Figure 6. Analysis of 23S 30 end processing by RNase protection. (A) The probe is indicated below the diagram of the 4.5S–5S region of the rrn operon. Sizes of protected RNAs found in (B) are indicated above the schematic. (B) Protected RNA from WT or from rnr1-3, as indicated above each lane, was separated in a 6% denaturing polyacrylamide gel. The positions of pro- tected bands corresponding to mature and pre-23S RNAs are indicated at right, and size standards are at left. moncistronic pre-4.5S transcripts. Signiﬁcant accumulation of both the 4.5S rRNA precursor and the 23S–4.5S transcripts is consistent with the view that processing of the 4.5S rRNA 30 end is a prerequisite to its cleavage from 23S rRNA, as has been previously suggested (4). This was tested by probing an RNA gel blot either with a 4.5S-speciﬁc oligonucleotide as a control (Figure 8A, left panel) or with a 4.5S rRNA precursor- speciﬁc oligonucleotide (Figure 8A, right panel). While the 4.5S-speciﬁc probe detected both dicistronic and mature 4.5S transcripts as expected, the precursor-speciﬁc probe detected only the dicistronic RNA, and not an 270 nt species. To better characterize the 23S–4.5S species, both 50 and 30 ends of this precursor were mapped by cRT–PCR. Two types of 50 ends were detected, both in WT and in rnr1 plants, and coincided either with the annotated mature 23S 50 end or to a position 72–73 nt upstream (Figure 8B). As expected, more Nucleic Acids Research, 2005, Vol. 33, No. 8 2757 clones corresponding to the mature 50 ends were detected in WT than in rnr1, but the lack of RNR1 did not seem to inﬂuence the location of these maturation sites. Interestingly, the 8 and 3 clones in WT and rnr1, respectively, which had mature 23S RNA 50 ends, also had mature 4.5S rRNA 30 ends (Figure 8C). This would represent the mature dicistronic inter- mediate prior to endonucleolytic cleavage between 23S and 4.5S rRNAs. However, due to the use of T4 RNA ligase during cRT–PCR it is not possible to exclude the ligation of mature 23S and mature 4.5S prior to cDNA synthesis. Nevertheless, all clones with unprocessed 23S rRNA 50 ends also had unpro- cessed 4.5S rRNA 30 ends, both in WT and in rnr1 (Figure 8C). Although the 30 ends of two clones from the mutant mapped downstream of the 5S rRNA, representing the 23S–4.5S–5S processing intermediate, most 30 ends of 23S–4.5S precursors mapped to a cluster of 4 nt situated 170 nt downstream of the 4.5S rRNA, as predicted by RNase protection (Figure 7C). This region likely represents an endonucleolytic cleavage site between the 4.5S and 5S rRNAs. Both RNA gel blot and cRT–PCR data suggested that 30 maturation of 4.5S rRNA precedes or occurs concomitantly with endonucleolytic cleavage of the dicistronic 23S–4.5S rRNA and, furthermore, that the impaired endonucleolytic processing of the 23S–4.5S dicistronic species in rnr1 plants is a pleiotropic effect of the lack of RNR1. Although it is tempting to suggest that incomplete 4.5S rRNA 30 processing is a primary defect of RNR1 deﬁciency, there is no direct evidence that this is the case, and accumulation of the 23S– 4.5S species has been observed in unrelated chloroplast bio- genesis mutants (2,6,8). To study the involvement of RNR1 in 5S rRNA 30 end processing more closely, both RNase protection and cRT– PCR experiments were performed. Figure 9B shows that a 5S antisense probe (Figure 9A) protected mature 5S rRNA from both WT and rnr1 plants, whereas rnr1 mutants alone accumulated a population of 5S precursor RNAs with short, heterogeneous 30 extensions averaging 10 nt in length, as well as a full-length precursor of 351 nt, which is most likely generated by RNase P cleavage at the 50 end of tRNAArg, and which was previously observed by agarose RNA gel blot analysis (Figure 4B, probe F). cRT–PCR was used to reveal the precise 30 ends of the pre-5S rRNAs. As shown in Figure 9C, all the clones obtained from WT RNA contained mature 50 and 30 ends, and although a few such clones were also obtained from rnr1 plants, most clones from the mutant contained small 30 extensions ranging from 1 to 11 nt. Together, these data indicate that RNR1 is involved in trim- ming these extensions, although since a small proportion of 5S rRNA mature 30 ends was observed, they can also be appar- ently generated by an RNR1-independent route. Purified RNR1 accurately processes pre-5S rRNA in vitro C-terminal-tagged RNR1 (RNR1-tag) was puriﬁed from a transgenic overexpressing line to check whether RNR1 was able to perform in vitro the 30 processing of 5S rRNA (Figure 10). A mock puriﬁcation was also performed from plants that did not overexpress tagged RNR1. Both fractions were incubated with RNA substrates corresponding to mature 5S rRNA, or to a precursor 5S rRNA containing a 20 nt