Microbiology (1999), 145, 3089–3100 Printed in Great Britain Absence of RNase III alters the pathway by which RNAI, the antisense inhibitor of ColE1 replication, decays Uta Binnie,† Kenny Wong,‡ Sean McAteer and Millicent Masters Author for correspondence : Millicent Masters. Tel : 44 131 650 5355. Fax: 44 131 650 8650. e-mail : M.Mastersed.ac.uk Institute of Cell and Molecular Biology, University of Edinburgh, The King’s Buildings, Mayfield Road, Edinburgh EH9 3JR, UK RNAI is a short RNA, 108 nt in length, which regulates the replication of the plasmid ColE1. RNAI turns over rapidly, enabling plasmid replication rate to respond quickly to changes in plasmid copy number. Because RNAI is produced in abundance, is easily extracted and turns over quickly, it has been used as a model for mRNA in studying RNA decay pathways. The enzymes polynucleotide phosphorylase, poly(A) polymerase and RNase E have been demonstrated to have roles in both messenger and RNAI decay ; it is reported here that these enzymes can work independently of one another to facilitate RNAI decay. The roles in RNAI decay of two further enzymes which facilitate mRNA decay, the exonuclease RNase II and the endonuclease RNase III, are also examined. RNase II does not appear to accelerate RNAI decay but it is found that, in the absence of RNase III, polyadenylated RNAI, unprocessed by RNase E, accumulates. It is also shown that RNase III can cut RNAI near nt 82 or 98 in vitro. An RNAI fragment corresponding to the longer of these can be found in extracts of an rnc M pcnB strain (which produces RNase III) but not of an rnc pcnB strain, suggesting that RNAI may be a substrate for RNase III in vivo.A possible pathway for the early steps in RNAI decay which incorporates this information is suggested. Keywords : Escherichia coli, plasmid replication, RNA degradation, ribonuclease III, RNAI INTRODUCTION ColE1 regulates its copy number with a short RNA counter-transcript, RNAI. RNAI can hybridize to the complementary portion of a longer RNA, RNAII, and thus prevent its maturation into the primer used to initiate ColE1 replication. RNAI is synthesized constitutively from a strong promoter and its steady- state level is high relative to that of RNAII. However, it turns over rapidly. As a result, its concentration alters quickly in response to a change in plasmid copy number and replication is immediately inhibited or stimulated ................................................................................................................................................. † Present address : Inveresk Research International Ltd, Tranent, East Lothian, UK. ‡ Present address : Asthma Genetics Group, Nuffield Department of Clinical Medicine, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DU, UK. Abbreviations : PAP I, poly(A) polymerase ; PNPase, polynucleotide phos- phorylase ; RIF, rifampicin. as appropriate (for reviews see Cesareni et al., 1991 ; Wagner & Simons, 1994). RNAI breakdown is rapid, and, like that of mRNAs (reviewed by Nierlich & Murakawa, 1996 ; Kushner, 1996), is accomplished by a combination of endo- nucleases and exonucleases. For these reasons, it has been studied as a model for mRNA decay. Major roles in RNAI decay have been demonstrated for a number of enzymes. These enzymes are listed in Table 1. RNase E (Tomcsa  nyi & Apirion, 1985) is an essential enzyme with endonucleolytic activity important in messenger decay (Kuwano et al., 1977) and rRNA processing (Ghora & Apirion, 1978). Lin-Chao & Cohen (1991) showed that it has a major role in RNAI decay. Polynucleotide phosphorylase (PNPase) is one of the two exonucleases implicated in mRNA degradation (Donovan & Kushner, 1986). Xu & Cohen (1995) reported on its role in RNAI decay. Poly(A) polymerase (PAP I), discovered as a consequence of its role in plasmid copy number maintenance (Lopilato et al., 0002-3432  1999 SGM 3089