Polyadenylation of three classes of chloroplast RNA in Chlamydomonas reinhardtii

Three classes of RNA, represented by atpB and petD mRNAs, Arg and Glu tRNAs , and 5S rRNA, were found to exist in polyadenylated form in Chlamydomonas reinhardtii chloroplasts. Sequence analysis of cDNA clones derived from rev erse transcriptase-polymerase chain reaction protocols used to select polya denylated RNAs revealed that, at least for the mRNAs and tRNAs, there are t hree apparent types of polyadenylation. In the first case, the poly(A) tail is added at or near the mature 3' end, even when this follows a strong sec ondary structure. In the second case, the tail is added to pre-mRNA or pre- tRNA, suggesting a possible competition between polyadenylation and RNA-pro cessing pathways. Finally, in all cases, the poly(A) tail can be added inte rnally, possibly as a part of an RNA-decay pathway. The tails found in Chla mydomonas chloroplasts differ from those of spinach chloroplasts in adenine content, being nearly homopolymeric (>98% adenine) versus 70% in spinach, and are similar in length to those of Escherichia coli, being mostly betwee n 20 and 50 nt. In vitro assays using a Chlamydomonas chloroplast protein e xtract showed that a 3' end A(25) tail was sufficient to stimulate rapid de gradation of atpB RNA in vitro, with a lesser effect for petD, and only min or effects on trnE. We therefore propose that polyadenylation contributes t o mRNA degradation in Chlamydomonas chloroplasts, but that its effect may v ary.