Skip to main content
NCBI home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1993 Dec 11;21(24):5775–5781. doi: 10.1093/nar/21.24.5775

Selection of a preribosomal RNA processing site by a nucleolar endoribonuclease involves formation of a stable complex.

D C Eichler 1, J A Liberatore 1, C M Shumard 1
PMCID: PMC310548  PMID: 8284228

Abstract

A nucleolar endoribonuclease from mouse Ehrlich ascites tumor cells, that has been implicated in the endonucleolytic cleavage of mouse precursor ribosomal RNA, specifically and stably binds an in vitro-derived rRNA transcript containing the +650 early processing site. The specificity of binding was demonstrated by mobility shift analysis, glycerol gradient velocity sedimentation analysis, and UV-crosslinking studies. Binding did not require Mg2+ and therefore was not dependent on cleavage; however, binding was dependent on the presence of the early +650 processing site since a pre-rRNA transcript with the +650 processing site deleted failed to compete in binding. A small nucleolar RNA component was not required for the formation of this stable complex or for the specific cleavage of a processing competent pre-rRNA transcript. UV crosslinking studies using 32P-labeled 5-azidouridine-substituted pre-rRNA with bound nucleolar endoribonuclease identified three closely sized polypeptides of approximately 50, approximately 48, and approximately 45 kDa, respectively, that specifically crosslinked to the processing competent rRNA transcript. These three polypeptides species were identified following ribonuclease digestion and electrophoresis on a SDS-polyacrylamide gel. An identical pattern of labeled polypeptides was also identified from gel mobility shift analysis where the specifically shifted material was U.V. crosslinked. The largest of these polypeptides corresponded to the estimated size of the nucleolar endoribonuclease, while the lower molecular weight species may represent partially proteolyzed enzyme. Overall, these results suggest that the unique specificity of the nucleolar endoribonuclease may, in part, be attributed to the formation of a stable complex at the +650 processing site for mouse preribosomal RNA, and that formation of this unique stable complex affords a means to specifically label the limited amount of available partially purified enzyme for sequence analysis.

Full text

PDF
5775

Images in this article

5777Image
on p.5777
5778Image
on p.5778
5778Image
on p.5778
5779Image
on p.5779
5779Image
on p.5779

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Craig N., Kass S., Sollner-Webb B. Nucleotide sequence determining the first cleavage site in the processing of mouse precursor rRNA. Proc Natl Acad Sci U S A. 1987 Feb;84(3):629–633. doi: 10.1073/pnas.84.3.629. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Eichler D. C., Eales S. J. Isolation and characterization of a single-stranded specific endoribonuclease from Ehrlich cell nucleoli. J Biol Chem. 1982 Dec 10;257(23):14384–14389. [PubMed] [Google Scholar]
  3. Glitz D. G. The nucleotide sequence at the 3'-linked end of bacteriophage MS2 ribonucleic acid. Biochemistry. 1968 Mar;7(3):927–932. doi: 10.1021/bi00843a007. [DOI] [PubMed] [Google Scholar]
  4. Hanna M. M. Photoaffinity cross-linking methods for studying RNA-protein interactions. Methods Enzymol. 1989;180:383–409. doi: 10.1016/0076-6879(89)80113-2. [DOI] [PubMed] [Google Scholar]
  5. Kass S., Craig N., Sollner-Webb B. Primary processing of mammalian rRNA involves two adjacent cleavages and is not species specific. Mol Cell Biol. 1987 Aug;7(8):2891–2898. doi: 10.1128/mcb.7.8.2891. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Kass S., Sollner-Webb B. The first pre-rRNA-processing event occurs in a large complex: analysis by gel retardation, sedimentation, and UV cross-linking. Mol Cell Biol. 1990 Sep;10(9):4920–4931. doi: 10.1128/mcb.10.9.4920. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Kass S., Tyc K., Steitz J. A., Sollner-Webb B. The U3 small nucleolar ribonucleoprotein functions in the first step of preribosomal RNA processing. Cell. 1990 Mar 23;60(6):897–908. doi: 10.1016/0092-8674(90)90338-f. [DOI] [PubMed] [Google Scholar]
  8. Maser R. L., Calvet J. P. U3 small nuclear RNA can be psoralen-cross-linked in vivo to the 5' external transcribed spacer of pre-ribosomal-RNA. Proc Natl Acad Sci U S A. 1989 Sep;86(17):6523–6527. doi: 10.1073/pnas.86.17.6523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  10. Melton D. A., Krieg P. A., Rebagliati M. R., Maniatis T., Zinn K., Green M. R. Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res. 1984 Sep 25;12(18):7035–7056. doi: 10.1093/nar/12.18.7035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Prestayko A. W., Tonato M., Busch H. Low molecular weight RNA associated with 28 s nucleolar RNA. J Mol Biol. 1970 Feb 14;47(3):505–515. doi: 10.1016/0022-2836(70)90318-9. [DOI] [PubMed] [Google Scholar]
  12. Raziuddin, Little R. D., Labella T., Schlessinger D. Transcription and processing of RNA from mouse ribosomal DNA transfected into hamster cells. Mol Cell Biol. 1989 Apr;9(4):1667–1671. doi: 10.1128/mcb.9.4.1667. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Savino R., Gerbi S. A. In vivo disruption of Xenopus U3 snRNA affects ribosomal RNA processing. EMBO J. 1990 Jul;9(7):2299–2308. doi: 10.1002/j.1460-2075.1990.tb07401.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Shumard C. M., Eichler D. C. Ribosomal RNA processing. Limited cleavages of mouse preribosomal RNA by a nucleolar endoribonuclease include the early +650 processing site. J Biol Chem. 1988 Dec 25;263(36):19346–19352. [PubMed] [Google Scholar]
  15. Shumard C. M., Torres C., Eichler D. C. In vitro processing at the 3'-terminal region of pre-18S rRNA by a nucleolar endoribonuclease. Mol Cell Biol. 1990 Aug;10(8):3868–3872. doi: 10.1128/mcb.10.8.3868. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Stroke I. L., Weiner A. M. The 5' end of U3 snRNA can be crosslinked in vivo to the external transcribed spacer of rat ribosomal RNA precursors. J Mol Biol. 1989 Dec 5;210(3):497–512. doi: 10.1016/0022-2836(89)90126-5. [DOI] [PubMed] [Google Scholar]
  17. Tyc K., Steitz J. A. U3, U8 and U13 comprise a new class of mammalian snRNPs localized in the cell nucleolus. EMBO J. 1989 Oct;8(10):3113–3119. doi: 10.1002/j.1460-2075.1989.tb08463.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Zieve G., Penman S. Small RNA species of the HeLa cell: metabolism and subcellular localization. Cell. 1976 May;8(1):19–31. doi: 10.1016/0092-8674(76)90181-1. [DOI] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES