2758 Nucleic Acids Research, 2005, Vol. 33, No. 8 Figure 7. Analysis of 4.5S and 30 end processing. (A) An aliquot of 1.5 mg of total RNA from WT, rnr1-1, rnr1-2 and rnr1-3 was separated on a 12% acrylamide gel and stained with ethidium bromide (left panel) or analyzed with a 4.5S rRNA-specific probe (right panel). Migration and size (nt) of molecular weight markers are indicated. (B) The probe used in this analysis is indicated by the heavy arrow below the schematic of the 4.5S–5S–tRNAArg region of the rrn operon. Sizes of protected RNAs seen in (C) are indicated above the schematic. (C) Protected RNA from WT or from rnr1-3, as indicated above each lane, was separated in a 6% denaturing polyacrylamide gel. The positions of protected bands corresponding to mature and pre-4.5S rRNAs are indicated at right, and size standards are at left. 30 extension (5S and 5S + 20, respectively, Figure 10). No RNase activity was observed with the mock fraction or in buffer alone, but tagged RNR1 efﬁciently digested the 20 nt extension to generate a mature 5S rRNA 30 end. These data conﬁrm the results obtained in rnr1 plants, i.e. RNR1 can trim the 30 ends of 5S rRNA precursors. Polysome association of mRNAs decreases in rnr1 chloroplasts Because rnr1 plants contained signiﬁcantly fewer mature ribosomal RNAs than WT plants, and because the mutants accumulated lower levels of chloroplast-encoded proteins, the association of chloroplast mRNAs with ribosomes was examined, as shown in Figure 11. Extracts from WT or rnr1 leaves were sedimented through 15–55% sucrose density gradients and following centrifugation, fractions were collec- ted and analyzed by RNA gel blot with the probes indicated in the right margin. As judged by the levels of 23S* and mature 16S transcripts in polysomal fractions ( 8–12), rnr1 chloroplasts contain signiﬁcantly fewer polysomes than WT chloroplasts. Polysome loading of mRNAs was differentially affected in rnr1 plants. The transcript encoding the psaA-psaB-rps14 transcript was shifted toward the top of the gradient, indicating that it was associated with fewer ribosomes in rnr1 than in WT plants. These results would presumably correlate with lower protein synthesis rates, although we did not measure protein synthesis directly. The rbcL, psbA and atpB/E tran- scripts, however, showed a similar pattern in WT and in rnr1, suggesting that these transcripts were being loaded under conditions where ribosomes might be limiting for translation. DISCUSSION RNR1 is a ubiquitous, dual-targeted exoribonuclease that is important for chloroplast rRNA processing. Here, we have presented a detailed analysis of rnr1 mutants, which has illuminated several facets of chloroplast rRNA maturation. The RNA phenotypes presented here are much more striking than those observed in mitochondria of one of the same mutant lines, rnr1-2 (14), raising the question of whether RNR1 is a minor player in mitochondria. Just as perplexing are the contradictory GFP targeting results which, rather than showing dual targeting in any of three laboratories which tested similar fusions [(14,15) and Supplementary Figure 1], generated either uniquely chloroplast or uniquely mitochon- drial targeting. While the fusions made in each case were slightly different and GFP is well-known to produce artifacts, such results are unusual but not unprecedented (26). Further- more, short nucleotide extensions were found in both