2760 Nucleic Acids Research, 2005, Vol. 33, No. 8 Figure 9. Analysis of 5S rRNA 30 end processing. (A) The probe is indicated by the heavy arrow below the diagram of the 4.5S–5S–tRNAArg region. Sizes of protected RNAs seen in (C) are indicated above the diagram. (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 protected bands corresponding to mature and pre-5S rRNAs are indicated at the right, and size standards are at the left. (C) Mapping of 5S rRNA 50 and 30 ends by cRT–PCR. The genomic sequence is shown at the top, with the mature 30 and 50 ends underlined. 5S rRNA 30 and 50 ends obtained for WT and rnr1 plants are indicated with the number on the right representing the number of clones for each sequence. Nucleotides that could be present either at 30 or at 50 ends are indicated in the middle column. Adenosines that are added post-transcriptionally are in lowercase. Figure 10. RNR1 accurately processes 5S rRNA 30 ends in vitro. Affinity- purified RNR1-tag and a fraction from a mock purification were incubated with substrates corresponding to the mature 5S rRNA (5S) or to the same RNA with a 20 nt 30 extension (5S + 20). Incubation times are indicated in minutes above each lane and the positions and putative secondary structures of substrate and product are indicated at the right and left, respectively. Arabidopsis dal1-2 (5,6), which are impaired in pre-16S 30 end maturation. Both dal1-2 and the tomato dcl-s mutant, which is defective in an unidentiﬁed step of ribosome assembly, accumulate abnormal levels of the 23S–4.5S processing inter- mediate (2,6). Therefore, in rnr1 mutants, the 23S–4.5S inter- mediate may accumulate simply because ribosome assembly is slowed and its accumulation may, therefore, be a pleiotropic effect of the mutation. Because some mature 4.5S and 23S rRNAs accumulate and because rnr1 chloroplasts are clearly not entirely bereft of translation, an RNR1-independent path- way for ribosome maturation, perhaps relying on PNPase, must operate in the mutant. 23S 30 end maturation is catalyzed by PNPase and by RNR1 Similar to plant chloroplasts, many eubacterial species encode a fragmented 23S rRNA, whose intervening sequences (IVSs) are removed by looping out and RNase III-directed cleavage of the intergenic spacer (37). In contrast, once incorporated into ribosomes, chloroplast 23S–4.5S rRNA pro- cessing includes an endonucleolytic cleavage to produce the monocistronic 4.5S rRNA and a 23S precursor rRNA, and two subsequent exonucleolytic events, PNPase-catalyzed generation of a precursor with 10–15 nt extensions (11), followed by RNR1 trimming. Removal of the pre-23S 30 extension is not required for ribosome assembly and trans- lation (11), which is also true of Salmonella typhimurium 23S IVS processing (38–40). Because defects in 23S RNA IVS processing are phenotypically silent, their physiological signiﬁcance is unknown.