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In vivo assembly of functional U7 snRNP requires RNA backbone flexibility within the Sm-binding site

Nature Structural & Molecular Biology volume 13, pages 347–353 (2006)Cite this article

Abstract

Most histone precursor mRNAs (pre-mRNAs) in metazoans are matured by 3′-end cleavage directed by the U7 small nuclear ribonucleoprotein (snRNP). RNA functional groups necessary for in vivo assembly and activity of the U7 snRNP were examined by nucleotide-analog interference mapping and mutagenesis using a chimeric mouse histone H4 pre-mRNA–U7 snRNA construct that is cleaved in cis in Xenopus laevis oocytes. Assembly of the unique U7 Sm protein core is rate limiting for processing in vivo and requires four conserved nucleotides within the U7 Sm-binding site, as well as the correct positioning and size of the U7 terminal stem-loop structure. To our surprise, pseudouridine substitution revealed a requirement for backbone flexibility at a particular position within the U7 Sm site, providing in vivo biochemical evidence that an unusual C2′-endo sugar conformation is necessary for assembly of the Sm ring.

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Figure 1: The histone pre-mRNA–U7 snRNA chimera.
Figure 2: U24Ψ interferes with U7 snRNP assembly in vivo.
Figure 3: The Y12 immunoprecipitation (IP) assay simultaneously assesses U7 Sm core assembly and processing efficiency of the chimera.
Figure 4: Spacing between the U7 terminal stem and the Sm site affects U7 snRNP assembly and processing.
Figure 5: Effects of single-base substitutions within the Sm-binding site on assembly and processing.

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References

  1. Strub, K., Galli, G., Busslinger, M. & Birnstiel, M.L. The cDNA sequences of the sea urchin U7 small nuclear RNA suggest specific contacts between histone mRNA precursor and U7 RNA during RNA processing. EMBO J. 3, 2801–2807 (1984).

    Article  CAS  Google Scholar 

  2. Mowry, K.L. & Steitz, J.A. Identification of the human U7 snRNP as one of several factors involved in the 3′ end maturation of histone premessenger RNA's. Science 238, 1682–1687 (1987).

    Article  CAS  Google Scholar 

  3. Dominski, Z., Yang, X.C., Purdy, M. & Marzluff, W.F. Cloning and characterization of the Drosophila U7 small nuclear RNA. Proc. Natl. Acad. Sci. USA 100, 9422–9427 (2003).

    Article  CAS  Google Scholar 

  4. Pillai, R.S. et al. Unique Sm core structure of U7 snRNPs: assembly by a specialized SMN complex and the role of a new component, Lsm11, in histone RNA processing. Genes Dev. 17, 2321–2333 (2003).

    Article  CAS  Google Scholar 

  5. Schümperli, D. & Pillai, R.S. The special Sm core structure of the U7 snRNP: far-reaching significance of a small nuclear ribonucleoprotein. Cell. Mol. Life Sci. 61, 2560–2570 (2004).

    Article  Google Scholar 

  6. Azzouz, T.N. et al. Toward an assembly line for U7 snRNPs: interactions of U7-specific Lsm proteins with PRMT5 and SMN complexes. J. Biol. Chem. 280, 34435–34440 (2005).

    Article  CAS  Google Scholar 

  7. Stefanovic, B., Hackl, W., Lührmann, R. & Schümperli, D. Assembly, nuclear import and function of U7 snRNPs studied by microinjection of synthetic U7 RNA into Xenopus oocytes. Nucleic Acids Res. 23, 3141–3151 (1995).

    Article  CAS  Google Scholar 

  8. Pillai, R.S., Will, C.L., Lührmann, R., Schümperli, D. & Müller, B. Purified U7 snRNPs lack the Sm proteins D1 and D2 but contain Lsm10, a new 14 kDa Sm D1-like protein. EMBO J. 20, 5470–5479 (2001).

    Article  CAS  Google Scholar 

  9. Grimm, C., Stefanovic, B. & Schümperli, D. The low abundance of U7 snRNA is partly determined by its Sm binding site. EMBO J. 12, 1229–1238 (1993).

    Article  CAS  Google Scholar 

  10. Wu, C.H., Murphy, C. & Gall, J.G. The Sm binding site targets U7 snRNA to coiled bodies (spheres) of amphibian oocytes. RNA 2, 811–823 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Marzluff, W.F. & Duronio, R.J. Histone mRNA expression: multiple levels of cell cycle regulation and important developmental consequences. Curr. Opin. Cell Biol. 14, 692–699 (2002).

    Article  CAS  Google Scholar 

  12. Schaufele, F., Gilmartin, G.M., Bannwarth, W. & Birnstiel, M.L. Compensatory mutations suggest that base-pairing with a small nuclear RNA is required to form the 3′ end of H3 messenger RNA. Nature 323, 777–781 (1986).

    Article  CAS  Google Scholar 

  13. Bond, U.M., Yario, T.A. & Steitz, J.A. Multiple processing-defective mutations in a mammalian histone pre-mRNA are suppressed by compensatory changes in U7 RNA both in vivo and in vitro. Genes Dev. 5, 1709–1722 (1991).

    Article  CAS  Google Scholar 

  14. Dominski, Z., Zheng, L.X., Sanchez, R. & Marzluff, W.F. Stem-loop binding protein facilitates 3′-end formation by stabilizing U7 snRNP binding to histone pre-mRNA. Mol. Cell. Biol. 19, 3561–3570 (1999).

    Article  CAS  Google Scholar 

  15. Kolev, N.G. & Steitz, J.A. Symplekin and multiple other polyadenylation factors participate in 3′-end maturation of histone mRNAs. Genes Dev. 19, 2583–2592 (2005).

    Article  CAS  Google Scholar 

  16. Dominski, Z., Yang, X.C. & Marzluff, W.F. The polyadenylation factor CPSF-73 is involved in histone-pre-mRNA processing. Cell 123, 37–48 (2005).

    Article  CAS  Google Scholar 

  17. Stefanovic, B., Wittop Koning, T.H. & Schümperli, D. A synthetic histone pre-mRNA-U7 small nuclear RNA chimera undergoing cis cleavage in the cytoplasm of Xenopus oocytes. Nucleic Acids Res. 23, 3152–3160 (1995).

    Article  CAS  Google Scholar 

  18. Ryder, S.P. & Strobel, S.A. Nucleotide analog interference mapping. Methods 18, 38–50 (1999).

    Article  CAS  Google Scholar 

  19. Ryder, S.P., Ortoleva-Donnelly, L., Kosek, A.B. & Strobel, S.A. Chemical probing of RNA by nucleotide analog interference mapping. Methods Enzymol. 317, 92–109 (2000).

    Article  CAS  Google Scholar 

  20. Szewczak, L.B., DeGregorio, S.J., Strobel, S.A. & Steitz, J.A. Exclusive interaction of the 15.5 kD protein with the terminal box C/D motif of a methylation guide snoRNP. Chem. Biol. 9, 1095–1107 (2002).

    Article  CAS  Google Scholar 

  21. Lerner, E.A., Lerner, M.R., Janeway, C.A., Jr. & Steitz, J.A. Monoclonal antibodies to nucleic acid-containing cellular constituents: probes for molecular biology and autoimmune disease. Proc. Natl. Acad. Sci. USA 78, 2737–2741 (1981).

    Article  CAS  Google Scholar 

  22. Fischer, U., Sumpter, V., Sekine, M., Satoh, T. & Lührmann, R. Nucleo-cytoplasmic transport of U snRNPs: definition of a nuclear location signal in the Sm core domain that binds a transport receptor independently of the m3G cap. EMBO J. 12, 573–583 (1993).

    Article  CAS  Google Scholar 

  23. Gilmartin, G.M., Schaufele, F., Schaffner, G. & Birnstiel, M.L. Functional analysis of the sea urchin U7 small nuclear RNA. Mol. Cell. Biol. 8, 1076–1084 (1988).

    Article  CAS  Google Scholar 

  24. Arnez, J.G. & Steitz, T.A. Crystal structure of unmodified tRNA(Gln) complexed with glutaminyl-tRNA synthetase and ATP suggests a possible role for pseudo-uridines in stabilization of RNA structure. Biochemistry 33, 7560–7567 (1994).

    Article  CAS  Google Scholar 

  25. Newby, M.I. & Greenbaum, N.L. Investigation of Overhauser effects between pseudouridine and water protons in RNA helices. Proc. Natl. Acad. Sci. USA 99, 12697–12702 (2002).

    Article  CAS  Google Scholar 

  26. Charette, M. & Gray, M.W. Pseudouridine in RNA: what, where, how, and why. IUBMB Life 49, 341–351 (2000).

    Article  CAS  Google Scholar 

  27. Davis, D.R. Stabilization of RNA stacking by pseudouridine. Nucleic Acids Res. 23, 5020–5026 (1995).

    Article  CAS  Google Scholar 

  28. Uesugi, S., Miki, H., Ikehara, M., Iwahashi, H. & Kyogoku, Y. A linear relationship between electronegativity of 2′-substituents and conformation of adenine nucleosides. Tetrahedr. Lett. 20, 4073–4076 (1979).

    Article  Google Scholar 

  29. Altona, C. & Sundaralingam, M. Conformational analysis of the sugar ring in nucleosides and nucleotides. Improved method for the interpretation of proton magnetic resonance coupling constants. J. Am. Chem. Soc. 95, 2333–2344 (1973).

    Article  CAS  Google Scholar 

  30. Streit, A., Koning, T.W., Soldati, D., Melin, L. & Schümperli, D. Variable effects of the conserved RNA hairpin element upon 3′ end processing of histone pre-mRNA in vitro. Nucleic Acids Res. 21, 1569–1575 (1993).

    Article  CAS  Google Scholar 

  31. Jones, M.H. & Guthrie, C. Unexpected flexibility in an evolutionarily conserved protein-RNA interaction: genetic analysis of the Sm binding site. EMBO J. 9, 2555–2561 (1990).

    Article  CAS  Google Scholar 

  32. McConnell, T.S., Lokken, R.P. & Steitz, J.A. Assembly of the U1 snRNP involves interactions with the backbone of the terminal stem of U1 snRNA. RNA 9, 193–201 (2003).

    Article  CAS  Google Scholar 

  33. Golembe, T.J., Yong, J. & Dreyfuss, G. Specific sequence features, recognized by the SMN complex, identify snRNAs and determine their fate as snRNPs. Mol. Cell. Biol. 25, 10989–11004 (2005).

    Article  CAS  Google Scholar 

  34. Jarmolowski, A. & Mattaj, I.W. The determinants for Sm protein binding to Xenopus U1 and U5 snRNAs are complex and non-identical. EMBO J. 12, 223–232 (1993).

    Article  CAS  Google Scholar 

  35. Hartmuth, K., Raker, V.A., Huber, J., Branlant, C. & Lührmann, R. An unusual chemical reactivity of Sm site adenosines strongly correlates with proper assembly of core U snRNP particles. J. Mol. Biol. 285, 133–147 (1999).

    Article  CAS  Google Scholar 

  36. Raker, V.A., Hartmuth, K., Kastner, B. & Lührmann, R. Spliceosomal U snRNP core assembly: Sm proteins assemble onto an Sm site RNA nonanucleotide in a specific and thermodynamically stable manner. Mol. Cell. Biol. 19, 6554–6565 (1999).

    Article  CAS  Google Scholar 

  37. Urlaub, H., Raker, V.A., Kostka, S. & Lührmann, R. Sm protein-Sm site RNA interactions within the inner ring of the spliceosomal snRNP core structure. EMBO J. 20, 187–196 (2001).

    Article  CAS  Google Scholar 

  38. Törö, I. et al. RNA binding in an Sm core domain: X-ray structure and functional analysis of an archaeal Sm protein complex. EMBO J. 20, 2293–2303 (2001).

    Article  Google Scholar 

  39. Thore, S., Mayer, C., Sauter, C., Weeks, S. & Suck, D. Crystal structures of the Pyrococcus abyssi Sm core and its complex with RNA. Common features of RNA binding in archaea and eukarya. J. Biol. Chem. 278, 1239–1247 (2003).

    Article  CAS  Google Scholar 

  40. Schumacher, M.A., Pearson, R.F., Møller, T., Valentin-Hansen, P. & Brennan, R.G. Structures of the pleiotropic translational regulator Hfq and an Hfq-RNA complex: a bacterial Sm-like protein. EMBO J. 21, 3546–3556 (2002).

    Article  CAS  Google Scholar 

  41. Inners, L.D. & Felsenfeld, G. Conformation of polyribouridylic acid in solution. J. Mol. Biol. 50, 373–389 (1970).

    Article  CAS  Google Scholar 

  42. Newby, M.I. & Greenbaum, N.L. Sculpting of the spliceosomal branch site recognition motif by a conserved pseudouridine. Nat. Struct. Biol. 9, 958–965 (2002).

    Article  CAS  Google Scholar 

  43. Cotten, M., Gick, O., Vasserot, A., Schaffner, G. & Birnstiel, M.L. Specific contacts between mammalian U7 snRNA and histone precursor RNA are indispensable for the in vitro 3′ RNA processing reaction. EMBO J. 7, 801–808 (1988).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank S. Strobel (Yale University) for generous gifts of 5′-O-(1-thio)-nucleoside analog triphosphates; R. Breaker, K. Tycowski and L. Weinstein-Szewczak for stimulating discussions; T. Steitz, K. Tycowski, S. Vasudevan and A. Alexandrov for critical reading of the manuscript; and A. Miccinello for secretarial help. This work was supported by US National Institutes of Health grant GM26154 to J.A.S. J.A.S. is an investigator of the Howard Hughes Medical Institute.

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  1. Department of Molecular Biophysics and Biochemistry, Howard Hughes Medical Institute, Yale University, New Haven, 06536, Connecticut, USA

    Nikolay G Kolev & Joan A Steitz

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Correspondence to Joan A Steitz.

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Supplementary information

Supplementary Fig. 1

Known and putative U7 snRNAs identified by BLAST search (PDF 29 kb)

Supplementary Fig. 2

Three-dimensional model of the hydrogen bond–mediated water bridge formed by Ψ in RNA (PDF 44 kb)

Supplementary Methods (PDF 38 kb)

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Kolev, N., Steitz, J. In vivo assembly of functional U7 snRNP requires RNA backbone flexibility within the Sm-binding site. Nat Struct Mol Biol 13, 347–353 (2006). https://doi.org/10.1038/nsmb1075

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