome assembly is initiated through recognition of the 5′ splice site by U1 snRNP and the polypyrimidine tract by the U2 small nuclear ribonucleoprotein particle (snRNP) auxiliary factor, U2AF (refs 1, 2). U2AF is a heterodimer comprising a large subunit, U2AF65, and a small subunit, U2AF35 (ref. 3). U2AF65 directly contacts the polypyrimidine tract and is required for splicing in vitro4. In comparison, the role of U2AF35 has been puzzling: U2AF35 is highly conserved5,6,7 and is required for viability6,7, but can be dispensed with for splicing in vitro4,8,9. Here we use site-specific crosslinking to show that very early during spliceosome assembly U2AF35 directly contacts the 3′ splice site. Mutational analysis and in vitro genetic selection indicate that U2AF35 has a sequence-specific RNA-binding activity that recognizes the 3′-splice-site consensus, AG/G. We show that for introns with weak polypyrimidine tracts, the U2AF35–3′-splice-site interaction is critical for U2AF binding and splicing. Our results demonstrate a new biochemical activity of U2AF35, identify the factor that initially recognizes the 3′ splice site, and explain why the AG dinucleotide is required for the first step of splicing for some but not all introns.

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Figure 1: Detection of U2AF35–AG-dinucleotide interaction by site-specific labelling and crosslinking.
Figure 2: In vitro genetic selection by U2AF heterodimer from HeLa cells.
Figure 3: Boundaries and AG-dinucleotide requirement for the U2AF35–3′-splice-site interaction.
Figure 4: The U2AF35–3′-splice-site interaction increases U2AF binding affinity.
Figure 5: Requirement of U2AF35–3′-splice-site interaction for splicing of AG-dependent introns.

References

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Acknowledgements

We thank S. DuPont, J. Varcarcel, R. Singh, J. Kan and P. Maroney for suggestions and reagents; R. Reed for β-globin derivatives; P. Zuo and T. Maniatis for U2AF35 antibody and baculovirus U2AF35 expression construct; M. Gama-Carvalho and M. Carmo-Fonseca for U2AF65 monoclonal antibody (MC3); L. Chiang and J. Wang for nuclear extracts. We thank T. Blumenthal for communicating results before publication. This work was supported in part by grants from the NIH to M.R.G. and T.W.N. M.R.G. is an investigator of the Howard Hughes Medical Institute.

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Correspondence to Michael R. Green.

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Wu, S., Romfo, C., Nilsen, T. et al. Functional recognition of the 3′ splice site AG by the splicing factor U2AF35. Nature 402, 832–835 (1999) doi:10.1038/45590

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