The Plant tRNA 3′ Processing Enzyme Has a Broad Substrate Spectrum ² Steffen Schiffer, ‡ Mark Helm, §,| Anne The ´obald-Dietrich, § Richard Giege ´, § and Anita Marchfelder* ,‡ Molekulare Botanik, UniVersita ¨ t Ulm, 89069 Ulm, Germany, Unite ´ Propre de Recherche 9002 du CNRS, Institut de Biologie Mole ´ culaire et Cellulaire, 15 rue Rene ´ Descartes, 67084 Strasbourg Cedex, France, and DiVision of Biology, California Institute of Technology, Pasadena, California 91125 ReceiVed January 30, 2001; ReVised Manuscript ReceiVed April 16, 2001 ABSTRACT: To elucidate the minimal substrate for the plant nuclear tRNA 3′ processing enzyme, we synthesized a set of tRNA variants, which were subsequently incubated with the nuclear tRNA 3′ processing enzyme. Our experiments show that the minimal substrate for the nuclear RNase Z consists of the acceptor stem and T arm. The broad substrate spectrum of the nuclear RNase Z raises the possibility that this enzyme might have additional functions in the nucleus besides tRNA 3′ processing. Incubation of tRNA variants with the plant mitochondrial enzyme revealed that the organellar counterpart of the nuclear enzyme has a much narrower substrate spectrum. The mitochondrial RNase Z only tolerates deletion of anticodon and variable arms and only with a drastic reduction in cleavage efficiency, indicating that the mitochondrial activity can only cleave bona fide tRNA substrates efficiently. Both enzymes prefer precursors containing short 3′ trailers over extended 3′ additional sequences. Determination of cleavage sites showed that the cleavage site is not shifted in any of the tRNA variant precursors. Generation of functional tRNA molecules from precursor RNAs includes removal of 5′ and 3′ additional sequences in all organisms. Whereas the maturation of the tRNA 5′ end by RNase P has been studied in great detail (for reviews, see refs 1 and 2), less is known about the activities involved in tRNA 3′ processing. In Escherichia coli, several enzymes are involved in the maturation of the tRNA 3′ end (3). An endonuclease cleaves the precursor downstream of the tRNA, and exonucleases remove the residual nucleotides up to the CCA triplet which is encoded in almost all bacterial tRNA genes (4). Using mutant strains of E. coli, it has been shown that several exonucleases are capable of removing the 3′ trailer, suggesting that there is no “tRNA-specific exo- nuclease” in E. coli (3). Nothing is known about tRNA 3′ end generation in archaea, beyond the observation that the 5′ end is processed before 3′ end maturation takes place (5). It is not clear whether endo- and/or exonucleases are involved in this process in archaea. The general mechanism for yielding the tRNA 3′ end in eukarya seems to be an endonucleolytic cut 3′ to the discriminator (6-10), although some exonucleolytic processing systems have also been reported (11-14). Purification of tRNA 3′ processing activi- ties from Saccharomyces cereVisiae resulted in the isolation of three exo- and two endonucleases (13). Yoo and Wolin made the observation that in S. cereVisiae tRNA precursors are cleaved by an endonuclease in the presence of the Lhp1p protein, while in the absence of Lhp1p they are processed by exonucleases (15). Thus, in vivo the exonucleolytic pathway may act as backup system for the endonucleolytic processing step. Organellar tRNA precursors are generated by endonucleases which cleave next to the discriminator (16-21). In this respect, tRNA 3′ processing in these endosymbionts is similar to nuclear tRNA 3′ processing and different from bacterial tRNA 3′ end maturation. Substrate specificities for the tRNA 5′ processing enzyme, RNase P, have been investigated intensively. The bacterial RNase P has a very broad substrate spectrum, the minimal substrate being a minihelix consisting of solely T and acceptor stems and the 3′ terminal CCA (22). In contrast, eukaryotic and organellar RNase P enzymes have a much narrower substrate specificity (23-25). Substrate specificities of tRNA 3′ processing enzymes have so far only been described in detail for nuclear enzymes from Drosophila and mammalia. Both nuclear enzymes process a minimal sub- strate consisting of the T arm, the acceptor stem, and a short connecting loop as efficiently as the wild-type substrate (25, 26). While the mammalian 3′ processing enzyme prefers short 3′ trailers, the Drosophila activity is not influenced by 3′ trailer length (27, 28). Here we report the substrate specificity analysis of the tRNA 3′ processing activity from plant nuclei. 1 In addition, we show that plant mitochondrial and nuclear RNase Z have different substrate specificities clearly distinguishing both enzymes. The mitochondrial RNase Z has a very narrow substrate spectrum, while the nuclear RNase Z also accepts several more degenerated substrates. Short 3′ trailers are preferred by both the mitochondrial and nuclear RNase Z. ² Work presented here was funded by the Anfangsfo ¨rderung Uni- versita ¨t Ulm, the Deutsche Forschungsgemeinschaft, and the Fonds der Chemischen Industrie. * Corresponding author. E-mail: anita.marchfelder@biologie. uni-ulm.de. Phone: +49-731-5022658. Fax: +49-731-5022626. ‡ Universita ¨t Ulm. § Institut de Biologie Mole ´culaire et Cellulaire. | California Institute of Technology. 1 We used a cytoplasmic extract from wheat embryos for our studies. Since it was shown previously that tRNA processing occurs in the nucleus and maybe in the nucleolus, we termed the activity nuclear RNase Z. 8264 Biochemistry 2001, 40, 8264-8272 10.1021/bi0101953 CCC: $20.00 © 2001 American Chemical Society Published on Web 06/21/2001