scholarly journals RNA Sequence and Base Pairing Effects on Insertion Editing in Trypanosoma brucei

2002 ◽  
Vol 22 (5) ◽  
pp. 1567-1576 ◽  
Author(s):  
Robert P. Igo ◽  
Sobomabo D. Lawson ◽  
Kenneth Stuart
Keyword(s):  
Trypanosoma Brucei ◽  
Rna Editing ◽  
Base Pair ◽  
Editing Site ◽  
Base Pairing ◽  
Editing Efficiency ◽  
Rna Sequence ◽  
Guide Rnas

ABSTRACT RNA editing inserts and deletes uridylates (U's) in kinetoplastid mitochondrial pre-mRNAs by a series of enzymatic steps. Small guide RNAs (gRNAs) specify the edited sequence. Editing, though sometimes extensive, is precise. The effects of mutating pre-mRNA and gRNA sequences in, around, and upstream of the editing site on the specificity and efficiency of in vitro insertion editing were examined. U's could be added opposite guiding pyrimidines, but guiding purines, particularly A's, were required for efficient ligation. A base pair between mRNA and gRNA immediately upstream of the editing site was not required for insertion editing, although it greatly enhanced its efficiency and accuracy. In addition, a gRNA/mRNA duplex upstream of the editing site enhanced insertion editing when it was close to the editing site, but prevented cleavage, and hence editing, when immediately adjacent to the editing site. Thus, several aspects of mRNA-gRNA interaction, as well as gRNA base pairing with added U's, optimize editing efficiency, although they are not required for insertion editing.

scholarly journals Distinct Functions of Two RNA Ligases in Active Trypanosoma brucei RNA Editing Complexes

2002 ◽  
Vol 22 (13) ◽  
pp. 4652-4660 ◽  
Author(s):  
Jorge Cruz-Reyes ◽  
Alevtina G. Zhelonkina ◽  
Catherine E. Huang ◽  
Barbara Sollner-Webb
Keyword(s):  
Genetic Analysis ◽  
Trypanosoma Brucei ◽  
Rna Editing ◽  
Protein Complex ◽  
Base Pairing ◽  
Guide Rnas ◽  
Single Protein ◽  
The Individual ◽  
Catalytic Functions

ABSTRACT Trypanosome RNA editing is a unique U insertion and U deletion process that involves cycles of pre-mRNA cleavage, terminal U addition or U removal, and religation. This editing can occur at massive levels and is directed by base pairing of trans-acting guide RNAs. Both U insertion and U deletion cycles are catalyzed by a single protein complex that contains only seven major proteins, band I through band VII. However, little is known about their catalytic functions, except that band IV and band V are RNA ligases and genetic analysis indicates that the former is important in U deletion. Here we establish biochemical approaches to distinguish the individual roles of these ligases, based on their distinctive ATP and pyrophosphate utilization. These in vitro analyses revealed that both ligases serve in RNA editing. Band V is the RNA editing ligase that functions very selectively to seal in U insertion (IREL), while band IV is the RNA editing ligase needed to seal in U deletion (DREL). In combination with our earlier findings about the cleavage and the U-addition/U-removal steps of U deletion and U insertion, these results show that all three steps of these editing pathways exhibit major differences and suggest that the editing complex could have physically separate regions for U deletion and U insertion.

scholarly journals Uridylate Addition and RNA Ligation Contribute to the Specificity of Kinetoplastid Insertion RNA Editing

2000 ◽  
Vol 20 (22) ◽  
pp. 8447-8457 ◽  
Author(s):  
Robert P. Igo ◽  
Setareh S. Palazzo ◽  
Moffett L. K. Burgess ◽  
Aswini K. Panigrahi ◽  
Kenneth Stuart
Keyword(s):  
Trypanosoma Brucei ◽  
Rna Editing ◽  
Editing Site ◽  
Rna Ligase ◽  
Correct Number ◽  
Guide Rnas ◽  
Rna Ligation

ABSTRACT RNA editing in Trypanosoma brucei inserts and deletes uridylates (U's) in mitochondrial pre-mRNAs under the direction of guide RNAs (gRNAs). We report here the development of a novel in vitro precleaved editing assay and its use to study the gRNA specificity of the U addition and RNA ligation steps in insertion RNA editing. The 5′ fragment of substrate RNA accumulated with the number of added U's specified by gRNA, and U addition products with more than the specified number of U's were rare. U addition up to the number specified occurred in the absence of ligation, but accumulation of U addition products was slowed. The 5′ fragments with the correct number of added U's were preferentially ligated, apparently by adenylylated RNA ligase since exogenously added ATP was not required and since ligation was eliminated by treatment with pyrophosphate. gRNA-specified U addition was apparent in the absence of ligation when the pre-mRNA immediately upstream of the editing site was single stranded and more so when it was base paired with gRNA. These results suggest that both the U addition and RNA ligation steps contributed to the precision of RNA editing.

scholarly journals Role of Uridylate-Specific Exoribonuclease Activity in Trypanosoma brucei RNA Editing

2002 ◽  
Vol 1 (1) ◽  
pp. 112-118 ◽  
Author(s):  
Robert P. Igo ◽  
David S. Weston ◽  
Nancy Lewis Ernst ◽  
Aswini K. Panigrahi ◽  
Reza Salavati ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Rna Editing ◽  
Base Pair ◽  
Exonuclease Activity ◽  
Atpase Subunit ◽  
Guide Rnas

ABSTRACT Editing of mitochondrial mRNAs in kinetoplastid protozoa occurs by a series of enzymatic steps that insert and delete uridylates (U's) as specified by guide RNAs (gRNAs). The characteristics of the 3" exonuclease activity that removes the U's following cleavage during deletion editing were determined by using an in vitro precleaved deletion assay that is based on ATPase subunit 6 pre-mRNA and gA6[14] gRNA. The exonuclease in partially purified editing complexes is specific for U's. The specificity occurs in the absence of gRNA, but its activity is enhanced by the presence of gRNA. The 3" pre-mRNA fragment enhances the specificity, but not the efficiency, of U removal. The activity is sensitive to the 5" phosphate of the 3" fragment, which is not required for U removal. The ability of the 3" U's to base pair with purines in the gRNA protects them from removal, suggesting that the U-specific 3" exonuclease (exoUase) is specific for U's which are not base paired. ExoUase is stereospecific and cannot remove (Rp )α-thio-U. The specificity of the exoUase activity thus contributes to the precision of RNA editing.

scholarly journals Trypanosoma brucei RNA Editing: Coupled Cycles of U Deletion Reveal Processive Activity of the Editing Complex

2008 ◽  
Vol 28 (7) ◽  
pp. 2437-2445 ◽  
Author(s):  
Vadim S. Alatortsev ◽  
Jorge Cruz-Reyes ◽  
Alevtina G. Zhelonkina ◽  
Barbara Sollner-Webb
Keyword(s):  
Sequence Analysis ◽  
Trypanosoma Brucei ◽  
Rna Editing ◽  
Base Pairing ◽  
The Common ◽  
Editing Process

ABSTRACT RNA editing in Trypanosoma brucei is posttranscriptional uridylate removal/addition, generally at vast numbers of pre-mRNA sites, but to date, only single editing cycles have been examined in vitro. We here demonstrate achieving sequential cycles of U deletion in vitro, with editing products confirmed by sequence analysis. Notably, the subsequent editing cycle is much more efficient and occurs far more rapidly than single editing cycles; plus, it has different recognition requirements. This indicates that the editing complex acts in a concerted manner and does not dissociate from the RNA substrate between these cycles. Furthermore, the multicycle substrate exhibits editing that is unexpected from a strictly 3′-to-5′ progression, reminiscent of the unexpected editing that has been shown to occur frequently in T. brucei mRNAs edited in vivo. This unexpected editing is most likely due to alternate mRNA:guide RNA (gRNA) alignment forming a hyphenated anchor; its having only a 2-bp proximal duplex helps explain the prevalence of unexpected editing in vivo. Such unexpected editing was not previously reported in vitro, presumably because the common use of artificially tight mRNA:gRNA base pairing precludes alternate alignments. The multicycle editing and unexpected editing presented in this paper bring in vitro reactions closer to reproducing the in vivo editing process.

scholarly journals RNA Editing in Trypanosoma brucei Requires Three Different Editosomes

2007 ◽  
Vol 28 (1) ◽  
pp. 122-130 ◽  
Author(s):  
Jason Carnes ◽  
James Raffaello Trotter ◽  
Adam Peltan ◽  
Michele Fleck ◽  
Kenneth Stuart
Keyword(s):  
Trypanosoma Brucei ◽  
Rna Editing ◽  
Editing Site ◽  
Unexpected Finding ◽  
Rnase Iii ◽  
Growth And Survival ◽  
Insertion And Deletion ◽  
Additional Level

ABSTRACT Trypanosoma brucei has three distinct ∼20S 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 ∼20S 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.

scholarly journals Dyskinetoplastic Trypanosoma brucei Contains Functional Editing Complexes

2003 ◽  
Vol 2 (3) ◽  
pp. 569-577 ◽  
Author(s):  
Gonzalo J. Domingo ◽  
Setareh S. Palazzo ◽  
Bingbing Wang ◽  
Brian Pannicucci ◽  
Reza Salavati ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Rna Editing ◽  
Trypanosoma Evansi ◽  
Wild Type ◽  
Single Class ◽  
Catalytic Activities ◽  
Guide Rnas ◽  
Insertion And Deletion ◽  
And Function

ABSTRACT Mitochondrial pre-mRNAs undergo posttranscriptional RNA editing as directed by small guide RNAs (gRNAs) to produce functional mRNAs in kinetoplastid protozoa. The pre-mRNAs and gRNAs are encoded in the maxicircle and minicircle components, respectively, of the kinetoplastid mitochondrial DNA (kDNA), and editing is catalyzed by a multienzyme protein complex. Trypanosoma evansi AnTat3/3, which lacks maxicircles but retains a single class of minicircles, and a dyskinetoplastic mutant of Trypanosoma brucei EATRO164, which is devoid of kDNA, were both shown to retain genes and proteins for the editing complex. The proteins are present in complexes that immunoprecipitate and sediment indistinguishably from wild-type complexes. The complexes catalyze precleaved insertion and deletion editing as well as full-round deletion editing in vitro. Thus, mutants which lack the natural substrates for RNA editing and all or most gRNAs retain editing complexes that contain the four primary catalytic activities of editing and function in editing, at least in vitro. Therefore neither pre-mRNA nor gRNA is required to form functional RNA-editing complexes.

scholarly journals Trypanosome RNA Editing: Simple Guide RNA Features Enhance U Deletion 100-Fold

2001 ◽  
Vol 21 (3) ◽  
pp. 884-892 ◽  
Author(s):  
Jorge Cruz-Reyes ◽  
Alevtina Zhelonkina ◽  
Laura Rusche ◽  
Barbara Sollner-Webb
Keyword(s):  
Rna Editing ◽  
Central Region ◽  
Marked Improvement ◽  
Base Pairing ◽  
Guide Rna ◽  
Guide Rnas ◽  
Evolutionary Forces

ABSTRACT Trypanosome RNA editing is a massive processing of mRNA by U deletion and U insertion, directed by trans-acting guide RNAs (gRNAs). A U deletion cycle and a U insertion cycle have been reproduced in vitro using synthetic ATPase (A6) pre-mRNA and gRNA. Here we examine which gRNA features are important for this U deletion. We find that, foremost, this editing depends critically on the single-stranded character of a few gRNA and a few mRNA residues abutting the anchor duplex, a feature not previously appreciated. That plus any base-pairing sequence to tether the upstream mRNA are all the gRNA needs to direct unexpectedly efficient in vitro U deletion, using either the purified editing complex or whole extract. In fact, our optimized gRNA constructs support faithful U deletion up to 100 times more efficiently than the natural gRNA, and they can edit the majority of mRNA molecules. This is a marked improvement of in vitro U deletion, in which previous artificial gRNAs were no more active than natural gRNA and the editing efficiencies were at most a few percent. Furthermore, this editing is not stimulated by most other previously noted gRNA features, including its potential ligation bridge, 3′ OH moiety, any U residues in the tether, the conserved structure of the central region, or proteins that normally bind these regions. Our data also have implications about evolutionary forces active in RNA editing.

scholarly journals Association of Two Novel Proteins, TbMP52 and TbMP48, with the Trypanosoma brucei RNA Editing Complex

2001 ◽  
Vol 21 (2) ◽  
pp. 380-389 ◽  
Author(s):  
Aswini K. Panigrahi ◽  
Steven P. Gygi ◽  
Nancy L. Ernst ◽  
Robert P. Igo ◽  
Setareh S. Palazzo ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Trypanosoma Brucei ◽  
Rna Editing ◽  
Gel Filtration ◽  
Novel Proteins ◽  
Guide Rnas ◽  
Its Gene ◽  
Editing Activity ◽  
Novel Protein

ABSTRACT RNA editing in kinetoplastid mitochondria inserts and deletes uridylates at multiple sites in pre-mRNAs as directed by guide RNAs. This occurs by a series of steps that are catalyzed by endoribonuclease, 3′-terminal uridylyl transferase, 3′-exouridylylase, and RNA ligase activities. A multiprotein complex that contains these activities and catalyzes deletion editing in vitro was enriched fromTrypanosoma brucei mitochondria by sequential ion-exchange and gel filtration chromatography, followed by glycerol gradient sedimentation. The complex size is approximately 1,600 kDa, and the purified fraction contains 20 major polypeptides. A monoclonal antibody that was generated against the enriched complex reacts with an ∼49-kDa protein and specifically immunoprecipitates in vitro deletion RNA editing activity. The protein recognized by the antibody was identified by mass spectrometry, and the corresponding gene, designated TbMP52, was cloned. Recombinant TbMP52 reacts with the monoclonal antibody. Another novel protein, TbMP48, which is similar to TbMP52, and its gene were also identified in the enriched complex. These results suggest that TbMP52 and TbMP48 are components of the RNA editing complex.

scholarly journals A Novel Member of the RNase D Exoribonuclease Family Functions in Mitochondrial Guide RNA Metabolism in Trypanosoma brucei

2011 ◽  
Vol 286 (12) ◽  
pp. 10329-10340 ◽  
Author(s):  
Sara L. Zimmer ◽  
Sarah M. McEvoy ◽  
Jun Li ◽  
Jun Qu ◽  
Laurie K. Read
Keyword(s):  
Trypanosoma Brucei ◽  
Rna Editing ◽  
Sequence Similarity ◽  
Mitochondrial Gene ◽  
Sequence Information ◽  
Rna Turnover ◽  
Guide Rna ◽  
Guide Rnas

RNA turnover and RNA editing are essential for regulation of mitochondrial gene expression in Trypanosoma brucei. RNA turnover is controlled in part by RNA 3′ adenylation and uridylation status, with trans-acting factors also impacting RNA homeostasis. However, little is known about the mitochondrial degradation machinery or its regulation in T. brucei. We have identified a mitochondrial exoribonuclease, TbRND, whose expression is highly up-regulated in the insect proliferative stage of the parasite. TbRND shares sequence similarity with RNase D family enzymes but differs from all reported members of this family in possessing a CCHC zinc finger domain. In vitro, TbRND exhibits 3′ to 5′ exoribonuclease activity, with specificity toward uridine homopolymers, including the 3′ oligo(U) tails of guide RNAs (gRNAs) that provide the sequence information for RNA editing. Several lines of evidence generated from RNAi-mediated knockdown and overexpression cell lines indicate that TbRND functions in gRNA metabolism in vivo. First, TbRND depletion results in gRNA tails extended by 2–3 nucleotides on average. Second, overexpression of wild type but not catalytically inactive TbRND results in a substantial decrease in the total gRNA population and a consequent inhibition of RNA editing. The observed effects on the gRNA population are specific as rRNAs, which are also 3′-uridylated, are unaffected by TbRND depletion or overexpression. Finally, we show that gRNA binding proteins co-purify with TbRND. In summary, TbRND is a novel 3′ to 5′ exoribonuclease that appears to have evolved a function highly specific to the mitochondrion of trypanosomes.

scholarly journals Recognition of RNA Editing Sites Is Directed by Unique Proteins in Chloroplasts: Biochemical Identification of cis-Acting Elements and trans-Acting Factors Involved in RNA Editing in Tobacco and Pea Chloroplasts

2002 ◽  
Vol 22 (19) ◽  
pp. 6726-6734 ◽  
Author(s):  
Tetsuya Miyamoto ◽  
Junichi Obokata ◽  
Masahiro Sugiura
Keyword(s):  
Rna Editing ◽  
Editing Site ◽  
Mrna Editing ◽  
Higher Plant ◽  
In Vitro System ◽  
Cis Acting ◽  
Biochemical Identification ◽  
Editing Activity

ABSTRACT RNA editing in higher-plant chloroplasts involves C-to-U conversions at specific sites. Although in vivo analyses have been performed, little is known about the biochemical aspects of chloroplast editing reactions. Here we improved our original in vitro system and devised a procedure for preparing active chloroplast extracts not only from tobacco plants but also from pea plants. Using our tobacco in vitro system, cis-acting elements were defined for psbE and petB mRNAs. Distinct proteins were found to bind specifically to each cis-element, a 56-kDa protein to the psbE site and a 70-kDa species to the petB site. Pea chloroplasts lack the corresponding editing site in psbE since T is already present in the DNA. Parallel in vitro analyses with tobacco and pea extracts revealed that the pea plant has no editing activity for psbE mRNAs and lacks the 56-kDa protein, whereas petB mRNAs are edited and the 70-kDa protein is also present. Therefore, coevolution of an editing site and its cognate trans-factor was demonstrated biochemically in psbE mRNA editing between tobacco and pea plants.

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