RNase protection Templates for the RNA probes used in this analysis were PCR- ampliﬁed from Arabidopsis total DNA. A 207 nt 23S template was ampliﬁed using the primers 23S-50 and 23S-30, a 374 nt 4.5S template was ampliﬁed using the primers 4.5S-50 and 4.5S-30, and a 376 nt 5S template was ampliﬁed using the primers 5S-50 and 5S-30. The resulting PCR products, which encode a T3 RNA polymerase promoter, were used to syn- thesize probes in vitro as described previously (21). S1 hybrid- ization buffer (40 mM PIPES, pH 6.4, 1 mM EDTA, 400 mM NaCl and 85% formamide), total RNA from WT or rnr1 plants, or yeast tRNA and either 23S, 4.5S or 5S RNA probes were mixed, and the assay was carried out as described by Monde et al. (22) with the exception that overnight hybrid- ization was carried out at 42˚C, and digestion of single- stranded RNA was carried out at 4˚C for 30 min. Reaction products were resolved in 6% denaturing polyacrylamide gels and were visualized and quantiﬁed using a Storm scanner (Molecular Dynamics, Sunnyvale, CA). Circular RT–PCR The precise 50 and 30 ends of the 16S rRNA, 5S rRNA and 23S–4.5S rRNA precursors were determined using circular RT–PCR (cRT–PCR) as described previously (19). Brieﬂy, total RNA was circularized using T4 RNA ligase (New England Biolabs), and cDNA spanning the junction of the 50 and 30 ligated ends was synthesized using gene-speciﬁc reverse primers (Table 1). This region was then ampliﬁed by PCR using gene-speciﬁc primers, cloned into pCR2.1 TOPO (Invitrogen) and sequenced. In vitro assay of purified RNR1 The puriﬁcation and assay of RNR1 were performed as described previously (14). Templates for in vitro transcription were generated by amplifying the 5S gene from Arabidopsis DNA using the primers T7 5S and 5S Reverse, to generate the mature 5S rRNA substrate, or T7 5S and 5S + 20 Reverse, to generate the pre-5S rRNA substrate containing a 20 nt extension. Protein isolation and immunoblotting Tissue from 40-day-old plants was ground to a ﬁne powder in liquid nitrogen and allowed to thaw in extraction buffer (10 mM Tris–HCl, 18% sucrose, 10 mM MgCl2, 1% 2-mercaptoethanol and 1% SDS). Debris was pelleted by cent- rifugation and an equal volume of 2· SDS–PAGE loading buffer was added to the supernatant. Loading of proteins was calibrated by measuring total absorption at 280 nm. RESULTS RNR1 T-DNA insertion mutant analysis The Arabidopsis nuclear gene At5g02250 encodes a 782 amino acid protein possessing an RNB domain (Pfam domain PF00773), which is found in at least 274 proteins related to RNase II and RNase R (23). The exoribonucleolytic activity of the At5g02250 gene product was conﬁrmed following the puriﬁcation of a C-terminal tagged version from overexpress- ing transgenic Arabidopsis plants (14,19). The At5g02250 Nucleic Acids Research, 2005, Vol. 33, No. 8 2753 product was ﬁrst named AtmtRNase II based on its mitochondrial localization as determined by transient expres- sion of a fusion of the N-terminal 192 amino acids to green ﬂuorescent protein (GFP), and the observation that an increased number of incompletely processed mitochondrial atp9 mRNA 30 ends accumulated in mutant plant, based on sequencing of cRT–PCR products (14). However, a chloroplast localization was subsequently pro- posed for this same protein, when a GFP fusion to 118 N- terminal amino acids was expressed, also in Arabidopsis (15). These workers further showed that a T-DNA insertion 36 bp upstream of the At5g02250 translation initiation codon gene decreased, but did not abolish expression, and resulted in plants exhibiting impaired photosynthesis, retarded growth and pale green leaves. Given that the GFP results and growth phentoypes suggested a role for At5g02250 in chloroplast biogenesis, the gene was re-named RNR1, a nomenclature that we have adopted here. However, the participation of RNR1 in cpRNA metabolism was not thoroughly explored. To pursue the analysis of RNR1 in the context of chloroplast biogenesis, we selected three T-DNA insertion alleles from the Salk SIGnAL collection, naming them rnr1-1 (SALK_138535), rnr1-2 (SALK_044726) and rnr1-3 (SALK_090294) (Figure 1A). Homozygous mutants from Figure 1. RNR1 T-DNA insertion mutants. (A) Diagram of the RNR1 gene showing the positions of three T-DNA insertion alleles used in this work. The positions of T-DNA insertions were confirmed by PCR. (B) WT and homo- zygous rnr1 mutants were germinated on sucrose-containing media for 15 days (top panels), or grown on sucrose-containing media for 40 days and transferred to soil for an additional 9 days (bottom panels). (C) RT–PCR showing expres- sion of the RNR1 gene in different tissues of WT Arabidopsis. Primers used in this analysis (RNR50 and RNR30) are indicated in (A).