Skip to main content
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
. 1979 Jun;76(6):2635–2639. doi: 10.1073/pnas.76.6.2635

Defective splicing of mitochondrial rRNA in cytochrome-deficient nuclear mutants of Neurospora crassa.

C A Mannella, R A Collins, M R Green, A M Lambowitz
PMCID: PMC383662  PMID: 156923

Abstract

Recent studies have shown that the gene encoding the large (25 S) mitochondrial rRNA of Neurospora crassa contains an intervening sequence of 2-2.5 kilobases that is not present in the mature 25S mitochondrial rRNA. Earlier studies had provided evidence that mitochondrial rRNAs in Neurospora are synthesized via a 32S precursor RNA that contains sequences for both the mature 19S and 25S RNA species. The present work shows that the intervening sequence is not present in 32S RNA. However, we have identified two temperature-sensitive nuclear mutants that fail to excise the intervening sequence at the nonpermissive temperature (37 degrees C). When grown at 37 degrees C, the mutants show decreased ratios of 25S to 19S RNA and accumulate a novel 35S RNA that appears to consist of 25S RNA plus most or all of the intervening sequence. The mutants are allelic but can be distinguished in temperature shift-up experiments, mitochondrial rRNA processing turning off more rapidly in one than in the other. These mutants should provide powerful new tools for studying RNA processing in eukaryotic cells.

Full text

PDF
2635

Images in this article

Selected References

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

  1. Berk A. J., Sharp P. A. Spliced early mRNAs of simian virus 40. Proc Natl Acad Sci U S A. 1978 Mar;75(3):1274–1278. doi: 10.1073/pnas.75.3.1274. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bernardi G. Intervening sequences in the mitochondrial genome. Nature. 1978 Dec 7;276(5688):558–559. doi: 10.1038/276558a0. [DOI] [PubMed] [Google Scholar]
  3. Bos J. L., Heyting C., Borst P., Arnberg A. C., Van Bruggen E. F. An insert in the single gene for the large ribosomal RNA in yeast mitochondrial DNA. Nature. 1978 Sep 28;275(5678):336–338. doi: 10.1038/275336a0. [DOI] [PubMed] [Google Scholar]
  4. Goldberg S., Weber J., Darnell J. E., Jr The definition of a large viral transcription unit late in Ad2 infection of HeLa cells: mapping by effects of ultraviolet irradiation. Cell. 1977 Apr;10(4):617–621. doi: 10.1016/0092-8674(77)90094-0. [DOI] [PubMed] [Google Scholar]
  5. Hopper A. K., Banks F. A yeast mutant which accumulates precursor tRNAs. Cell. 1978 Jun;14(2):211–219. doi: 10.1016/0092-8674(78)90108-3. [DOI] [PubMed] [Google Scholar]
  6. Horowitz M., Laub O., Bratosin S., Aloni Y. Splicing of SV40 late mRNA is a post-transcriptional process. Nature. 1978 Oct 12;275(5680):558–559. doi: 10.1038/275558a0. [DOI] [PubMed] [Google Scholar]
  7. Hutchison H. T., Hartwell L. H., McLaughlin C. S. Temperature-sensitive yeast mutant defective in ribonucleic acid production. J Bacteriol. 1969 Sep;99(3):807–814. doi: 10.1128/jb.99.3.807-814.1969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Knapp G., Beckmann J. S., Johnson P. F., Fuhrman S. A., Abelson J. Transcription and processing of intervening sequences in yeast tRNA genes. Cell. 1978 Jun;14(2):221–236. doi: 10.1016/0092-8674(78)90109-5. [DOI] [PubMed] [Google Scholar]
  9. Kourilsky P., Marcaud L., Sheldrick P., Luzzati D., Gros F. Studies of the messenger RNA of bacteriophage lambda, I. Various species synthesized early after induction of the prophage. Proc Natl Acad Sci U S A. 1968 Nov;61(3):1013–1020. doi: 10.1073/pnas.61.3.1013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Kuriyama Y., Luck D. J. Ribosomal RNA synthesis in mitochondria of Neurospora crassa. J Mol Biol. 1973 Feb 5;73(4):425–437. doi: 10.1016/0022-2836(73)90091-0. [DOI] [PubMed] [Google Scholar]
  11. LUCK D. J. Formation of mitochondria in Neurospora crassa. A quantitative radioautographic study. J Cell Biol. 1963 Mar;16:483–499. doi: 10.1083/jcb.16.3.483. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. LaPolla R. J., Lambowitz A. M. Mitochondrial ribosome assembly in Neurospora crassa. Chloramphenicol inhibits the maturation of small ribosomal subunits. J Mol Biol. 1977 Oct 25;116(2):189–205. doi: 10.1016/0022-2836(77)90212-1. [DOI] [PubMed] [Google Scholar]
  13. Lambowitz A. M., Chua N. H., Luck D. J. Mitochondrial ribosome assembly in Neurospora. Preparation of mitochondrial ribosomal precursor particles, site of synthesis of mitochondrial ribosomal proteins and studies on the poky mutant. J Mol Biol. 1976 Nov 5;107(3):223–253. doi: 10.1016/s0022-2836(76)80003-4. [DOI] [PubMed] [Google Scholar]
  14. Lambowitz A. M., Luck D. J. Studies on the poky mutant of eurospora crassa. Fingerprint analysis of mitochondrial ribosomal RNA. J Biol Chem. 1976 May 25;251(10):3081–3095. [PubMed] [Google Scholar]
  15. Lambowitz A. M. Preparation and analysis of mitochondrial ribosomes. Methods Enzymol. 1979;59:421–433. doi: 10.1016/0076-6879(79)59103-4. [DOI] [PubMed] [Google Scholar]
  16. Lizardi P. M., Luck D. J. Absence of a 5S RNA complnent in the mitochondrial ribosomes of Neurospora crassa. Nat New Biol. 1971 Feb 3;229(5):140–142. doi: 10.1038/newbio229140a0. [DOI] [PubMed] [Google Scholar]
  17. Mannella C. A., Lambowitz A. Interaction of wild-type and poky mitochondrial DNA in heterokaryons of Neurospora. Biochem Biophys Res Commun. 1978 Feb 14;80(3):673–679. doi: 10.1016/0006-291x(78)91621-2. [DOI] [PubMed] [Google Scholar]
  18. McDonell M. W., Simon M. N., Studier F. W. Analysis of restriction fragments of T7 DNA and determination of molecular weights by electrophoresis in neutral and alkaline gels. J Mol Biol. 1977 Feb 15;110(1):119–146. doi: 10.1016/s0022-2836(77)80102-2. [DOI] [PubMed] [Google Scholar]
  19. McMaster G. K., Carmichael G. G. Analysis of single- and double-stranded nucleic acids on polyacrylamide and agarose gels by using glyoxal and acridine orange. Proc Natl Acad Sci U S A. 1977 Nov;74(11):4835–4838. doi: 10.1073/pnas.74.11.4835. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. O'Farrell P. Z., Cordell B., Valenzuela P., Rutter W. J., Goodman H. M. Structure and processing of yeast precursor tRNAs containing intervening sequences. Nature. 1978 Aug 3;274(5670):438–445. doi: 10.1038/274438a0. [DOI] [PubMed] [Google Scholar]
  21. Peacock A. C., Dingman C. W. Molecular weight estimation and separation of ribonucleic acid by electrophoresis in agarose-acrylamide composite gels. Biochemistry. 1968 Feb;7(2):668–674. doi: 10.1021/bi00842a023. [DOI] [PubMed] [Google Scholar]
  22. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  23. Spohr G., Mirault M. E., Imaizumi T., Scherrer K. Molecular-weight determination of animal-cell RNA by electrophoresis in formamide under fully denaturing conditions on exponential polyacrylamide gels. Eur J Biochem. 1976 Feb 16;62(2):313–322. doi: 10.1111/j.1432-1033.1976.tb10163.x. [DOI] [PubMed] [Google Scholar]
  24. Stanley W. M., Jr, Bock R. M. Isolation and physical properties of the ribosomal ribonucleic acid of Escherichia coli. Biochemistry. 1965 Jul;4(7):1302–1311. doi: 10.1021/bi00883a014. [DOI] [PubMed] [Google Scholar]
  25. Sümegi J., Breedveld D., Hossenlopp P., Chambon P. A rapid procedure for purification of EcoRI endonuclease. Biochem Biophys Res Commun. 1977 May 9;76(1):78–85. doi: 10.1016/0006-291x(77)91670-9. [DOI] [PubMed] [Google Scholar]
  26. Terpstra P., Holtrop M., Kroon A. M. The ribosomal RNA genes on Neurospora crassa mitochondrial DNA are adjacent. Biochim Biophys Acta. 1977 Sep 20;478(2):146–155. doi: 10.1016/0005-2787(77)90178-2. [DOI] [PubMed] [Google Scholar]
  27. Tilghman S. M., Curtis P. J., Tiemeier D. C., Leder P., Weissmann C. The intervening sequence of a mouse beta-globin gene is transcribed within the 15S beta-globin mRNA precursor. Proc Natl Acad Sci U S A. 1978 Mar;75(3):1309–1313. doi: 10.1073/pnas.75.3.1309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Wellauer P. K., Dawid I. B. Secondary structure maps of RNA: processing of HeLa ribosomal RNA. Proc Natl Acad Sci U S A. 1973 Oct;70(10):2827–2831. doi: 10.1073/pnas.70.10.2827. [DOI] [PMC free article] [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