Purification and Characterization of MAR1

Abstract

A relatively thermostable 22-kDa endoribonuclease (MAR1) was purified more than 10,000-fold from a mitochondrial 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 substrate 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 recombinant enzyme from L. tarentolae was identical to that of the native enzyme. 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 Saccharomyces cerevisiae mitochondria, 5Ј end maturation of tRNAs is carried out by a mitochondrial RNase P (3), and RNA degradation may involve a 3Ј-5Ј exonuclease activity (4). The 3Ј 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 3Ј ends of protein-coding genes serve as processing signals for 3Ј end maturation (6), and RNase Z has been identified as the nuclease responsible for the specific processing of the 3Ј ends of tRNAs (7).In the kinetoplast mitochondrion of the trypanosomatid protozoa, Leishmania tarentolae and Trypanosoma brucei, the maxicircle DNA encodes two rRNA genes and 18 potential protein-coding genes, 12 of which are cryptogenes whose transcripts are edited by the insertion and deletion of uridine residues usually within coding regions (8 -11). RNA editing reactions appear to be initiated by specific cleavages of the preedited mRNAs, mediated by base pairing with specific cognate guide RNAs (gRNAs) (12, 13). Little is known about the processing and turnover of mitochondrial RNAs in these cells, either in terms of specific cis-acting signals or enzymatic activities. 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 predicted by the enzyme cascade model for RNA editing (12). Another activity, which sedimented at 15 S and was independent of added gRNA for cleavage, might correspond to an endoribonuclease 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 th...

Mammalian mitochondrial RNAs are degraded in the mitochondrial intermembrane space by RNASET2

“…However, it is not a unique feature. Same characteristic is shared by ribonucleases in Leishmania tarentolae mitochondria (Alfonzo et al, 1998 ; Simpson et al, 1992 ).…”

Proteomic Analysis of Rat Liver Peroxisome

“…CGI-135 shows similarity to Fis1p, which is required for Dnm1p GTPase-mediated mitochondrial fission (26). Tumor-related protein (gi 14572526) is homologous to CGI-111, which shows some similarity to MAR1, a mitochondrial-associated ribonuclease (27). In Leishmania tarentolae in which the enzyme was characterized, it is an endoribonuclease and the 18 amino acid sequence of N-terminal shows characteristics of an uncleaved mitochondrial sequence.…”

Mitochondrial RNA processing in trypanosomes

“…Several nucleases have been investigated, but none could be unambiguously assigned to a specific processing function. A mitochondrial-associated (endo)ribonuclease (MAR1) has been purified and the gene cloned from L. tarentolae (Alfonzo et al, 1998). In addition, three distinct 3′-5′ exonuclease activities were characterized in L. tarentolae : a U-specific exonuclease, a processive non-specific enzyme and an exonuclease with preference for 3′ phosphorylated RNAs (Aphasizhev and Simpson, 2001).…”