Purification and Characterization of MAR1 A MITOCHONDRIAL ASSOCIATED RIBONUCLEASE FROM LEISHMANIA TARENTOLAE* (Received for publication, April 9, 1998, and in revised form, August 12, 1998) Juan D. Alfonzo‡, Otavio H. Thiemann§, and Larry Simpson‡§¶ From the ‡Howard Hughes Medical Institute, the §Department of Molecular, Cell, and Developmental Biology, UCLA and the ¶Department of Medical Microbiology, Immunology, and Molecular Genetics, UCLA School of Medicine, Los Angeles, California 90095-1662 A relatively thermostable 22-kDa endoribonuclease (MAR1) was purified more than 10,000-fold from a mito- chondrial extract of Leishmania tarentolae and the gene cloned. The purified nuclease has a K m of 100 –145  33 nM and a V max of 1.8 –2.9  2 nmol/min, depending on the RNA substrate, and yields a 3-OH and a 5-phosphate. Cleavage was limited to several specific sites in the sub- strate RNAs tested, but cleavage of pre-edited RNAs was generally independent of the addition of cognate guide RNA. The MAR1 gene was expressed in Escherichia coli or in L. tarentolae cells, and the recombinant protein was affinity-purified. The cleavage specificity of the re- combinant enzyme from L. tarentolae was identical to that of the native enzyme. The single copy MAR1 gene maps to an 820-kilobase pair chromosome and contains an open reading frame of 579 nucleotides. The 18-amino acid N-terminal sequence shows characteristics of an uncleaved mitochondrial targeting sequence. Data base searching revealed two homologues of MAR1 corre- sponding to unidentified open reading frames in Cae- norhabditis elegans (GenBank TM accession number Z69637) and Archaeoglobus fulgidus (GenBank TM acces- sion number AE000943). The function of MAR1 in mito- chondrial RNA metabolism in L. tarentolae remains to be determined. The processing of mitochondrial RNAs shows great variation between species. In mammalian mitochondria, both DNA strands are completely transcribed, and the transcripts are processed by the excision of interspersed tRNAs (1, 2). In Sac- charomyces cerevisiae mitochondria, 5' end maturation of tRNAs is carried out by a mitochondrial RNase P (3), and RNA degradation may involve a 3'-5' exonuclease activity (4). The 3' end processing of the cytochrome b (Cyb) 1 mRNA in yeast is mediated by the product of the nuclear gene CBT1 (5). In plant mitochondria, inverted repeats at the 3' ends of protein-coding genes serve as processing signals for 3' end maturation (6), and RNase Z has been identified as the nuclease responsible for the specific processing of the 3' ends of tRNAs (7). In the kinetoplast mitochondrion of the trypanosomatid pro- tozoa, Leishmania tarentolae and Trypanosoma brucei, the maxicircle DNA encodes two rRNA genes and 18 potential protein-coding genes, 12 of which are cryptogenes whose tran- scripts are edited by the insertion and deletion of uridine res- idues usually within coding regions (8 –11). RNA editing reac- tions appear to be initiated by specific cleavages of the pre- edited mRNAs, mediated by base pairing with specific cognate guide RNAs (gRNAs) (12, 13). Little is known about the proc- essing and turnover of mitochondrial RNAs in these cells, ei- ther in terms of specific cis-acting signals or enzymatic activi- ties. Three different endoribonuclease activities, separable by sedimentation or anion exchange chromatography, have been identified in a mitochondrial extract from T. brucei (14). One of these activities, which sedimented at 20 S in glycerol gradients, exhibited a gRNA-dependent cleavage at the first mismatch upstream of a duplex RNA region (14 –16), precisely as pre- dicted by the enzyme cascade model for RNA editing (12). Another activity, which sedimented at 15 S and was independ- ent of added gRNA for cleavage, might correspond to an en- doribonuclease activity that has been described previously in crude mitochondrial extracts from both L. tarentolae and T. brucei (17, 18). The endoribonuclease activities in the crude extracts were both shown to cleave pre-edited Cyb mRNAs two nucleotides upstream of the first editing site. However, the T. brucei activity had specificity for the pre-edited Cyb sequence and did not to cleave the mature edited sequence. In addition to a gRNA-dependent editing endoribonuclease, additional nucleases are presumably necessary for kinetoplas- tid mitochondrial mRNA maturation. There is some evidence for polycistronic transcription of the maxicircle (16, 19 –22). Primary transcripts are then subjected to 5' processing and 3' end cleavage, followed by 3' polyadenylation and polyuridyly- lation. In addition, many of the mitochondrial protein-coding genes have overlapping 5' and 3' ends, and the maturation of such transcripts could represent an additional level of gene regulation (19). Clearly, multiple specific ribonucleases must be required for the processing and turnover of rRNAs, mRNAs, gRNAs, and tRNAs in the mitochondria of these organisms. However, to date, the only ribonuclease purified to homogene- ity from kinetoplastid mitochondria is RNase H (23, 24). In this paper we describe the isolation and characterization of MAR1 (for Mitochondrial Associated Ribonuclease) from a mitochondrial extract of L. tarentolae. MAR1 was purified to homogeneity and the MAR1 gene cloned and expressed. Fur- ther biochemical characterization and genetic analysis should help elucidate the role of this nuclease in the processing of RNAs within the mitochondrion of Leishmania. * This work was supported in part by Grant AI09102 from the Na- tional Institutes of Health (to L. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM /EBI Data Bank with accession number(s) AF083881.  To whom correspondence and reprint requests should be addressed. Tel.: 310-825-4215; Fax: 310-206-8967; E-mail: simpson@hhmi.ucla.edu. 1 The abbreviations used are: Cyb, cytochrome b; gRNA, guide RNA; MSB, mitochondrial storage-breakage buffer; CHAPS, 3[(3-cholamido- propyl)dimethylammonio]-1-propanesulfonic acid; Tricine, N-[2-hy- droxy-1,1-bis(hydroxymethyl)ethyl]glycine; DTT, dithiothreitol; RT- PCR, reverse transcription-polymerase chain reaction; slRNA, spliced leader RNA; bp, base pair(s); kb, kilobase(s); CHEF, contour-clamped homogeneous field electrophoresis; PER, pre-edited region. THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 273, No. 45, Issue of November 6, pp. 30003–30011, 1998 © 1998 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. This paper is available on line at http://www.jbc.org 30003 by guest on May 6, 2020 http://www.jbc.org/ Downloaded from