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

Article metrics

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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

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.

References

  1. 1

    Burge, C. B., Tuschl, T. & Sharp, P. A. in The RNA World II (eds Gesteland, R. R., Cech, T. R. & Atkins, J. F.) 525–560 (Cold Spring Laboratory Harbor Press, Cold Spring Harbor, 1999)

  2. 2

    Séraphin, B. & Rosbash, M. Identification of functional U1 snRNA–pre-mRNA complexes committed to spliceosome assembly and splicing. Cell 59, 349–358 (1989)

  3. 3

    Michaud, S. & Reed, R. An ATP-independent complex commits pre-mRNA to the mammalian spliceosome assembly pathway. Genes Dev. 5, 2534–2546 (1991)

  4. 4

    Lerner, M. R., Boyle, J. A., Mount, S. M., Wolin, S. & Steitz, J. A. Are snRNPs involved in splicing? Nature 283, 220–224 (1980)

  5. 5

    Zhuang, Y. & Weiner, A. M. A compensatory base change in U1 snRNA suppresses a 5′ splice site mutation. Cell 46, 827–835 (1986)

  6. 6

    Séraphin, B., Kretzner, L. & Rosbash, M. A U1 snRNA:pre-mRNA base pairing interaction is required early in yeast spliceosome assembly but does not uniquely define the 5′ cleavage site. EMBO J. 7, 2533–2538 (1988)

  7. 7

    Siliciano, P. G. & Guthrie, C. 5′ splice site selection in yeast: genetic alterations in base-pairing with U1 reveal additional requirements. Genes Dev. 2, 1258–1267 (1988)

  8. 8

    Zhang, D. & Rosbash, M. Identification of eight proteins that cross-link to pre-mRNA in the yeast commitment complex. Genes Dev. 13, 581–592 (1999)

  9. 9

    Puig, O., Gottschalk, A., Fabrizio, P. & Séraphin, B. Interaction of the U1 snRNP with nonconserved intronic sequences affects 5′ splice site selection. Genes Dev. 13, 569–580 (1999)

  10. 10

    Chen, J. Y. et al. Specific alterations of U1-C protein or U1 small nuclear RNA can eliminate the requirement of Prp28p, an essential DEAD box splicing factor. Mol. Cell 7, 227–232 (2001)

  11. 11

    Du, H. & Rosbash, M. Yeast U1 snRNP-pre-mRNA complex formation without U1snRNA–pre-mRNA base pairing. RNA 7, 133–142 (2001)

  12. 12

    Lund, M. & Kjems, J. Defining a 5′ splice site by functional selection in the presence and absence of U1 snRNA 5′ end. RNA 8, 166–179 (2002)

  13. 13

    Heinrichs, V., Bach, M., Winkelmann, G. & Lührmann, R. U1-specific protein C needed for efficient complex formation of U1 snRNP with a 5′ splice site. Science 247, 69–72 (1990)

  14. 14

    Jamison, S. F. et al. U1 snRNP–ASF/SF2 interaction and 5′ splice site recognition: characterization of required elements. Nucleic Acids Res. 23, 3260–3267 (1995)

  15. 15

    Tang, J., Abovich, N., Fleming, M., Séraphin, B. & Rosbash, M. Identification and characterization of a yeast homolog of U1 snRNP-specific protein C. EMBO J. 16, 4082–4091 (1997)

  16. 16

    Traub, P. & Nomura, M. Structure and function of Escherichia coli ribosomes. VI. Mechanism of assembly of 30S ribosomes studied in vitro. J. Mol. Biol. 40, 391–413 (1969)

  17. 17

    Crispino, J. D., Blencowe, B. J. & Sharp, P. A. Complementation by SR proteins of pre-mRNA splicing reactions depleted of U1 snRNP. Science 265, 1866–1869 (1994)

  18. 18

    Tarn, W. Y. & Steitz, J. A. SR proteins can compensate for the loss of U1 snRNP functions in vitro. Genes Dev. 8, 2704–2717 (1994)

  19. 19

    Konforti, B. B. & Konarska, M. M. A short 5′ splice site RNA oligo can participate in both steps of splicing in mammalian extracts. RNA 1, 815–827 (1995)

  20. 20

    Crispino, J. D. & Sharp, P. A. A U6 snRNA:pre-mRNA interaction can be rate-limiting for U1-independent splicing. Genes Dev. 9, 2314–2323 (1995)

  21. 21

    Crispino, J. D., Mermoud, J. E., Lamond, A. I. & Sharp, P. A. Cis-acting elements distinct from the 5′ splice site promote U1-independent pre-mRNA splicing. RNA 2, 664–673 (1996)

  22. 22

    Valcarcel, J., Gaur, R. K., Singh, R. & Green, M. R. Interaction of U2AF65 RS region with pre-mRNA branch point and promotion of base pairing with U2 snRNA. Science 273, 1706–1709 (1996)

  23. 23

    Abovich, N. & Rosbash, M. Cross-intron bridging interactions in the yeast commitment complex are conserved in mammals. Cell 89, 403–412 (1997)

  24. 24

    Berglund, J. A., Chua, K., Abovich, N., Reed, R. & Rosbash, M. The splicing factor BBP interacts specifically with the pre-mRNA branchpoint sequence UACUAAC. Cell 89, 781–787 (1997)

  25. 25

    Herschlag, D. Implications of ribozyme kinetics for targeting the cleavage of specific RNA molecules in vivo: more isn't always better. Proc. Natl Acad. Sci. USA 88, 6921–6925 (1991)

  26. 26

    Rossi, F. et al. Involvement of U1 small nuclear ribonucleoproteins (snRNP) in 5′ splice site–U1 snRNP interaction. J. Biol. Chem. 271, 23985–23991 (1996)

  27. 27

    Reyes, J. L., Kois, P., Konforti, B. B. & Konarska, M. M. The canonical GU dinucleotide at the 5′ splice site is recognized by p220 of the U5 snRNP within the spliceosome. RNA 2, 213–225 (1996)

  28. 28

    Maroney, P. A., Romfo, C. M. & Nilsen, T. W. Functional recognition of 5′ splice site by U4/U6.U5 tri-snRNP defines a novel ATP-dependent step in early spliceosome assembly. Mol. Cell 6, 317–328 (2000)

  29. 29

    Johnson, T. L. & Abelson, J. Characterization of U4 and U6 interactions with the 5′ splice site using a S. cerevisiae in vitro trans-splicing system. Genes Dev. 15, 1957–1970 (2001)

Download references

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.

Author information

Correspondence to Michael Rosbash.

Ethics declarations

Competing interests

The authors declare that they have no competing financial interests.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

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) doi:10.1038/nature00947

Download citation

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.