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

Aphasizhev, Ruslan; Aphasizheva, Inna; Nelson, Robert E.; Simpson, Larry

doi:10.1261/rna.2134303

Cited by 101 publications

(125 citation statements)

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“…Tb REH1 was detected by mass spectrometry analysis in an MP63-immunoprecipitated sample and also in a RECC preparation isolated by ion-exchange chromatography (32)(33)(34). However, REH1 was not detected in REN1-, REN2-, or REN3-TAP pull-downs from T. brucei (32,35), nor in REL1-TAP and MP44-TAP pull-downs from L. tarentolae (7,11). These results could be explained by our finding that REH1 is associated with the RECC by RNA linkers, as are several other editingassociated complexes (6,11), and that this linkage is easily disrupted during the isolation.…”

Section: Discussionmentioning

confidence: 93%

Trypanosome REH1 is an RNA helicase involved with the 3′–5′ polarity of multiple gRNA-guided uridine insertion/deletion RNA editing

Herrera

Zhou

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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Uridine insertion/deletion RNA editing in kinetoplastid mitochondria corrects encoded frameshifts in mRNAs. The genetic information for editing resides in small guide RNAs (gRNAs), which form anchor duplexes just downstream of an editing site and mediate editing within a single editing "block." Many mRNAs require multiple gRNAs; the observed overall 3′ to 5′ polarity of editing is determined by the formation of upstream mRNA anchors by downstream editing. Hel61, a mitochondrial DEAD-box protein, was previously shown to be involved in RNA editing, but the functional role was not clear. Here we report that down-regulation of Hel61 [renamed REH1 (RNA editing helicase 1)] expression in Trypanosoma brucei selectively affects editing mediated by two or more overlapping gRNAs but has no effect on editing within a single block. Down-regulation produces an increased abundance of the gRNA/edited mRNA duplex for the first editing block of the A6 mRNA. Recombinant REH1 has an ATP-dependent double strand RNA unwinding activity in vitro with a model gRNA-mRNA duplex. These data indicate that REH1 is involved in gRNA displacement either directly by unwinding the gRNA/edited mRNA duplex or indirectly, to allow the 5′ adjacent upstream gRNA to form an anchor duplex with the edited mRNA to initiate another block of editing. Purified tagged REH1 is associated with the RNA editing core complex by RNA linkers and a colocalization of REH1, REL1, and two kinetoplast ribosomal proteins with the kinetoplast DNA was observed by immunofluorescence, suggesting that editing, transcription, and translation may be functionally linked.Leishmania | RNAi | tandem affinity purification | streptavidin binding and protein A purification

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Section: Discussionmentioning

confidence: 93%

Trypanosome REH1 is an RNA helicase involved with the 3′–5′ polarity of multiple gRNA-guided uridine insertion/deletion RNA editing

Herrera

Zhou

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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“…The GP-complex interacts with the L-complex through RNA (7), and we have previously described a third complex with RNA annealing activity that also interacts with the L-complex and the GP-complex through RNA (11). The nature of the RNA linker is unknown, but bound gRNA is a likely candidate.…”

Section: Discussionmentioning

confidence: 99%

“…The L-complex depends solely on protein-protein interactions for stability and exists in a dimeric or tetrameric configuration. The interaction of the L-complex with RET1 and with an RNA-binding protein complex is RNA dependent (11). To dissect the biological roles of the two TUTases in RNA editing, we have compared the effects of RET1 and RET2 down-regulation by RNAi on mitochondrial mRNAs, gRNAs, and on in vitro editing activities of RNA-editing complexes.…”

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confidence: 99%

A tale of two TUTases

Aphasizhev

Aphasizheva

Simpson

2003

Proc. Natl. Acad. Sci. U.S.A.

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The insertion and deletion of U residues at specific sites in mRNAs in trypanosome mitochondria is thought to involve 3 terminal uridylyl transferase (TUTase) activity. TUTase activity is also required to create the nonencoded 3 oligo[U] tails of the transacting guide RNAs (gRNAs). We have described two TUTases, RET1 (RNA editing TUTase 1) and RET2 (RNA editing TUTase 2) as components of different editing complexes. Tandem affinity purificationtagged Trypanosoma brucei RET2 (TbRET2) was expressed and localized to the cytosol in Leishmania tarentolae cells by removing the mitochondrial signal sequence. Double-affinity isolation yielded tagged TbRET2, together with a few additional proteins. This material exhibits a U-specific transferase activity in which a single U is added to the 3 end of a single-stranded RNA, thereby confirming that RET2 is a 3 TUTase. We also found that RNA interference of RET2 expression in T. brucei inhibits in vitro Uinsertion editing and has no effect on the length of the 3 oligo[U] tails of the gRNAs, whereas down-regulation of RET1 has a minor effect on in vitro U-insertion editing, but produces a decrease in the average length of the oligo[U] tails. This finding suggests that RET2 is responsible for U-insertions at editing sites and RET1 is involved in gRNA 3 end maturation, which is essential for creating functional gRNAs. From these results we have functionally relabeled the previously described TUT-II complex containing RET1 as the guide RNA processing complex.U -insertion͞deletion RNA editing in trypanosome mitochondria involves the annealing of a guide RNA (gRNA) to an mRNA, endonuclease cleavage at a non-base-paired editing site adjacent to the RNA duplex, addition or deletion of Us to or from the 3Ј end of the 5Ј cleavage fragment, and religation of the two mRNA fragments (1-4). Each gRNA mediates the editing of 1-5 sites in a 3Ј to 5Ј polarity, and multiple overlapping gRNAs mediate the editing of an entire domain, also in a 3Ј to 5Ј polarity (5).We previously isolated the RNA editing terminal uridylyl transferase (TUTase) 1 (RET1) and showed it to be present both as a free tetramer and as a component of a high molecular weight complex (TUT II) (6). This complex migrates on a native gel at Ϸ700 kDa and interacts in an RNase-sensitive manner with the larger ligase-containing complex (L-complex) (7). Downregulation of RET1 expression in Trypanosoma brucei by conditional RNA interference (RNAi) caused a decrease in the steady-state abundance of edited mRNAs without any effect on transcription (6). The core L-complex and associated proteins, capable of both U-insertion and U-deletion editing activity in vitro, was isolated from Leishmania tarentolae mitochondrial extract by tandem affinity purification (TAP) (8), followed by glycerol gradient sedimentation (7). The L-complex contains two adenylatable RNA ligases, REL1 and REL2, three related zinc finger-containing proteins, two proteins with RNase III motifs, two proteins with an AP endonuclease exonuclease phosphatase motif, a se...

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“…This fragment is highly conserved among RET1 and RET2 sequences, and, given the major differences in oligomeric structure and RNA substrates of these enzymes in vivo, it is unlikely to be involved in either oligomerization or RNA binding. The extended RNA-binding region of RET1 may be relevant to the finding that in the mitochondrial extract, RET1 interacts via an RNA linker with the ϳ16-polypeptide L-complex (14) and the MRP1/2 RNA chaperone complex (26).…”

Section: Discussionmentioning

confidence: 99%

RNA-editing Terminal Uridylyl Transferase 1

Aphasizheva

Aphasizhev

Simpson

2004

Journal of Biological Chemistry

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The catalytic, RNA-binding and oligomerization domains of the RNA-editing terminal uridylyl transferase 1 (RET1) from Leishmania tarentolae mitochondria were characterized by mutational analysis. Significant N-and C-terminal portions of the protein were found to be dispensable for UTP polymerization in vitro. Changes of conserved amino acids in the active site demonstrated a general similarity of sugar-phosphate moiety recognition of the incoming ribonucleotide triphosphate by RET1 and eukaryotic poly(A) polymerases. Overlapping RNA-binding and oligomerization regions were mapped to the C-terminal region, which is conserved only among trypanosomatid RET1 enzymes. In the absence of an RNA primer, RET1 can use UTP itself to initiate nucleotide transfer and produce poly(U) molecules of several hundred nucleotides. An N-terminal zinc finger motif is essential for enzyme activity; deletion of this motif or chelation of zinc inhibits activity.Transfer of a nucleotide to an acceptor hydroxyl group is a fundamental chemical reaction involved in a variety of biological processes. Among many enzyme superfamilies that perform this reaction, the DNA polymerase ␤-type group of nucleotidyltransferases is one of most widespread. Members of this group are involved in such diverse pathways as DNA repair, RNA processing, antibiotic resistance, and signal transduction (1). They include poly(A) polymerase (PAP), 1 CCA-adding enzymes, terminal deoxynucleotidyl transferase, kanamycin nucleotidyltransferase, and others. A characteristic feature of the superfamily is the presence of conserved catalytic domain with three metal-coordinating invariant carboxylates and a helical turn motif hG(G/S) 9 -13 XDh(D/E)h (where X represents any amino acid, and h is a hydrophobic amino acid) (2). Two classifications have been proposed based on sequence similarities. The class I subfamily includes the archaeal CCA-adding enzyme, eukaryotic PAP, DNA polymerase ␤, terminal deoxynucleotidyltransferase, and kanamycin nucleotidyltransferase, whereas the class II subfamily contains eubacterial and eukaryotic CCA-adding enzymes and eubacterial PAP (3). Aravind and Koonin (1) expanded the DNA polymerase ␤-type nucleotidyltransferase superfamily to contain several more groups of proteins and divided the enlarged set into nine families.Until recently, PAPs were the only known template-independent enzymes that exhibited specificity toward a particular nucleotide in an otherwise typical polymerization reaction. Atomic structures have been solved for yeast (4) and bovine (5) PAP, and several conserved amino acids were proposed to direct the specificity of the enzyme for ATP, albeit not by direct contacts between the nucleotide base and active center residues.Terminal uridylyltransferases (TUTases), enzymatic activities that add UMP residues to the 3Ј-hydroxyl group of RNA, have been described in mammalian cells (6 -8), plants (9), and trypanosomes (10, 11). No such activity has yet been found in prokaryotes. A biochemical analysis of protein complexes involved i...

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

Cited by 101 publications

References 42 publications

Trypanosome REH1 is an RNA helicase involved with the 3′–5′ polarity of multiple gRNA-guided uridine insertion/deletion RNA editing

Trypanosome REH1 is an RNA helicase involved with the 3′–5′ polarity of multiple gRNA-guided uridine insertion/deletion RNA editing

A tale of two TUTases

RNA-editing Terminal Uridylyl Transferase 1

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