Skip to main content
SpringerLink
Log in

In vivo analysis of plant RNA structure: soybean 18S ribosomal and ribulose-1,5-bisphosphate carboxylase small subunit RNAs

  • Published:
Plant Molecular Biology Aims and scope Submit manuscript

Abstract

A method to investigate the structure of RNA molecules within intact plant tissues has been developed. The RNA structures are analyzed using dimethyl sulfate (DMS), which modifies substituents of adenine and cytosine residues within single-stranded regions of RNA molecules. Reactive sites are identified by primer extension analysis. Using this procedure, an analysis of the secondary structure of the cytoplasmic 18S ribosomal RNA in soybean seedling leaves has been completed. DMS modification data are in good agreement with the phylogenetic structure predicted for soybean 18S rRNA. However, there are a few notable exceptions where residues thought to be involved in double-stranded regions in all 18S rRNAs are strongly modified in soybean leaf samples. These data taken together with the phylogenetic structure suggest that alternate structures may exist in vivo.

The further applicability of this technique is demonstrated by comparing the modification pattern obtained in vivo to that obtained in vitro for a particular mRNA molecule encoding the small subunit of ribulose-1,5-bisphosphate carboxylase. The results obtained are compared to a predicted minimum energy secondary structure. The data indicate that the conformation of RNA molecules within the cell may not be reflected in a structural analysis of purified mRNA molecules.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Berry JO, Breiding DE, Klessig DF: Light-mediated control of translational initiation of ribulose-1,5-bisphosphate carboxylase in amaranth cotyledons. Plant Cell 2: 795–803 (1990).

    Google Scholar 

  2. Berry-Lowe SL, Meagher RB: The nucleotide sequence, expression, and evolution of one member of a multigene family encoding the small subunit of ribulose-1,5-bisphosphate carboxylase in soybean. J Mol Appl Genet 1: 483–498 (1982).

    Google Scholar 

  3. Berry-Lowe SL, Meagher RB: Transcriptional regulation of a gene encoding the small subunit of ribulose-1,5-bisphosphate carboxylase in soybean tissue is linked to the phytochrome response. Mol Cell Biol 5: 1910–1917 (1985).

    Google Scholar 

  4. Capasso O, Bleecker GC, Heintz N: Sequences controlling histone H4 mRNA abundance. EMBO J 6: 1825–1831 (1987).

    Google Scholar 

  5. Chan Y-L, Gutell R, Noller HF, Wool IG: The nucleotide sequence of a rat 18S ribosomal ribonucleic acid gene and a proposal for the secondary structure of 18S ribosomal ribonucleic acid. J Biol Chem 259: 224–230 (1984).

    Google Scholar 

  6. Choi YD, Dreyfuss G. Isolation of the heterogeneous nuclear RNA-ribonucleoprotein complex (hnRNP): A unique supramolecular assembly. Proc Natl Acad Sci USA 81: 7471–7475 (1984).

    Google Scholar 

  7. Condit CM, Meagher RB: Expression of a gene encoding a glycine-rich protein in petunia. Mol Cell Biol 7: 4273–4279 (1987).

    Google Scholar 

  8. Cone KC, Steege DA: Functional analysis of lac repressor restart sites in translational initiation and reinitiation. J Mol Biol 186: 733–742 (1985).

    Google Scholar 

  9. Cone KC, Steege DA: Messenger RNA conformation and ribosome selection of translational reinitiation sites in the lac repressor mRNA. J Mol Biol 186: 725–732 (1985).

    Google Scholar 

  10. Devereaux J, Haeberli P, Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucl Acids Res 12: 387–395 (1984).

    Google Scholar 

  11. Dickey LF, Wang Y-H, Shull GE, WortmanIII IA, Theil EC: The importance of the 3′-untranslated region in the translational control of ferritin mRNA. J Biol Chem 263: 3071–3074 (1988).

    Google Scholar 

  12. Dreyfuss G, Swanson MS, Pino-Roma S: Heterogeneous nuclear ribonucleoprotein particles and the pathway of mRNA formation. Trends Biochem Sci 13: 86–91 (1988).

    Google Scholar 

  13. Ehresmann C, Baudin F, Mougel M, Romby P, Ebel J-P, Ehresmann B. Probing the structure of RNAs in solution. Nucl Acids Res 15: 9109–9128 (1987).

    Google Scholar 

  14. Eperon LR, Graham IR, Griffiths AD, Eperon IC: Effects of RNA secondary structure on alternative splicing of pre-mRNA: Is folding limited to a region behind the transcribing RNA polymerase? Cell 54: 393–401 (1988).

    Google Scholar 

  15. Grandbastien MA, Berry-Lowe SL, Shirley BW, Meagher RB: Two soybean ribulose-1,5-bisphosphate carboxylase small subunit genes share extensive homology even in distant flanking sequences. Plant Mol Biol 7: 451–465 (1986).

    Google Scholar 

  16. Hill WE, Dahlberg A, Garrett RA, Moore PB, Schlessinger D, Warner JR. The Ribosome. American Society for Microbiology, Washington, DC (1990).

  17. Inou T, Cech TR: Secondary structure of the circular form of the Tetrahymena rRNA intervening sequence: A technique for RNA structure analysis using chemical probes and reverse transcriptase. Proc Natl Acad Sci USA 82: 648–652 (1985).

    Google Scholar 

  18. Jones TR, Cole MD: Rapid cytoplasmic turnover of c-myc mRNA: Requirement of the 3′ untranslated sequences. Mol Cell Biol 7: 4513–4521 (1987).

    Google Scholar 

  19. Kozak M: Possible role of flanking nucleotides in recognition of the AUG initiator codon by eukaryotic ribosomes. Nucl Acids Res 9: 5233–5252 (1981).

    Google Scholar 

  20. Kozak M: Influences of mRNA secondary structure on initiation by eukaryotic ribosomes. Proc Natl Acad Sci USA 83: 2850–2854 (1986).

    Google Scholar 

  21. Kozak M: Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell 44: 283–292 (1986).

    Google Scholar 

  22. Kozak M: At least six nucleotides preceding the AUG initiator codon enhance translation in mammalian cells. J Mol Biol 196: 947–950 (1987).

    Google Scholar 

  23. Kozak M: Leader length and secondary structure modulate mRNA function under conditions of stress. Mol Cell Biol 8: 2737–2744 (1988).

    Google Scholar 

  24. Kozak M: A profusion of controls. J Cell Biol 107: 1–7 (1988).

    Google Scholar 

  25. Kozak M: Circumstances and mechanisms of inhibition of translation by secondary structure in eucaryotic mRNAs. Mol Cell Biol 9: 5134–5142 (1989).

    Google Scholar 

  26. Lempereur L, Nicoloso M, Riehl N, Ehresmann C, Ehresmann B, Bachellerie J-P: Conformation of yeast 18S rRNA. Direct chemical probing of the 5′ domain in ribosomal subunits and in deproteinized RNA by reverse transcriptase mapping of dimethyl sulfate-accessible sites. Nucl Acids Res 13: 8339–8357 (1985).

    Google Scholar 

  27. Maden BEH: Methylation map of Xenopus laevis ribosomal RNA. 288: 293–296 (1980).

    Google Scholar 

  28. Maden BEH, Reeder RH: Partial mapping of methylated sequences in Xenopus laevis ribosomal RNA by preparative hybridization to cloned fragments of ribosomal DNA. Nucl Acids Res 6: 817–830 (1979).

    Google Scholar 

  29. Mayford M, Weisblum B: Conformation alterations in the ermC transcript in vivo during induction. EMBO J 8: 4307–4314 (1989).

    Google Scholar 

  30. Moazed D, Stern S, Noller HF: Rapid chemical probing of conformation in 16S ribosomal RNA and 30S ribosomal subunits using primer extension. J Mol Biol 187: 399–416 (1986).

    Google Scholar 

  31. Moine H, Romby P, Springer M, Grunberg-Manago M, Ebel JP, Ehresmann C, Ehresmann B: Messenger RNA structure and gene regulation at the translational level in Escherichia coli: The case of threonine:tRNAThr ligase. Proc Natl Acad Sci USA 85: 7892–7896 (1988).

    Google Scholar 

  32. Mougel M, Eyermann L, Westhof L, Romby P, Expert-Bezancon A, Ebel J-P, Ehresmann B, Ehresmann C: Binding of Escherichi coli ribosomal protein S8 to 16 S rRNA: A model for the interaction and tertiary structure of the RNA binding site. J Mol Biol 198: 91–107 (1987).

    Google Scholar 

  33. Noller HF: Structure of ribosomal RNA. Annu Rev Biochem 53: 119–162 (1984).

    Google Scholar 

  34. Pandey NB, Marzluff WF: The stem-loop structure at the 3′ end of histone mRNA is necessary and sufficient for regulation of histone mRNA stability. Mol Cell Biol 7: 4557–4559 (1987).

    Google Scholar 

  35. Rabbitts PH, Forster A, Stinson MA, Rabbitts TH: Truncation of exon 1 from the c-myc gene results in prolonged c-myc mRNA stability. EMBO J 4: 3727–3733 (1985).

    Google Scholar 

  36. Rairkar A, Rubino HM, Lockard RE: Chemical probing of adenine residues within the secondary structure of rabbit 18LS ribosomal RNA. Biochemistry 27: 528–592 (1988).

    Google Scholar 

  37. Ross J, Kobs G: H4 histone messenger RNA decay in cell-free extracts initiates at or near the 3′ terminus and proceeds 3′ to 5′. J Mol Biol 188: 579–593 (1986).

    Google Scholar 

  38. Ross J, Peltz SW, Kobs G, Brewer G: Histone mRNA degradation in vivo: the first detectable step occurs at or near the 3′ terminus. Mol Cell Biol 6: 4362–4371 (1986).

    Google Scholar 

  39. Saito H, Hayday AC, Wiman K, Hayward WS, Tonegawa S: Activation of the c-myc gene by translocation: A model for translational control. Proc Natl Acad Sci USA 80: 7476–7480 (1983).

    Google Scholar 

  40. Senecoff JF, Meagher RB: In vivo analysis of plant 18S rRNA structure: Evolution of plant 18S rRNA. Meth Enzymol, in press (1991).

  41. Senecoff JF, Rossmeissl PJ, Cox MM: DNA recognition by the FLP recombinase of the yeast 2μ plasmid. A mutational analysis of the FLP binding site. J Mol Biol 201: 405–421 (1988).

    Google Scholar 

  42. Shatkin AJ: mRNA cap binding proteins: essential factors for initiating translation. Cell 40: 223–224 (1985).

    Google Scholar 

  43. Shirley BW, Berry-Lowe SL, Rogers SG, Flick JS, Horsch R, Fraley RT, Meagher RB. 5′ proximal sequences of a soybean ribulose-1,5-bisphosphate carboxylase small subunit gene direct light and phytochrome controlled transcription. Nucl Acids Res 15: 6501–6514. (1987).

    Google Scholar 

  44. Shirley BW, Ham DP, Senecoff JF, Berry-Lowe SL, Zurfluh LL, Shah DM, Meagher RB: Comparison of the expression of two highly homologous members of the soybean ribulose-1,5-bisphosphate carboxylase small subunit gene family. Plant Mol Biol 14: 909–925 (1990).

    Google Scholar 

  45. Shirley BW, Meagher RB: A potential role for RNA turnover in the light regulation of plant gene expression: ribulose-1,5-bisphosphate carboxylase small subunit gene in soybean. Nucl Acids Res (1990).

  46. Silverthorne J, Tobin EM: Phytochrome regulation of nuclear gene expression. BioEssays 7: 18–23 (1987).

    Google Scholar 

  47. Smith CJS, Watson CF, Ray J, Bird CR, Morris PC, Schuch W, Grierson D: Antisense RNA inhibition of polygalacturonase gene expression in transgenic tomatoes. Nature 334: 724–726 (1988).

    Google Scholar 

  48. Solnick D: Alternative splicing caused by RNA secondary structure. Cell 43: 667–676 (1985).

    Google Scholar 

  49. Solnick D, Lee SI: Amount of RNA secondary structure required to induce an alternative splice. Mol Cell Biol 7: 3194–3198 (1987).

    Google Scholar 

  50. Spena A, Krause E, Dobberstein B: Translational efficiency of zein mRNA is reduced by hybrid formation between the 5′ and 3′ untranslated region. EMBO J 4: 2153–2158 (1985).

    Google Scholar 

  51. Stern S, Wilson RC, Noller HF: Localization of the binding site for protein S4 on 16S ribosomal RNA by chemical and enzymatic probing and primer extension. J Mol Biol 192: 101–110 (1986).

    Google Scholar 

  52. Thompson DM, Meagher RB: Transcriptional and post-transcriptional processes regulate expression of RNA encoding the small subunit of ribulose-1,5-bisphosphate carboxylase differently in petunia and in soybean. Nucl Acids Res 18: 3621–3629 (1990).

    Google Scholar 

  53. van der Krol AR, Mol JNM, Stuitje AR: Modulation of eukaryotic gene expression by complementary RNA or DNA sequences. Biotechniques 6: 958–976 (1988).

    Google Scholar 

  54. Van Stolk BJ, Noller HF. Use of RNA-DNA hybridization to facilitate chemical probing of large RNA molecules. Meth Enzymol, preprint (1987).

  55. Zuker M, Stiegler P: Optimal computer folding of large RNA sequences using thermodynamics and auxilliary information. Nucl Acids Res 9: 133–148 (1981).

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Genetics, University of Georgia, 30602, Athens, GA, USA

    Julie F. Senecoff & Richard B. Meagher

Authors
  1. Julie F. Senecoff
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Richard B. Meagher
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Senecoff, J.F., Meagher, R.B. In vivo analysis of plant RNA structure: soybean 18S ribosomal and ribulose-1,5-bisphosphate carboxylase small subunit RNAs. Plant Mol Biol 18, 219–234 (1992). https://doi.org/10.1007/BF00034951

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00034951

Key words

Search

Navigation