The mechanism of preferential degradation of polyadenylated RNA in the chloroplast. The exoribonuclease 100RNP/polynucleotide phosphorylase displays high binding affinity for poly(A) sequence

• Published in: The Journal of biological chemistry Vol. 272; no. 28; pp. 17648 - 17653
• Main Authors: Lisitsky, I, Kotler, A, Schuster, G
• Format: Journal Article
• Language: English
• Published: United States 11-07-1997
• Subjects: Binding Sites; Chloroplasts - metabolism; Exoribonucleases - metabolism; Plant Proteins - metabolism; Poly A - metabolism; Polyribonucleotide Nucleotidyltransferase - metabolism; RNA, Messenger - metabolism; RNA, Plant - metabolism; Spinacia oleracea; Substrate Specificity
• ISSN: 0021-9258
• DOI: 10.1074/jbc.272.28.17648

Polyadenylation of mRNA in the chloroplast has recently been shown to target the RNA molecule for rapid exonucleolytic degradation. A model has been suggested in which the degradation of chloroplast mRNA is initiated by endonucleolytic cleavage(s) followed by the addition of poly(A)-rich sequences and rapid exonucleolytic degradation. When in vitro transcribed RNAs were incubated with chloroplast protein extract, competition between polyadenylated and non-polyadenylated RNAs for degradation resulted in the rapid degradation of the polyadenylated molecules and stabilization of their non-polyadenylated counterparts. To elucidate the molecular mechanism governing this effect, we determined whether the chloroplast exoribonuclease 100RNP/polynucleotide phosphorylase (PNPase) preferably degrades polyadenylated RNA. When separately incubated with each molecule, isolated 100RNP/PNPase degraded polyadenylated and non-polyadenylated RNAs at the same rate. However, when both molecules were mixed together, the polyadenylated RNA was degraded, whereas the non-polyadenylated RNA was stabilized. In RNA binding experiments, 100RNP/PNPase bound the poly(A) sequence with much higher affinity than other RNA molecules, thereby defining the poly(A)-rich RNA as a preferential substrate for the enzyme. 100RNP/PNPase may therefore be involved in a mechanism in which post-transcriptional addition of poly(A)-rich sequence targets the chloroplast RNA for rapid exonucleolytic degradation.