Mitochondrial Glutamate Dehydrogenase from Leishmania tarentolae Is a Guide RNA-Binding Protein

Abstract

To identify specific proteins interacting with guide RNAs (gRNAs) in mitochondrial ribonucleoprotein complexes from Leishmania tarentolae, fractionated and unfractionated mitochondrial extracts were subjected to UV cross-linking with added labeled gRNA and also with [␣-32 P]UTP-labeled endogenous RNA. An abundant 110-kDa protein (p110) localized in the T-V complex, which sediments in glycerol gradients at the leading edge of the 10S terminal uridylyltransferase peak, was found to interact with both types of labeled RNAs. The p110 protein was gel isolated and subjected to microsequence analysis, and the gene was cloned. The sequence revealed significant similarity with mitochondrial glutamate dehydrogenases. A polyclonal antiserum was raised against a recombinant fragment of the p110 gene and was used to demonstrate a stable and specific gRNA-binding activity by coimmunoprecipitation and competitive gel shift analyses. Complex formation was strongly inhibited by competition with poly(U) or by deletion or substitution of the gRNA 3 oligo(U) tail. Also, addition of a 3 oligo(U) tail to an unrelated transcript was sufficient for p110 binding. Both the gRNA-binding activity of the p110 protein and in vitro gRNA-independent and gRNA-dependent uridine insertion activities in the mitochondrial extract were inhibited by high concentrations of dinucleotides.RNA editing in trypanosomatid mitochondria involves the insertion and deletion of uridine (U) residues at multiple sites mainly within the coding regions of transcripts of more than half of the structural genes in the maxicircle genome (2, 24, 53, 57). The sequence information required for editing is provided by guide RNAs (gRNAs), which are small RNAs (40 to 70 nucleotides) that are complementary (if G:U and, rarely, C:A pairs are allowed) to edited blocks of the mature RNA (3, 42). The gRNAs also possess 3Ј-terminal nonencoded oligo(U) tails (4) that are created by the action of a mitochondrial terminal uridylyltransferase (TUTase) (1).Several models for the mechanism of the U insertion-deletion type of RNA editing have been proposed (3,5,12). Recent results from partial in vitro systems with both Trypanosoma brucei (28,48,49) and Leishmania tarentolae (10,15,20,21) suggest that the original enzyme cascade model is essentially correct. This model postulated an initial cleavage of the preedited mRNA at the first editing site, followed by an addition of U residues to the 3Ј end of the mRNA 5Ј fragment. It was initially proposed that the number of U's added was directly determined by base pairing with the guide A or G nucleotides in the cognate gRNA (3), but recent evidence suggests that multiple U's are first added to the 5Ј fragment and those terminal U's not base paired to the gRNA are then deleted by a 3Ј exonuclease (10,18,28,48). The final step is a ligation of the 5Ј fragment and the 3Ј fragment. Guide RNAdependent U deletions would involve the removal of all of the added U's and the encoded U's due to the lack of guiding nucleotides in the gRNA at that site. Rel...

A glycolytic enzyme, enolase, is recruited as a cofactor of tRNA targeting toward mitochondria in Saccharomyces cerevisiae

“…Interaction of yeast Eno1/2p with single-stranded DNA was reported previously (al-Giery and Brewer 1992). Numerous other proteins, including glycolytic enzymes, were already found to have RNA-or tRNA-binding capacities (Singh and Green 1993;Hentze 1994;Bringaud et al 1997;Pioli et al 2002). In the majority of cases, there was no direct evidence of the functional importance of these RNA-binding capacities of well known enzymes, but additional roles in RNA turnover and compartmentalization were suggested.…”

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

“…IP from mitochondrial extract was performed using Sepharose G beads coated with affinity-purified mouse polyclonal antibodies against recombinant Ltp26, recombinant Ltp28, and L. tarentolae 3Ј TUTase (Ltp26-IP, Ltp28-IP, and TUT-IP lanes). Immunodetection was performed with rabbit antibodies raised against purified Ltp26/Ltp28 complex, recombinant glutamate dehydrogenase (GDH) (Bringaud et al 1997), and TUTase. Ext, mitochondrial extract; Pre, proteins bound to G-beads coated with preimmune serum; Ab, proteins bound to G-beads coated with specific antibodies.…”

“…This approach has had varied success. For example, in L. tarentolae, three proteins, p18, p51, and p110, were identified by RNA binding and then discovered to be the mitochondrial metabolic enzymes ATPase subunit b, aldehyde dehydrogenase, and glutamate dehydrogenase, respectively (Bringaud et al 1995(Bringaud et al , 1997, and a double knockout of the glutamate dehydrogenase gene had no effect on editing (Estévez et al 1999). TBRGG1, a mitochondrial protein from T. brucei that binds poly [U] and gRNAs, was cloned by homology to nucleolin and shown to cosediment with in vitro editing activity, but no role in editing has been ascribed to this protein (Leegwater et al 1995;Vanhamme et al 1998).…”

Different Antigenic Reactivities of Bovine Brain Glutamate Dehydrogenase Isoproteins

“…Actually, other roles of GDHs have been reported. For instance, a membrane-bound form of GDH possesses a microtubule-binding activity (Rajas et al, 1996), and GDH reacts as an RNA-binding protein and shows a possible role in the regulation of transcription (Preiss et al, 1993;McDaniel, 1995;Bringaud et al, 1997). Recently, Cavallaro et al (1997) have identified GDH as one of the late memory-related genes in the hippocampus, and Frattini et al (1997) have identified GDH as a new member of the ring finger gene family in It would appear that we have just begun to unravel the mystery of GDHs and their role in a biological system.…”