The mechanism of preferential degradation of polyadenylated RNA in the chloroplast. The exoribonuclease 100RNP/polynucleotide phosphorylase displays high binding affinity for poly(A) sequence.
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.
Lisitsky
I
I
Department of Biology, Technion-Israel Institute of Technology, Haifa 32000, Israel.
Kotler
A
A
Schuster
G
G
D001665:Binding Sites; D002736:Chloroplasts / Q000378:metabolism*; D005095:Exoribonucleases / Q000378:metabolism*; D010940:Plant Proteins / Q000378:metabolism; D011061:Poly A / Q000378:metabolism*; D011117:Polyribonucleotide Nucleotidyltransferase / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism*; D018749:RNA, Plant / Q000378:metabolism; D018724:Spinacia oleracea; D013379:Substrate Specificity
D010940:Plant Proteins; D012333:RNA, Messenger; D018749:RNA, Plant; D011061:Poly A; D011117:Polyribonucleotide Nucleotidyltransferase; D005095:Exoribonucleases; C107125:100RNP-polynucleotide phosphorylase