Polyadenylation in Arabidopsis and Chlamydomonas organelles: The input of nucleotidyltransferases, poly(A) polymerases and polynucleotide phosphorylase

The polyadenylation-stimulated RNA degradation pathway takes place in plant and algal organelles, yet the identities of the enzymes that catalyze the addition of the tails remain to be clarified. In a search for the enzymes responsible for adding poly(A) tails in Chlamydomonas and Arabidopsis organelles, reverse genetic and biochemical approaches were employed. The involvement of candidate enzymes including members of the nucleotidyltransferase (Ntr) family and polynucleotide phosphorylase (PNPase) was examined. For several of the analyzed nuclear-encoded proteins, mitochondrial localization was established and possible dual targeting to mitochondria and chloroplasts could be predicted. We found that certain members of the Ntr family, when expressed in bacteria, displayed poly(A) polymerase (PAP) activity and partially complemented an Escherichia coli strain lacking the endogenous PAP1 enzyme. Other Ntr proteins appeared to be specific for tRNA maturation. When the expression of PNPase was down-regulated by RNAi in Chlamydomonas, very few poly(A) tails were detected in chloroplasts for the atpB transcript, suggesting that this enzyme may be solely responsible for chloroplast polyadenylation activity in this species. Depletion of PNPase did not affect the number or sequence of mitochondrial mRNA poly(A) tails, where unexpectedly we found, in addition to polyadenylation, poly(U)-rich tails. Together, our results identify several Ntr-PAPs and PNPase in organelle polyadenylation, and reveal novel poly(U)-rich sequences in Chlamydomonas mitochondria.