Molecular and General Genetics MGG

, Volume 234, Issue 3, pp 390–400

Multiple plant RNA binding proteins identified by PCR: expression of cDNAs encoding RNA binding proteins targeted to chloroplasts in Nicotiana plumbaginifolia

  • Maja Mieszczak
  • Ulrich Klahre
  • Jonathan H. Levy
  • Gregory J. Goodall
  • Witold Filipowicz
Article

Summary

Pre-mRNA processing in eukaryotic cells requires the participation of multiple protein factors and ribonucleoprotein particles. One class of proteins involved in this process are RNA-binding proteins, which contain a domain of ca. 90 amino acids with a characteristic ribonucleoprotein consensus sequence (RNP-CS). A PCR approach that is suitable for the characterization of RNP-CS-type proteins is described. Fifteen different RNA-binding domains were amplified from Nicotiana tabacum (tobacco) using oligonucleotide primers specific for the sequences (K/R)G(F/Y)(G/A)FVX(F/Y) and (L/I/V)(F/Y)(V/I)(G/K)(N/G)L, which are conserved in known RNP-CS proteins. Using the tobacco domains as probes, cDNAs encoding two RNA-binding proteins, each containing two RNP-CS-type domains, were characterized in N. plumbaginifolia. The proteins, designated CP-RBP30 and CP-RBP31, are targeted to chloroplasts as demonstrated by expression of epitope-tagged cDNAs in transfected protoplasts, followed by indirect immunofluorescence. High levels of mRNA for each protein were found in leaves but not in roots, and expression of the CP-RBP31 mRNA was strongly regulated by light. The N. plumbaginifolia proteins described in this work are distinct from chloroplast RNA-binding proteins characterized recently in tobacco and spinach.

Key words

Immunofluorescence Polymerase chain reaction Protein sorting Protein/RNA interactions Protoplast transfection 

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Copyright information

© Springer-Verlag 1992

Authors and Affiliations

  • Maja Mieszczak
    • 1
  • Ulrich Klahre
    • 1
  • Jonathan H. Levy
    • 1
  • Gregory J. Goodall
    • 1
  • Witold Filipowicz
    • 1
  1. 1.Friedrich Miescher-InstitutBaselSwitzerland
  2. 2.Hanson Centre for Cancer ResearchInstitute of Medical and Veterinary ScienceAdelaideSouth Australia

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