The Mitochondrial RNA Ligase from Leishmania tarentolae Can Join RNA Molecules Bridged by a Complementary RNA

Abstract

A biochemical characterization was performed with a partially purified RNA ligase from isolated mitochondria of Leishmania tarentolae. This ligase has a K m of 25 ؎ 0.75 nM and a V max of 1.0 ؋ 10 -4 ؎ 2.4 ؋ 10 ؊4 nmol/ min when ligating a nicked double-stranded RNA substrate. Ligation was negatively affected by a gap between the donor and acceptor nucleotides. The catalytic efficiency of the circularization of a single-stranded substrate was 5-fold less than that of the ligation of a nicked substrate. These properties of the mitochondrial RNA ligase are consistent with an expected in vivo role in the process of uridine insertion/deletion RNA editing, in which the mRNA cleavage fragments are bridged by a cognate guide RNA.RNA ligases are present in a large variety of organisms, but only a few have been implicated in specific metabolic pathways. For example, T4 RNA ligase repairs nicks in the anticodon domains of tRNAs in T4-infected Escherichia coli cells (1). In eukaryotes and Archea, a tRNA ligase is involved in tRNA splicing; this RNA ligase contributes to the maturation of tRNAs by joining tRNA half molecules generated by the removal of introns from tRNA precursors (2). Recently, a 2Ј-5Ј RNA ligase has been characterized in uninfected bacteria; the function of this RNA ligase remains unclear, but it may reveal an unexpected step in E. coli RNA metabolism (3). An RNA ligase has also been invoked for the final step of uridine insertion/deletion RNA editing in mitochondria of kinetoplastid protozoa (4).RNA editing in kinetoplastid mitochondria is a posttranscriptional maturation of pre-edited mRNA (5). This modification process consists of insertions and, to a lesser extent, deletions of U residues in the pre-edited mRNA, usually within coding regions. The information for the specific insertions or deletions is present as complementary sequences (allowing G-U base pairs) in short guide RNA molecules (gRNAs).1 The gRNAs are encoded by both the maxicircle and minicircle components of the mitochondrial DNA (kinetoplast DNA) of trypanosomatids (4). Evidence to date suggests that the modified enzyme cascade model (4 -9) is essentially correct, but many details remain to be established. A specific gRNA first forms a duplex region just downstream of the editing site. This is followed by a precise endonucleolytic cleavage at the first mismatched base (10, 11), addition of Us to the 3Ј end of the 5Ј cleavage fragment, trimming of non-base-paired Us by a 3Ј to 5Ј exonuclease, and finally a religation of the two cleavage fragments. An RNA ligase activity has been detected in isolated mitochondria of Leishmania tarentolae (12) and Trypanosoma brucei (13). Ligase activity sedimented as a major 20 S peak and a minor 10 S peak in glycerol gradients of mitochondrial extract from L. tarentolae. In T. brucei, mitochondrial ligase activity sedimented as two peaks of equal size (14). Two proteins of 50 and 45 kDa in both species were detected comigrating with the ligase activities. These two proteins could be adenylated by [␣-32 P...

RNA Substrate Specificity and Structure-guided Mutational Analysis of Bacteriophage T4 RNA Ligase 2

“…T4 Rnl2 is clearly not restricted to an RNA-templated substrate. Second, although neither the RELs nor Rnl2 can ligate across a 3-nucleotide gap, the RELs are more tolerant of 2-nucleotide gaps than is Rnl2 (24,25).…”

“…The kinetoplastid REL enzymes have been studied biochemically either in the context of a native mitochondrial editosome complex or as translation products synthesized in vitro in a coupled transcription-translation system (24,25). Rnl2 displays certain similarities to the RELs with respect to substrate and cofactor specificity, including a requirement for ATP, a common preference for a perfectly paired nicked substrate, and loss of activity when gaps are introduced between the reactive termini.…”

“…Studies of the kinetoplastid RNA-editing ligases (which we classify as Rnl2-like enzymes) do indicate that the RELs prefer to join RNA termini that are splinted together by a bridging RNA template strand (24,25). It is notable that the specificity for RNA versus DNA as the template bridge differed between recombinant REL and the REL present in the native "editosome" complex, suggesting that editosome components associated with REL can alter its substrate preference (25).…”

Uridine insertion/deletion RNA editing in trypanosome mitochondria: A complex business

“…This is not surprising, considering the gRNA-dependent specific addition of U's by the recombinant 121 kD 3Ј TUTase (Aphasizhev et al 2002b), but it suggests that the specificity of the ligation step may determine the precise number of U's in the final product by base pairing with guiding A's or G's. The propensity of one or both of the RNA ligases to seal nicked, rather than gapped or bulged bridged substrates was shown previously using a partially purified enzyme preparation, probably containing both REL1 and REL2 (Blanc et al 1999). The advantage of an upstream duplex tether (an RNA-RNA duplex formed by the 3Ј portion of the synthetic gRNA and the 5Ј portion of the pre-edited mRNA) for efficient U-insertion editing was also demonstrated (Igo et al 2002b), as had been reported previously for the Leishmania tarentolae system (Kapushoc and Simpson 1999).…”

Kinetic mechanism of nick sealing by T4 RNA ligase 2 and effects of 3′-OH base mispairs and damaged base lesions

“…Rnl2 is also adept at sealing hybrid RNA:DNA substrates in which the 3 ′ -OH strand at the nick is RNA and the 5 ′ -PO 4 strand and template strand are DNA . The Rnl2 enzyme family includes the RNA-editing ligases (RELs) of the protozoan parasites Trypanosoma and Leishmania (Blanc et al 1999;Schnaufer et al 2001;Palazzo et al 2003;Deng et al 2004). RELs are essential for kinetoplastid mRNA editing, which involves RNA-guided mRNA incision, RNA templated gap filling by UMP additions to form a duplex nick, and then nick sealing by the ligase.…”