Isolation and Characterization of a U-specific 3′-5′-Exonuclease from Mitochondria of Leishmania tarentolae

Abstract

We have purified a 3-5-exoribonuclease from mitochondrial extract of Leishmania tarentolae over 4000-fold through six column fractionations. This enzyme digested RNA in a distributive manner, showed a high level of specificity for 3-terminal Us, and was blocked by a terminal dU; there was slight exonucleolytic activity on a 3-terminal A or C but no activity on a 3-terminal G residue. The enzyme preferred single-stranded 3-oligo(U) overhangs and did not digest duplex RNA. Two other 3-5-exoribonuclease activities were also detected in the mitochondrial extract, one of which was stimulated by a 3-phosphate and the other of which degraded RNAs with a 3-OH to mononucleotides in a processive manner. The properties of the distributive U-specific 3-5-exoribonuclease suggest an involvement in the U-deletion RNA editing reaction that occurs in the mitochondrion of these cells.3Ј-5Ј-Exoribonucleases have been shown to play important roles in the maturation and degradation of RNAs, including the 3Ј-end maturation of the 5.8 S rRNA (1) and pre-tRNAs (2) and both deadenylation-dependent (3) and deadenylation-independent mRNA decay in eukaryotes (4). An exonuclease activity specific for removal of the post-transcriptionally added (5) 3Ј-oligo(U) tail of U6 small nuclear RNA has been partially purified and characterized (6). A number of 3Ј-5Ј-exoribonucleases have also been described in bacteria and shown to be involved in RNA processing. For example, RNase E from Escherichia coli is a site-specific endoribonuclease that also has a 3Ј-5Ј-exonuclease activity on poly(A) and poly(U) tails (7). There is conservation of some exoribonucleases between bacteria and eukaryotes. In yeast, four of the five components of the "exosome" complex required for the 3Ј-end processing of the 5.8 S rRNA are homologous to bacterial 3Ј-5Ј-exoribonucleases that have distributive, processive, and phosphorolytic activities (8). In addition, a conserved 3Ј-5Ј-exonuclease domain that is similar to the 3Ј-5Ј-proofreading domain of DNA polymerases has been identified by hidden Markov model and phylogenetic analysis (9).The presence of a U-specific 3Ј-5Ј-exoribonuclease in mitochondria of kinetoplastid protozoa was predicted by the enzyme cascade model of U-insertion/deletion RNA editing (10). This model involves an initial base-pairing interaction of specific guide RNAs with the pre-edited mRNAs just downstream of the first editing site, and a cleavage occurs at the first mismatched base of the mRNA. Then, in the case of U-deletion editing, a proposed U-specific 3Ј-5Ј-exonuclease removes non-base-paired Us from the 3Ј-end of the 5Ј-cleavage fragment, which is followed by ligation of the two mRNA cleavage fragments. Evidence from analysis of in vitro editing systems from the trypanosomatids Trypanosoma brucei (11-13) and Leishmania tarentolae (14, 15) has provided strong evidence for this model. All of the proposed enzymatic activities have been detected in mitochondrial extracts, including a U-specific 3Ј-5Ј-exoribonuclease activity (16). This activity has...

The specificity of nucleotide removal during RNA editing in Trypanosoma brucei

“…Because base pairing immediately 59 to ES1 does not appear to be required for precise deletion of uridines from ES1, another step(s) must direct removal of the precise number of Us specified by gRNA+ One possibility is that the exoribonuclease that removes nucleotides from an ES may be specific to Us+ Although that has been a presumed characteristic of the exonuclease (Cruz-Reyes & Sollner-Webb, 1996), and although a 39 exoUase has been partially purified in Leishmania tarentolae mitochondria (Aphasizhev & Simpson, 2001), the U specificity of nucleotide removal has not been systematically tested in an in vitro editing context+ Following the endoribonucleolytic cleavage, such an exonuclease would be predicted to remove the Us from the 39 end of the 59 cleavage product until it encounters non-U nucleotides, after which the 59 and 39 cleavage products would be ligated to complete one round of editing+ To test this possibility, we constructed a series of pre-mRNA substrates in which the Us in the ES were replaced one by one with a non-U nucleotide (Fig+ 3A) and analyzed their processing in vitro+ Two gRNAs, gA6[14]⌬G and wt gA6 [14] gRNA, were used to verify the gRNA-directed processing of the mutated premRNAs+ Processing was compared to previously characterized editing of A6U5 pre-mRNA, which is edited by the removal of four or three Us using the gA6[14]⌬G or wt gRNAs, respectively (Fig+ 3B,C, lanes 11 and 12; also see Seiwert & Stuart, 1994)+ The pre-mRNA 39 cleavage product directed by wt gRNA is 1 nt longer than with gA6[14]⌬G, because the anchor duplex is 1 bp longer, which shifts the cleavage site 1 nt upstream+…”

“…The 39 exonuclease that is associated with RNA editing removes nucleotides from the 39 terminus of the premRNA 59 cleavage product as previously described (Seiwert et al+, 1996; Rusché et al+, 1997)+ We show here that this activity, in the context of full-round deletion editing, is specific for Us+ The replacement of any U in the ES with A, C, or G results in edited mRNA in which only the Us that are 39 to the non-U nucleotide are removed+ Consequently, we suggest a designation for this activity as the editing-associated 39 terminal exouridylylase (39 exoUase)+ The characteristics of the 39 exoUase other than its specificity for Us (Fig+ 3; Aphasizhev & Simpson, 2001) are not yet known+ The observed populations of 59 cleavage products with varying number of Us (Fig+ 3C; Seiwert et al+, 1996) are consistent with the distributive action of purified exoUase from L. tarentolae (Aphasizhev & Simpson, 2001)+ One report suggests that the activity that removes Us during editing is distinct from that which adds Us based on sensitivity to pyrophosphate (Cruz-Reyes & SollnerWebb, 1996), and Us removed are released as free UMP (Rusché et al+, 1997), whereas TUTase uses UTP as a substrate+ Thus, U addition and removal are unlikely to be catalyzed by the same enzyme+…”

A 100-kD complex of two RNA-binding proteins from mitochondria of Leishmania tarentolae catalyzes RNA annealing and interacts with several RNA editing components

“…A smaller RNA ligase (REL2) and several unidentified proteins have also been isolated as components of a large "editing complex" . A gRNA-directed mitochondrial endoribonuclease has been partially purified from T. brucei Rusche et al 1997) and a 3Ј-5Ј Uspecific mitochondrial exonuclease has been partially purified from Leishmania tarentolae (Aphasizhev and Simpson 2001). The REAP-1 mitochondrial protein from T. brucei was isolated as a component of high-molecular-weight complexes, and an inhibition of in vitro editing by anti-REAP-1 antibodies was reported (Madison-Antenucci et al 1998), but the specific role of this protein remains to be identified.…”

Guide RNAs of the recently isolated LEM125 strain of Leishmania tarentolae: An unexpected complexity

“…The mitochondrial genome of trypanosomatid protozoa is composed of a single giant network containing two forms of catenated circular DNA molecules, maxicircles and minicircles (Simpson, 1987)+ The minicircles are present in approximately 10,000 molecules per network and the maxicircles in approximately 20-50 copies per network+ The maxicircle contains two rRNA genes and 18 structural genes+ Transcripts of 12 of the 18 genes of the maxicircle contain variable numbers of frameshifts that must be corrected for translation to occur (Horváth et al+, 2000)+ The transcripts are edited by the insertion and occasional deletion of U residues at specific sites within the gene, thereby creating open reading frames (Estévez & Simpson, 1999)+ The information for editing is contained in a class of small usually trans-acting RNAs known as guide RNAs (gRNAs), which are encoded both in the maxicircle and minicircle components of the mitochondrial DNA Sturm & Simpson, 1991)+ The gRNAs consist of a 59 anchor region that forms a duplex with the pre-edited mRNA just downstream of the first editing site, a central guiding region that contains guiding A and G nucleotides that can base pair with the inserted Us, and a posttranscriptionally added oligo[U] tail ranging from 5-30 nt in length of uncertain function )+ The mechanism of editing involves hybrid-ization of a specific gRNA forming the anchor duplex, endonuclease cleavage of the mRNA at the first mismatched base, addition of Us to the 39 end of the 59 cleavage fragment by a 39 terminal uridylyl transferase, base pairing of the added Us to the guiding nucleotides in the gRNA, and ligation of the two cleavage fragments Cruz-Reyes & Sollner-Webb, 1996;Kable et al+, 1996;Seiwert et al+, 1996)+ The editing machinery then moves upstream to the next editing site on the mRNA and the cycle is repeated+ Deletion editing involves removal of unpaired Us from the 59 cleavage fragment by a U-specific 39-59 exonuclease (Aphasizhev & Simpson, 2001) prior to ligation+ A single gRNA mediates the editing of a specific single block of editing sites in an mRNA+ In some cases, editing creates anchor sequences for hybridization of other overlapping gRNAs for editing of adjacent upstream blocks, thereby determining the observed overall 39 to 59 polarity of editing (Maslov & Simpson, 1992)+ A DEAD box protein with possible RNA helicase activity (Missel et al+, 1997) and a putative RNA ligase (McManus et al+, 2001;Rusché et al+, 2001;Schnaufer et al+, 2001) are the only components of the editing machinery established by gene knockout analysis+ In Leishmania tarentolae, the minicircle is approximately 900 bp in size and consists of a conserved region and a variable region+ The entire conserved regions are conserved in different minicircles within a trypanosomatid species and short segments within these regions are conserved between species and between genera+ The three short conserved sequences (CSB-1, -2, and -3; Ray, 1989) fo...…”