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. 1980 Nov 1;87(2):398–403. doi: 10.1083/jcb.87.2.398

Intracellular distribution of low molecular weight RNA species in HeLa cells

PMCID: PMC2110751  PMID: 6159360

Abstract

Intracellular distributions of the low molecular weight RNA species of HeLa cells were determined by a nonaqueous method of cell fractionation, in which lyophilized cells were homogenized and centrifuged in anhydrous glycerol. The nonaqueous method was used to avoid artifactual extraction of weakly bound nuclear RNA during cell fractionation. We found that the mature small RNA species K, A, C, and D were almost entirely (greater than 95%) nuclear, and that mature 4S tRNA was partially (5-10%) nuclear. Our results gave higher nuclear content of the mature species K, A, C, and 4S than was shown previously with conventional aqueous cell fractionation. The nonaqueous method also gave higher nuclear proportions of some short-lived precursors to mature small RNAs. We found that approximately one-half of recently synthesized pre-4S RNA and more than one-half of recently synthesized 5S RNA were nuclear, whereas these species had been thought to be cytoplasmic from previous work. The species C' and D', precursors to the stable nuclear species C and D respectively, were found to be partially nuclear, also in contrast to earlier work. The stable cytoplasmic species L (oncornavirus 7S RNA) was found to be mostly nuclear shortly after synthesis.

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Selected References

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

  1. Bonner W. M., Laskey R. A. A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels. Eur J Biochem. 1974 Jul 1;46(1):83–88. doi: 10.1111/j.1432-1033.1974.tb03599.x. [DOI] [PubMed] [Google Scholar]
  2. Burdon R. H. Processing of tRNA precursors in higher organisms. Brookhaven Symp Biol. 1975 Jul;(26):138–153. [PubMed] [Google Scholar]
  3. Deimel B., Louis C. H., Sekeris C. E. The presence of small molecular weight RNAs in nuclear ribonucleoprotein particles carrying HnRNA. FEBS Lett. 1977 Jan 15;73(1):80–84. [PubMed] [Google Scholar]
  4. Eliceiri G. L., Gurney T., Jr Subcellular location of precursors to small nuclear RNA species C and D and of newly synthesized 5 S RNA in HeLa cells. Biochem Biophys Res Commun. 1978 Apr 14;81(3):915–919. doi: 10.1016/0006-291x(78)91438-9. [DOI] [PubMed] [Google Scholar]
  5. Eliceiri G. L., Sayavedra M. S. Small RNAs in the nucleus and cytoplasm of HeLa cells. Biochem Biophys Res Commun. 1976 Sep 20;72(2):507–512. doi: 10.1016/s0006-291x(76)80070-8. [DOI] [PubMed] [Google Scholar]
  6. Eliceiri G. L. Sensitivity of low molecular weight RNA synthesis to UV radiation. Nature. 1979 May 3;279(5708):80–81. doi: 10.1038/279080a0. [DOI] [PubMed] [Google Scholar]
  7. Eliceiri G. L. Short-lived, small RNAs in the cytoplasm of HeLa cells. Cell. 1974 Sep;3(1):11–14. doi: 10.1016/0092-8674(74)90031-2. [DOI] [PubMed] [Google Scholar]
  8. Eliceiri G. L. The ribosomal RNA of hamster-mouse hybrid cells. J Cell Biol. 1972 Apr;53(1):177–184. doi: 10.1083/jcb.53.1.177. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Foster D. N., Gurney T., Jr Nuclear location of mammalian DNA polymerase activities. J Biol Chem. 1976 Dec 25;251(24):7893–7898. [PubMed] [Google Scholar]
  10. Gallinaro H., Jacob M. An evaluation of small nuclear RNA in hnRNP. FEBS Lett. 1979 Aug 1;104(1):176–182. doi: 10.1016/0014-5793(79)81110-2. [DOI] [PubMed] [Google Scholar]
  11. Guimont-Ducamp C., Sri-Widada J., Jeanteur P. Occurrence of small molecular weight RNAs in Hela nuclear ribonucleoprotein particles containing HnRNA. Biochimie. 1977;59(8-9):755–758. doi: 10.1016/s0300-9084(77)80259-9. [DOI] [PubMed] [Google Scholar]
  12. Gurney T., Jr, Foster D. N. Nonaqueous isolation of nuclei from cultured cells. Methods Cell Biol. 1977;16:45–68. doi: 10.1016/s0091-679x(08)60091-6. [DOI] [PubMed] [Google Scholar]
  13. Hamada H., Muramatsu M., Urano Y., Onishi T., Kominami R. In vitro synthesis of a 5S RNA precursor by isolated nuclei of rat liver and HeLa cells. Cell. 1979 May;17(1):163–173. doi: 10.1016/0092-8674(79)90304-0. [DOI] [PubMed] [Google Scholar]
  14. Howard E. F. Small nuclear RNA molecules in nuclear ribonucleoprotein complexes from mouse erythroleukemia cells. Biochemistry. 1978 Aug 8;17(16):3228–3236. doi: 10.1021/bi00609a009. [DOI] [PubMed] [Google Scholar]
  15. Kavenoff R., Zimm B. H. Chromosome-sized DNA molecules from Drosophila. Chromosoma. 1973;41(1):1–27. doi: 10.1007/BF00284071. [DOI] [PubMed] [Google Scholar]
  16. Kirsch W. M., Leitner J. W., Gainey M., Schulz D., Lasher R., Nakane P. Bulk isolation in nonaqueous media of nuclei from lyophilized cells. Science. 1970 Jun 26;168(3939):1592–1595. doi: 10.1126/science.168.3939.1592. [DOI] [PubMed] [Google Scholar]
  17. Knight E., Jr, Darnell J. E. Distribution of 5 s RNA in HeLa cells. J Mol Biol. 1967 Sep 28;28(3):491–502. doi: 10.1016/s0022-2836(67)80099-8. [DOI] [PubMed] [Google Scholar]
  18. Laskey R. A., Mills A. D. Quantitative film detection of 3H and 14C in polyacrylamide gels by fluorography. Eur J Biochem. 1975 Aug 15;56(2):335–341. doi: 10.1111/j.1432-1033.1975.tb02238.x. [DOI] [PubMed] [Google Scholar]
  19. Leibowitz R. D., Weinberg R. A., Penman S. Unusual metabolism of 5 S RNA in HeLa cells. J Mol Biol. 1973 Jan;73(1):139–144. doi: 10.1016/0022-2836(73)90166-6. [DOI] [PubMed] [Google Scholar]
  20. Lerner M. R., Boyle J. A., Mount S. M., Wolin S. L., Steitz J. A. Are snRNPs involved in splicing? Nature. 1980 Jan 10;283(5743):220–224. doi: 10.1038/283220a0. [DOI] [PubMed] [Google Scholar]
  21. Loening U. E. The fractionation of high-molecular-weight ribonucleic acid by polyacrylamide-gel electrophoresis. Biochem J. 1967 Jan;102(1):251–257. doi: 10.1042/bj1020251. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lönn U. Exclusive nuclear location of precursor 4 S RNA in vivo. J Mol Biol. 1977 Jun 5;112(4):661–666. doi: 10.1016/s0022-2836(77)80172-1. [DOI] [PubMed] [Google Scholar]
  23. PUCK T. T., MARCUS P. I., CIECIURA S. J. Clonal growth of mammalian cells in vitro; growth characteristics of colonies from single HeLa cells with and without a feeder layer. J Exp Med. 1956 Feb 1;103(2):273–283. doi: 10.1084/jem.103.2.273. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Pederson T., Bhorjee J. S. Evidence for a role of RNA in eukaryotic chromosome structure. Metabolically stable, small nuclear RNA species are covalently linked to chromosomal DNA in HeLa cells. J Mol Biol. 1979 Mar 15;128(4):451–480. doi: 10.1016/0022-2836(79)90288-2. [DOI] [PubMed] [Google Scholar]
  25. Reddy R., Henning D., Busch H. Nucleotide sequence of nucleolar U3B RNA. J Biol Chem. 1979 Nov 10;254(21):11097–11105. [PubMed] [Google Scholar]
  26. Reddy R., Ro-Choi T. S., Henning D., Busch H. Primary sequence of U-1 nuclear ribonucleic acid of Novikoff hepatoma ascites cells. J Biol Chem. 1974 Oct 25;249(20):6486–6494. [PubMed] [Google Scholar]
  27. Rein A. The small molecular weight monodisperse nuclear RNA's in mitotic cells. Biochim Biophys Acta. 1971 Mar 11;232(2):306–313. doi: 10.1016/0005-2787(71)90583-1. [DOI] [PubMed] [Google Scholar]
  28. Rogers J., Wall R. A mechanism for RNA splicing. Proc Natl Acad Sci U S A. 1980 Apr;77(4):1877–1879. doi: 10.1073/pnas.77.4.1877. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Seifert H., Scheurlen M., Northemann W., Heinrich P. C. Low molecular weight RNAs as components of nuclear ribonucleoprotein particles containing heterogeneous nuclear RNA. Biochim Biophys Acta. 1979 Aug 29;564(1):55–66. doi: 10.1016/0005-2787(79)90188-6. [DOI] [PubMed] [Google Scholar]
  30. Shibata H., Ro-Choi T. S., Reddy R., Choi Y. C., Henning D., Busch H. The primary nucleotide sequence of nuclear U-2 ribonucleic acid. The 5'-terminal portion of the molecule. J Biol Chem. 1975 May 25;250(10):3909–3920. [PubMed] [Google Scholar]
  31. Studier F. W. Analysis of bacteriophage T7 early RNAs and proteins on slab gels. J Mol Biol. 1973 Sep 15;79(2):237–248. doi: 10.1016/0022-2836(73)90003-x. [DOI] [PubMed] [Google Scholar]
  32. Walker T. A., Pace N. R., Erikson R. L., Erikson E., Behr F. The 7S RNA common to oncornaviruses and normal cells is associated with polyribosomes. Proc Natl Acad Sci U S A. 1974 Sep;71(9):3390–3394. doi: 10.1073/pnas.71.9.3390. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Weinberg R. A. Nuclear RNA metabolism. Annu Rev Biochem. 1973;42:329–354. doi: 10.1146/annurev.bi.42.070173.001553. [DOI] [PubMed] [Google Scholar]
  34. Weinberg R. A., Penman S. Small molecular weight monodisperse nuclear RNA. J Mol Biol. 1968 Dec;38(3):289–304. doi: 10.1016/0022-2836(68)90387-2. [DOI] [PubMed] [Google Scholar]
  35. Williams J. F. Enhancement of adenovirus plaque formation on HeLa cells by magnesium chloride. J Gen Virol. 1970 Dec;9(3):251–255. doi: 10.1099/0022-1317-9-3-251. [DOI] [PubMed] [Google Scholar]
  36. Zieve G., Benecke B. J., Penman S. Synthesis of two classes of small RNA species in vivo and in vitro. Biochemistry. 1977 Oct 4;16(20):4520–4525. doi: 10.1021/bi00639a029. [DOI] [PubMed] [Google Scholar]
  37. 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]
  38. Zimmerman S. B., Sandeen D. The ribonuclease activity of crystallized pancreatic deoxyribonuclease. Anal Biochem. 1966 Feb;14(2):269–277. doi: 10.1016/0003-2697(66)90137-0. [DOI] [PubMed] [Google Scholar]

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