Nucleic Acids Research, 2005, Vol. 33, No. 8 2751–2763 doi:10.1093/nar/gki576 RNR1, a 30–50 exoribonuclease belonging to the RNR superfamily, catalyzes 30 maturation of chloroplast ribosomal RNAs in Arabidopsis thaliana Thomas J. Bollenbach, Heike Lange 1 , Ryan Gutierrez, Mathieu Erhardt1, David B. Stern* and Dominique Gagliardi1 Boyce Thompson Institute for Plant Research, Cornell University, Tower Rd., Ithaca, NY 14853, USA and 1Institut de Biologie Mole´ culaire des Plantes, CNRS UPR2357, 12 rue du ge´ ne´ ral Zimmer, 67084 Strasbourg cedex, France Received March 23, 2005; Revised and Accepted April 26, 2005 ABSTRACT Arabidopsis thaliana chloroplasts contain at least two 30 to 50 exoribonucleases, polynucleotide phospho- rylase (PNPase) and an RNase R homolog (RNR1). PNPase has been implicated in both mRNA and 23S rRNA 30 processing. However, the observed maturation defects do not affect chloroplast translation, suggest- ing that the overall role of PNPase in maturation of chloroplast rRNA is not essential. Here, we show that this role can be largely ascribed to RNR1, for which homozygous mutants germinate only on sucrose- containing media, and have white cotyledons and pale green rosette leaves. Accumulation of chloroplast- encoded mRNAs and tRNAs is unaffected in such mutants, suggesting that RNR1 activity is either unne- cessary or redundant for their processing and turnover. However, accumulation of several chloroplast rRNA species is severely affected. High-resolution RNA gel blot analysis, and mapping of 50 and 30 ends, revealed that RNR1 is involved in the maturation of 23S, 16S and 5S rRNAs. The 30 extensions of the accumulating 5S rRNA precursors can be efficiently removed in vitro by purified RNR1, consistent with this view. Our data suggest that decreased accumulation of mature chloro- plast ribosomal RNAs leads to a reduction in the num- ber of translating ribosomes, ultimately compromising chloroplast protein abundance and thus plant growth and development. INTRODUCTION Development of chloroplasts from proplastids and expression of the plastid genome are intimately linked processes, which are ultimately coordinated by nucleus-encoded proteins and enzymes. Chloroplast gene expression is regulated at several steps, one of these being post-transcriptional RNA proces- sing. This includes endonucleolytic cleavage of long polycis- tronic transcripts, and 50 and 30 end processing of precursor RNAs (1). A heavily processed transcript in the plastids of ﬂowering plants is the polycistronic primary transcript emanating from the ribosomal RNA (rrn) operon. This highly conserved gene cluster encodes the 16S, 23S, 4.5S and 5S rRNAs and three tRNAs. Maturation of the primary transcript occurs via a series of endo- and exonucleolytic steps. The primary precursor is initially processed by excision of the tRNAs and by additional endonucleolytic cleavages to generate 16S and 5S rRNA pre- cursors, and a dicistronic 23S–4.5S processing intermediate. Subsequent endonucleolytic processing of the 23S–4.5S rRNA to generate monocistronic 23S and 4.5S rRNAs appears to occur on the ribosome [(2), reviewed in (3)] and is thought to require prior 30 end maturation of 4.5S rRNA (4). The 16S, 23S and 5S rRNA precursors generated by endonucleolytic cleavage require further processing to establish mature 50 and 30 ends. While the molecular nature of rRNA processing has been gradually elucidated, the nature of the enzymatic machinery has remained elusive, in part because certain rRNA defects can represent pleiotropic effects where plastid biogenesis has been otherwise impaired (5). Several higher plant mutations, how- ever, appear to have a primary defect in the processing of plastid rRNAs. For example, the maize mutant hcf 7 is impaired in 16S rRNA 50 and 30 end processing, and the Arabidopsis mutant dal1 is impaired in the processing of 16S rRNA 30 ends and accumulates the 23S–4.5S dicistronic pro- cessing intermediate (5,6). The Arabidopsis mutant wco is also affected at least in 16S rRNA processing, but this defect is observed only in cotyledons (7). The DCL gene is essential for the accumulation of mature chloroplast ribosomes in *To whom correspondence should be addressed. Tel: +1 607 254 1306; Fax: +1 607 254 6779; Email: ds28@cornell.edu The Author 2005. Published by Oxford University Press. All rights reserved. The online version of this article has been published under an open access model. 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