The specificity of nucleotide removal during RNA editing in Trypanosoma brucei

Lawson, Sobomabo D.; Igo, Robert P.; Salavati, Reza; Stuart, Kenneth

doi:10.1017/s135583820101055x

Cited by 17 publications

(15 citation statements)

References 38 publications

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“…When both mRNAs were hybridized with gA6-14, gA6-14sU, and gA6-14-C6, the ES and the sequence immediately upstream (6-8 nt) were inaccessible to single-strand specific probes and only accessible to the double-strand-or stacking-specific V1. This suggests that while the scissile base is not involved in Watson-Crick pairing, in agreement with predictions by Seiwert et al (1996), Cruz-Reyes et al (2001), and Lawson et al (2001), it is contained within a highly organized region. This finding was not surprising, because nucleotides in singlestranded areas in a secondary structure are often paired in long-range tertiary interactions, single-strand base stacking, or cross-strand base stacking (Peterson and Feigon 1996;Butcher et al 1997;Zimmermann et al 1997;Znosko et al 2004).…”

Section: Discussionsupporting

confidence: 86%

“…In addition, to facilitate the probing, the mRNAs were shortened at their 59-ends (sequence that does not interact with the U-tail), based on our U-tail cross-linking results described above. Again, shortening of the A6 substrate at its 59-end has no effect on its ability to undergo editing when incubated with glycerol gradient purified editosomes (Burgess et al 1999;Cruz-Reyes et al 2001;Lawson et al 2001;Igo et al 2002;Cifuentes-Rojas et al 2005; data not shown).…”

Section: Solution Structure Probingmentioning

confidence: 89%

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Trypanosoma brucei ATPase subunit 6 mRNA bound to gA6-14 forms a conserved three-helical structure

Reifur¹,

Koslowsky²

2008

RNA

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T. brucei survival relies on the expression of mitochondrial genes, most of which require RNA editing to become translatable. In trypanosomes, RNA editing involves the insertion and deletion of uridylates, a developmentally regulated process directed by guide RNAs (gRNAs) and catalyzed by the editosome, a complex of proteins. The pathway for mRNA/gRNA complex formation and assembly with the editosome is still unknown. Work from our laboratory has suggested that distinct mRNA/gRNA complexes anneal to form a conserved core structure that may be important for editosome assembly. The secondary structure for the apocytochrome b (CYb) pair has been previously determined and is consistant with our model of a three-helical structure. Here, we used cross-linking and solution structure probing experiments to determine the structure of the ATPase subunit 6 (A6) mRNA hybridized to its cognate gA6-14 gRNA in different stages of editing. Our results indicate that both unedited and partially edited A6/gA6-14 pairs fold into a three-helical structure similar to the previously characterized CYb/ gCYb-558 pair. These results lead us to conclude that at least two mRNA/gRNA pairs with distinct editing sites and distinct primary sequences fold to a three-helical secondary configuration that persists through the first few editing events.

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Section: Discussionsupporting

confidence: 86%

Section: Solution Structure Probingmentioning

confidence: 89%

Trypanosoma brucei ATPase subunit 6 mRNA bound to gA6-14 forms a conserved three-helical structure

Reifur¹,

Koslowsky²

2008

RNA

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“…5), indicating that recognition by KREN3 does not depend on structural features resulting from a cis guiding sequence. Nevertheless, the tertiary structure of the interacting gRNA/mRNAs may be critical to editosome endonuclease specificity (32,33). Indeed, cleavage site specificity by other RNase IIIs is influenced by tertiary structure, particularly internal loops (36,47,66).…”

Section: Discussionmentioning

confidence: 99%

RNA Editing in Trypanosoma brucei Requires Three Different Editosomes

Carnes

Trotter

Peltan

et al. 2008

Molecular and Cellular Biology

105

150

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Trypanosoma brucei has three distinct ϳ20S editosomes that catalyze RNA editing by the insertion and deletion of uridylates. Editosomes with the KREN1 or KREN2 RNase III type endonucleases specifically cleave deletion and insertion editing site substrates, respectively. We report here that editosomes with KREPB2, which also has an RNase III motif, specifically cleave cytochrome oxidase II (COII) pre-mRNA insertion editing site substrates in vitro. Conditional repression and mutation studies also show that KREPB2 is an editing endonuclease specifically required for COII mRNA editing in vivo. Furthermore, KREPB2 expression is essential for the growth and survival of bloodstream forms. Thus, editing in T. brucei requires at least three compositionally and functionally distinct ϳ20S editosomes, two of which distinguish between different insertion editing sites. This unexpected finding reveals an additional level of complexity in the RNA editing process and suggests a mechanism for how the selection of sites for editing in vivo is controlled.RNA editing recodes most of the mitochondrial mRNAs in trypanosomatids by the insertion and deletion of uridine nucleotides (U's) using information provided by guide RNAs (gRNAs) (58). RNA editing is developmentally regulated and is required for parasites that cycle between insect vector and mammalian host (54). Proteins that perform catalytic steps of RNA editing have been identified: endonucleases KREN1 or KREN2 cleave at deletion or insertion sites, respectively; terminal uridylyl transferase (TUTase) KRET2 adds U's at insertion sites; U specific exoribonuclease (exoUase) KREX1 removes U's at deletion sites; and ligases KREL1 or KREL2 rejoin mRNA fragments after U addition or removal (5,13,25,30,34,55,61). These catalytic steps are coordinated by the multiprotein editosomes that sediment at ϳ20S on glycerol gradients (9, 43). KREPB2 was identified as an editosome component by mass spectrometry, and found to contain an RNase III motif harboring amino acids that are critical for catalysis, a U1 Zn 2ϩ finger, and double-stranded RNA binding. It has sequence similarity to KREN1 and KREN2, which also contain these three motifs (23,41,64). These data strongly suggest an RNA editing endonuclease role for KREPB2.KREN1 and KREN2 were shown to be RNA editing endonucleases using both RNA interference (RNAi) and conditional knockout cell lines (5, 61). Both KREN1 and KREN2 are essential for the normal growth of PF and BF cells, and repression of their expression by either method led to dramatic growth defects or death, respectively. Such repression of KREN1 eliminated in vitro cleavage by ϳ20S editosomes of a deletion site in a synthetic substrate modeled on ATPase subunit 6 (A6) pre-mRNA but did not alter cleavage of an insertion site in a substrate derived from the same mRNA. These studies and other data have shown that KREN1 is an editing endonuclease with a preference for sites from which U's are deleted (29). Similarly, repression of KREN2 eliminated in vitro cleavage at insertion ed...

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“…This activity was also separated from TUTase, definitely showing that the exonuclease activity is not a reversal of the TUTase activity. In T. brucei (Rusché et al 1997) a similar 3Ј-exonuclease activity copurified with an editing complex (Rusché et al 1997;Lawson et al 2001;Igo et al 2002a). In both species, digestion of 3Ј-terminal U-stretches is single-strand specific and does not proceed past a G, A, or C residue, consistent with its proposed function during editing.…”

Section: U-specific Exonucleasementioning

confidence: 67%

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

Simpson¹,

Sbicego²,

Aphasizhev³

2003

RNA

154

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The basic mechanism of uridine insertion/deletion RNA editing in mitochondria of kinetoplastid protists has been established for some time but the molecular details remained largely unknown. Recently, there has been significant progress in defining the molecular components of the editing reaction. A number of factors have been isolated from trypanosome mitochondria, some of which have been definitely implicated in the uridine insertion/deletion RNA editing reaction and others of which have been circumstantially implicated. Several protein complexes have been isolated which exhibit some editing activities, and the macromolecular organization of these complexes is being analyzed. In addition, there have been several important technical advances in the in vitro analysis of editing. In this review we critically examine the various factors and complexes proposed to be involved in RNA editing.

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The specificity of nucleotide removal during RNA editing in Trypanosoma brucei

Cited by 17 publications

References 38 publications

Trypanosoma brucei ATPase subunit 6 mRNA bound to gA6-14 forms a conserved three-helical structure

Trypanosoma brucei ATPase subunit 6 mRNA bound to gA6-14 forms a conserved three-helical structure

RNA Editing in Trypanosoma brucei Requires Three Different Editosomes

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

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