Altering the 3 ' UTR endonucleolytic cleavage site of a Chlamydomonas chloroplast mRNA affects 3 '-end maturation in vitro but not in vivo

The 3' ends of chloroplast mRNAs are produced by the processing of longer p recursors. The 3' ends of most plastid mRNAs are located at, or several nuc leotides downstream of, stem-loop structures, which act as 3'-end-processin g signals and RNA stability elements. In chloroplasts of the green alga Chl amydomonas reinhardtii, 3'-end maturation of atpB mRNA involves endonucleol ytic cleavage of the pre-mRNA at an AU-rich site located about 10 nucleotid es downstream of the stem-loop structure. This cleavage is followed by exon ucleolytic resection to generate the mature 3' end. In order to define crit ical nucleotides of the endonucleolytic cleavage site, we mutated its seque nce. Incubation of synthetic atpB pre-RNAs containing these mutations in a chloroplast protein extract resulted in the accumulation of 3'-end-processe d products. However, in two cases where the AU-rich sequence of this site w as replaced with a GC-rich one, the 3' end of the stable processing product differed from that of the wild-type product. To examine whether these muta tions affected atpB mRNA processing or accumulation in vivo, the endogenous 3' UTR was replaced with mutated sequences by biolistic transformation of Chlamydomonas chloroplasts. Analysis of the resulting strains revealed that the accumulation of atpB mRNA was approximately equal to that of wild-type cells, and that a wild-type atpB 3' end was generated. These results imply that Chlamydomonas atpB 3' processing parallels the situation with other e ndonucleases such as Escherichia coli RNAse E, where specific sequences are required for correct in vitro processing, but in vivo these mutations can be overcome.