Multiple plant RNA binding proteins identified by PCR: expression of cDNAs encoding RNA binding proteins targeted to chloroplasts in Nicotiana plumbaginifolia.
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.
Mieszczak
M
M
Friedrich Miescher-Institut, Basel, Switzerland.
Klahre
U
U
Levy
J H
JH
Goodall
G J
GJ
Filipowicz
W
W
D000595:Amino Acid Sequence; D001483:Base Sequence; D002736:Chloroplasts / Q000502:physiology*; D016384:Consensus Sequence; D004247:DNA / Q000235:genetics; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D008027:Light; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D010947:Plants, Toxic; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics; D016601:RNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D012261:Ribonucleoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D014026:Nicotiana / Q000235:genetics*
D009838:Oligodeoxyribonucleotides; D010940:Plant Proteins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D012261:Ribonucleoproteins; D004247:DNA