Accurate processing of a eukaryotic precursor ribosomal RNA by ribonuclease MRP in vitro

Science

Volumn 272, Issue 5259, 1996, Pages 268-270

  Lygerou, Zoi ^b   Allmang, Christine ^b   Tollervey, David ^b   Séraphin, Bertrand ^b  

^a EUROPEAN MOLECULAR BIOLOGY LABORATORY   (Germany)

^b NONE

Author keywords

[No Author keywords available]

Indexed keywords

PRIMER RNA; RIBONUCLEASE; RIBONUCLEOPROTEIN; RIBOSOME RNA;

ARTICLE; CONTROLLED STUDY; MITOCHONDRION; NONHUMAN; NUCLEOLUS; PRIORITY JOURNAL; RNA CLEAVAGE; RNA PROCESSING; RNA PURIFICATION;

EID: 0029981894     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.272.5259.268     Document Type: Article

References (34)

1. J. Venema and D. Tollervey, Yeast 11, 1629 (1995).
2. D. D. Chang and D. A. Clayton, Science 235, 1178 (1987); Cell 56, 131 (1989).
3. D. D. Chang and D. A. Clayton, Science 235, 1178 (1987); Cell 56, 131 (1989).
4. T. Kiss and W. Filipowicz, Cell 70, 11 (1992); J. N. Topper, J. L. Bennett, D. A. Clayton, ibid., p. 16.
5. T. Kiss and W. Filipowicz, Cell 70, 11 (1992); J. N. Topper, J. L. Bennett, D. A. Clayton, ibid., p. 16.
6. J. Côté and A. Ruiz-Carrillo, Science 261, 765 (1993).
7. K. Shuai and J. W. Warner, Nucleic Acids Res. 19, 5059 (1991); L Lindahl, R. H. Archer, J. M. Zengal, ibid 20, 295 (1992), S Chu, R. H. Archer, J. M. Zengel, L Lindahl, Proc Natl. Acad. Sci. U.S.A. 91, 659 (1994).
8. K. Shuai and J. W. Warner, Nucleic Acids Res. 19, 5059 (1991); L Lindahl, R. H. Archer, J. M. Zengal, ibid 20, 295 (1992), S Chu, R. H. Archer, J. M. Zengel, L Lindahl, Proc Natl. Acad. Sci. U.S.A. 91, 659 (1994).
9. K. Shuai and J. W. Warner, Nucleic Acids Res. 19, 5059 (1991); L Lindahl, R. H. Archer, J. M. Zengal, ibid 20, 295 (1992), S Chu, R. H. Archer, J. M. Zengel, L Lindahl, Proc Natl. Acad. Sci. U.S.A. 91, 659 (1994).
10. M. E Schmitt and D. A Clayton, Mol Cell. Biol 13, 7935 (1993).
11. Z. Lygerou, P Mitchell, E. Petfalski, B. Séraphin, D. Tollervey, Genes Dev. 8, 1423 (1994).
12. Y. Henry et al , EMBO J. 13, 2452 (1994).
13. S. C. Darr, J W Brown, N. R Pace, Trends Biochem. Sci 17, 178 (1992); S. Altman, L. Kirsebom, S. Talbot, FASEB J. 7, 7 (1993).
14. S. C. Darr, J W Brown, N. R Pace, Trends Biochem. Sci 17, 178 (1992); S. Altman, L. Kirsebom, S. Talbot, FASEB J. 7, 7 (1993).
15. 2 to a final concentration of 4 mM Before addition of the substrate, EGTA was added to a final concentration of 32 mM. For the control, EGTA was added to the IgG precipitates before addition of the micrococcal nuclease. Reaction products were fractionated by gel electrophoresis (25).
16. J. Y. Lee, C. E. Rohlman, L. A. Molony, D. R. Engelke, Mol. Cell. Biol. 11, 721 (1991), A. J. Tranguch, D. W. Kindelberger, C. E. Rohlman, J. Y. Lee, D. R. Engelke, Biochemistry 33, 1778 (1994).
17. J. Y. Lee, C. E. Rohlman, L. A. Molony, D. R. Engelke, Mol. Cell. Biol. 11, 721 (1991), A. J. Tranguch, D. W. Kindelberger, C. E. Rohlman, J. Y. Lee, D. R. Engelke, Biochemistry 33, 1778 (1994).
18. G. Krupp, D. Kahle, T. Vogt, S. Char, J. Mol. Biol. 217, 637 (1991).
19. Z. Lygerou, C. Allmang, D. Tollervey, B. Séraphin, data not shown.
20. D. Pearson et al., Mol. Cell. Biol. 5, 808 (1985).
21. Note that the use of ProtA-Pop1p precipitates was essential for the detection of in vitro processing. No activity was detected in complete extracts (13), probably because of the presence of inhibitors or the occurrence of competing reactions.
22. 4 (pH 7), 50 mM NaCl, 0.2 mM EDTA, 10% glycerol, 0.5 mM DTT, 0 5 mM PMSF, and 2 mM benzamidine]. RNase P remained in the flowthrough (P2), whereas RNase MRP eluted in a 0.4-M NaCl step (M2). During the fractionation, we followed the RNase P and MRP RNAs by slot blot hybridizations and ProtA-Pop1p by protein immunoblotting (25). The RNase P and MRP peak fractions were affinity-selected with IgG agarose. Less than 0.01 % of the starting protein was present in fractions M2 and P2. Because of the high amount of IgGs present on the beads, it is not possible to directly evaluate the final level of purification after the affinity selection. The RNase MRP present in fraction P1 is a minor amount of the total RNase MRP present in extracts, which is poorly functional possibly because it is missing some protein component. This is in agreement with its aberrant chromatographic behavior compared with the bulk of RNase MRP.
23. B Séraphin and M. Rosbash, Cell 59, 349 (1989).
24. 3. Processing at both sites is micrococcal nuclease-sensitive (13)
25. T. Potuschak, W. Rossmanith, R. Karwan, Nucleic Acids Res. 21, 3239 (1993).
26. J. P. Morrissey and D. Tollervey, Trends Biochem. Sci. 20, 78 (1995).
27. G. J. Hannon et al., Mol. Cell. Biol. 9, 4422 (1989); M. T. Yip and M. J. Holland, J. Biol. Chem. 264, 4045 (1989); S Kass, K. Tyc, J A. Steitz, B. Sollner-Webb, Cell 60, 897 (1990).
28. G. J. Hannon et al., Mol. Cell. Biol. 9, 4422 (1989); M. T. Yip and M. J. Holland, J. Biol. Chem. 264, 4045 (1989); S Kass, K. Tyc, J A. Steitz, B. Sollner-Webb, Cell 60, 897 (1990).
29. G. J. Hannon et al., Mol. Cell. Biol. 9, 4422 (1989); M. T. Yip and M. J. Holland, J. Biol. Chem. 264, 4045 (1989); S Kass, K. Tyc, J A. Steitz, B. Sollner-Webb, Cell 60, 897 (1990).
30. R.-J. Lin, A J Newman,S.-C Cheng, J. Abelson, J. Biol. Chem. 260, 14780 (1985).
31. B. Séraphin, L. Kretzner, M. Rosbash, EMBO J 7, 2533 (1988).
32. D. Drainas, S Zimmerly, I Willis, D Soll, FEBS Lett. 251, 84 (1989).
33. J Sambrook, E. F. Fritsch, T. Maniatis, Molecular Cloning (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989).
34. We thank D. A Clayton, M. E. Schmitt, J. Morrissey, J. Mermoud, I. Willis, and D. Soll for the gift of yeast strains, plasmids, and enzymes, D. Engelke for the exchange of unpublished information, and J Lewis for helpful suggestions. We thank our colleagues at the European Molecular Biology Laboratory for careful reading of the manuscript and their help C A. is supported by a fellowship from the European Union. B.S. is on leave from Centre National de la Recherche Scientifique