Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1991 Nov 1;88(21):9784–9788. doi: 10.1073/pnas.88.21.9784

Two-step affinity purification of U7 small nuclear ribonucleoprotein particles using complementary biotinylated 2'-O-methyl oligoribonucleotides.

H O Smith 1, K Tabiti 1, G Schaffner 1, D Soldati 1, U Albrecht 1, M L Birnstiel 1
PMCID: PMC52805  PMID: 1835087

Abstract

U7 snRNP is a low-abundance small nuclear ribonucleoprotein particle essential for 3' processing of replication-dependent histone pre-mRNA. We have developed a two-step purification of the particle from TB21 mouse mastocytoma cell nuclear extracts, with about a 20% overall yield, using affinity binding to 2'-O-methyl oligoribonucleotides. The purified particle is homogeneous with respect to RNA content. SDS/PAGE of the U7 snRNP proteins revealed a full complement of the standard core proteins (B, DD', E, F, and G) found in the majority of snRNPs. In addition, two U7-specific polypeptides of 14 kDa and 50 kDa were identified. Summation of the molecular masses of the identified components of the U7 particle yields a particle mass of 249 kDa, in approximate agreement with estimates from sucrose gradient sedimentation (261 kDa) and nondenaturing gradient PAGE (217 kDa).

Full text

PDF

Images in this article

9785Image
on p.9785
9786Image
on p.9786
9786Image
on p.9786
9787Image
on p.9787
9787Image
on p.9787
9788Image
on p.9788
9788Image
on p.9788

Selected References

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

  1. Adolf G. R., Bodo G., Swetly P. Production of monoclonal antibodies to human IFN-alpha and their use for analysis of the antigenic composition of various natural interferons. J Cell Physiol Suppl. 1982;2:61–68. doi: 10.1002/jcp.1041130511. [DOI] [PubMed] [Google Scholar]
  2. Andersson L. -O., Borg H., Mikaelsson M. Molecular weight estimations of proteins by electrophoresis in polyacrylamide gels of graded porosity. FEBS Lett. 1972 Feb 1;20(2):199–202. doi: 10.1016/0014-5793(72)80793-2. [DOI] [PubMed] [Google Scholar]
  3. Barabino S. M., Sproat B. S., Ryder U., Blencowe B. J., Lamond A. I. Mapping U2 snRNP--pre-mRNA interactions using biotinylated oligonucleotides made of 2'-OMe RNA. EMBO J. 1989 Dec 20;8(13):4171–4178. doi: 10.1002/j.1460-2075.1989.tb08602.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  5. Christofori G., Keller W. 3' cleavage and polyadenylation of mRNA precursors in vitro requires a poly(A) polymerase, a cleavage factor, and a snRNP. Cell. 1988 Sep 9;54(6):875–889. doi: 10.1016/s0092-8674(88)91263-9. [DOI] [PubMed] [Google Scholar]
  6. 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. 1988 Mar;7(3):801–808. doi: 10.1002/j.1460-2075.1988.tb02878.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cotten M., Oberhauser B., Brunar H., Holzner A., Issakides G., Noe C. R., Schaffner G., Wagner E., Birnstiel M. L. 2'-O-methyl, 2'-O-ethyl oligoribonucleotides and phosphorothioate oligodeoxyribonucleotides as inhibitors of the in vitro U7 snRNP-dependent mRNA processing event. Nucleic Acids Res. 1991 May 25;19(10):2629–2635. doi: 10.1093/nar/19.10.2629. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dignam J. D., Lebovitz R. M., Roeder R. G. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 1983 Mar 11;11(5):1475–1489. doi: 10.1093/nar/11.5.1475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. England T. E., Bruce A. G., Uhlenbeck O. C. Specific labeling of 3' termini of RNA with T4 RNA ligase. Methods Enzymol. 1980;65(1):65–74. doi: 10.1016/s0076-6879(80)65011-3. [DOI] [PubMed] [Google Scholar]
  10. Fan B. R., Nguyen T., Waring A., Taeusch W. Staining properties of bovine low molecular weight hydrophobic surfactant proteins after polyacrylamide gel electrophoresis. Anal Biochem. 1990 Apr;186(1):41–45. doi: 10.1016/0003-2697(90)90569-u. [DOI] [PubMed] [Google Scholar]
  11. Gick O., Krämer A., Keller W., Birnstiel M. L. Generation of histone mRNA 3' ends by endonucleolytic cleavage of the pre-mRNA in a snRNP-dependent in vitro reaction. EMBO J. 1986 Jun;5(6):1319–1326. doi: 10.1002/j.1460-2075.1986.tb04362.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gick O., Krämer A., Vasserot A., Birnstiel M. L. Heat-labile regulatory factor is required for 3' processing of histone precursor mRNAs. Proc Natl Acad Sci U S A. 1987 Dec;84(24):8937–8940. doi: 10.1073/pnas.84.24.8937. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Harrison B., Zimmerman S. B. Stabilization of T4 polynucleotide kinase by macromolecular crowding. Nucleic Acids Res. 1986 Feb 25;14(4):1863–1870. doi: 10.1093/nar/14.4.1863. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Inoue H., Hayase Y., Imura A., Iwai S., Miura K., Ohtsuka E. Synthesis and hybridization studies on two complementary nona(2'-O-methyl)ribonucleotides. Nucleic Acids Res. 1987 Aug 11;15(15):6131–6148. doi: 10.1093/nar/15.15.6131. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Kohanski R. A., Lane M. D. Monovalent avidin affinity columns. Methods Enzymol. 1990;184:194–200. doi: 10.1016/0076-6879(90)84274-k. [DOI] [PubMed] [Google Scholar]
  17. Konarska M. M. Analysis of splicing complexes and small nuclear ribonucleoprotein particles by native gel electrophoresis. Methods Enzymol. 1989;180:442–453. doi: 10.1016/0076-6879(89)80116-8. [DOI] [PubMed] [Google Scholar]
  18. Krämer A. Fractionation of HeLa cell nuclear extracts reveals minor small nuclear ribonucleoprotein particles. Proc Natl Acad Sci U S A. 1987 Dec;84(23):8408–8412. doi: 10.1073/pnas.84.23.8408. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  20. Lüscher B., Stauber C., Schindler R., Schümperli D. Faithful cell-cycle regulation of a recombinant mouse histone H4 gene is controlled by sequences in the 3'-terminal part of the gene. Proc Natl Acad Sci U S A. 1985 Jul;82(13):4389–4393. doi: 10.1073/pnas.82.13.4389. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Neuhoff V., Arold N., Taube D., Ehrhardt W. Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G-250 and R-250. Electrophoresis. 1988 Jun;9(6):255–262. doi: 10.1002/elps.1150090603. [DOI] [PubMed] [Google Scholar]
  22. Parker K. A., Steitz J. A. Structural analysis of the human U3 ribonucleoprotein particle reveal a conserved sequence available for base pairing with pre-rRNA. Mol Cell Biol. 1987 Aug;7(8):2899–2913. doi: 10.1128/mcb.7.8.2899. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. Retamal C., Babul J. Determination of the molecular weight of proteins by electrophoresis in slab gels with a transverse pore gradient of crosslinked polyacrylamide in the absence of denaturing agents. Anal Biochem. 1988 Dec;175(2):544–547. doi: 10.1016/0003-2697(88)90581-7. [DOI] [PubMed] [Google Scholar]
  25. Schaer J. C., Schindler R. The requirement of mammalian cell cultures for serum proteins. Growth-promoting activity of pepsin-digested serum albumin in different media. Biochim Biophys Acta. 1967 Sep 19;147(1):154–161. doi: 10.1016/0005-2795(67)90098-0. [DOI] [PubMed] [Google Scholar]
  26. 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. 1986 Oct 30-Nov 5Nature. 323(6091):777–781. doi: 10.1038/323777a0. [DOI] [PubMed] [Google Scholar]
  27. Sproat B. S., Lamond A. I., Beijer B., Neuner P., Ryder U. Highly efficient chemical synthesis of 2'-O-methyloligoribonucleotides and tetrabiotinylated derivatives; novel probes that are resistant to degradation by RNA or DNA specific nucleases. Nucleic Acids Res. 1989 May 11;17(9):3373–3386. doi: 10.1093/nar/17.9.3373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Stunnenberg H. G., Birnstiel M. L. Bioassay for components regulating eukaryotic gene expression: a chromosomal factor involved in the generation of histone mRNA 3' termini. Proc Natl Acad Sci U S A. 1982 Oct;79(20):6201–6204. doi: 10.1073/pnas.79.20.6201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Vasserot A. P., Schaufele F. J., Birnstiel M. L. Conserved terminal hairpin sequences of histone mRNA precursors are not involved in duplex formation with the U7 RNA but act as a target site for a distinct processing factor. Proc Natl Acad Sci U S A. 1989 Jun;86(12):4345–4349. doi: 10.1073/pnas.86.12.4345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Zieve G. W., Sauterer R. A. Cell biology of the snRNP particles. Crit Rev Biochem Mol Biol. 1990;25(1):1–46. doi: 10.3109/10409239009090604. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES