Separate Insertion and Deletion Subcomplexes of the Trypanosoma brucei RNA Editing Complex

ABSTRACT RNA editing produces mature mitochondrial mRNAs in trypanosomatids by the insertion and deletion of uridylates. It is catalyzed by a multiprotein complex, the editosome. We identified TbMP44 among the components of enriched editosomes by a combination of mass spectrometry and DNA sequence database analysis. Inactivation of an ectopic TbMP44 allele in cells in which the endogenous alleles were disrupted abolished RNA editing, inhibited cell growth, and was eventually lethal to bloodstream form trypanosomes. Loss of TbMP44 mRNA was followed initially by a reduction in the editosome sedimentation coefficient and then by the absence of other editosome proteins despite the presence of the mRNA. Reactivation of TbMP44 gene expression resulted in the resumption of cell growth and the reappearance of editosomes. These data indicate that TbMP44 is a component of the editosome that is essential for editing and critical for the structural integrity of the editosome.

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The Two RNA Ligases of the Trypanosoma brucei RNA Editing Complex: Cloning the Essential Band IV Gene and Identifying the Band V Gene

Molecular and Cellular Biology ◽

10.1128/mcb.21.4.979-989.2001 ◽

2001 ◽

Vol 21 (4) ◽

pp. 979-989 ◽

Cited By ~ 63

Author(s):

Laura N. Rusché ◽

Catherine E. Huang ◽

Kenneth J. Piller ◽

Michael Hemann ◽

Elizabeth Wirtz ◽

...

Keyword(s):

Trypanosoma Brucei ◽

Rna Editing ◽

Mrna Levels ◽

Mitochondrial Targeting ◽

Terminal Sequence ◽

Partial Amino Acid Sequence ◽

Rna Ligase ◽

E Coli ◽

Insertion And Deletion ◽

Mitochondrial Transcripts

ABSTRACT Kinetoplastid RNA editing is a posttranscriptional insertion and deletion of U residues in mitochondrial transcripts that involves RNA ligase. A complex of seven different polypeptides purified fromTrypanosoma brucei mitochondria that catalyzes accurate RNA editing contains RNA ligases of ∼57 kDa (band IV) and ∼50 kDa (band V). From a partial amino acid sequence, cDNA and genomic clones of band IV were isolated, making it the first cloned component of the minimal RNA editing complex. It is indeed an RNA ligase, for when expressed inEscherichia coli, the protein autoadenylylates and catalyzes RNA joining. Overexpression studies revealed that T. brucei can regulate of total band IV protein at the level of translation or protein stability, even upon massively increased mRNA levels. The protein's mitochondrial targeting was confirmed by its location, size when expressed in T. brucei and E. coli, and N-terminal sequence. Importantly, genetic knockout studies demonstrated that the gene for band IV is essential in procyclic trypanosomes. The band IV and band V RNA ligases of the RNA editing complex therefore serve different functions. We also identified the gene for band V RNA ligase, a protein much more homologous to band IV than to other known ligases.

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RNA Editing in Trypanosoma brucei Requires Three Different Editosomes

Molecular and Cellular Biology ◽

10.1128/mcb.01374-07 ◽

2007 ◽

Vol 28 (1) ◽

pp. 122-130 ◽

Cited By ~ 70

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.

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In Trypanosoma brucei RNA Editing, Band II Enables Recognition Specifically at Each Step of the U Insertion Cycle

Molecular and Cellular Biology ◽

10.1128/mcb.25.7.2785-2794.2005 ◽

2005 ◽

Vol 25 (7) ◽

pp. 2785-2794 ◽

Cited By ~ 8

Author(s):

Julie A. Law ◽

Catherine E. Huang ◽

Sean F. O'Hearn ◽

Barbara Sollner-Webb

Keyword(s):

Rna Interference ◽

Trypanosoma Brucei ◽

Rna Editing ◽

Cell Lines ◽

Protein Recognition ◽

Essential Nature ◽

Insertion And Deletion ◽

Insertion Sites

ABSTRACT Trypanosome RNA editing is the posttranscriptional insertion and deletion of uridylate (U) residues, often to a massive extent, through cycles of cleavage, U addition or U removal, and ligation. These editing cycles are catalyzed by a complex that we purified to seven major proteins (bands I through VII). Here we analyze the role of band II using extracts of clonal band II RNA interference (RNAi) cell lines prepared by a rapid protocol that enables retention of activities that are lost during traditional extract preparation. By individually scoring each step of editing, we show that band II is critical for all steps of U insertion but is not important for any of the steps of U deletion or for their coordination into the U deletion cycle. This specificity supports the long- standing model that U-insertional and U-deletional activities are separated within the editing complex. Furthermore, by assaying the basic activities of the enzymes that catalyze the steps of U insertion, independent of their action in editing, we show that band II is not any of those enzymes. Rather, band II enables endonuclease action at authentic U insertion sites, terminal-uridylyl-transferase (TUTase) action at cleaved U insertion sites, and U-insertion-specific ligase (band V/IREL) action in the editing complex. Thus, band II facilitates each step of U insertion by providing proper RNA and/or protein recognition. We propose that band II (TbMP81) be called IRER, indicating its essential nature in U-insertional RNA editing recognition.

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Dyskinetoplastic Trypanosoma brucei Contains Functional Editing Complexes

Eukaryotic Cell ◽

10.1128/ec.2.3.569-577.2003 ◽

2003 ◽

Vol 2 (3) ◽

pp. 569-577 ◽

Cited By ~ 20

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.

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The KREPA3 Zinc Finger Motifs and OB-Fold Domain Are Essential for RNA Editing and Survival of Trypanosoma brucei

Molecular and Cellular Biology ◽

10.1128/mcb.01115-08 ◽

2008 ◽

Vol 28 (22) ◽

pp. 6939-6953 ◽

Cited By ~ 18

Author(s):

Xuemin Guo ◽

Nancy Lewis Ernst ◽

Kenneth D. Stuart

Keyword(s):

Trypanosoma Brucei ◽

Rna Editing ◽

Mutational Analysis ◽

Zinc Fingers ◽

Double Knockout ◽

Insertion And Deletion ◽

Life Cycle Stages ◽

Ob Fold

ABSTRACT Three types of editosomes, each with an identical core containing six related KREPA proteins, catalyze the U insertion and deletion RNA editing of mitochondrial mRNAs in trypanosomes. Repression of expression of one of these, KREPA3 (also known as TbMP42), shows that it is essential for growth and in vivo editing in both procyclic (PF) and bloodstream (BF) life cycle stages of Trypanosoma brucei. RNA interference knockdown results in editosome disruption and altered in vitro editing in PFs, while repression by regulatable double knockout results in almost complete loss of editosomes in BFs. Mutational analysis shows that the KREPA3 zinc fingers and OB-fold domain are each essential for growth and in vivo editing. Nevertheless, KREPA3 with mutated zinc fingers incorporates into editosomes that catalyze in vitro editing and thus is not essential for editosome integrity, although stability is affected. In contrast, the OB-fold domain is essential for editosome integrity. Overall, KREPA3, especially its OB-fold, functions in editosome integrity, and its zinc fingers are essential for editing in vivo but not for the central catalytic steps. KREPA3 may function in editosome organization and/or RNA positioning.

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Trypanosoma brucei RNA Editing Complex

Molecular and Cellular Biology ◽

10.1128/mcb.23.21.7909-7919.2003 ◽

2003 ◽

Vol 23 (21) ◽

pp. 7909-7919 ◽

Cited By ~ 16

Author(s):

Sean F. O'Hearn ◽

Catherine E. Huang ◽

Mike Hemann ◽

Alevtina Zhelonkina ◽

Barbara Sollner-Webb

Keyword(s):

Trypanosoma Brucei ◽

Rna Editing ◽

Protein Pair ◽

Normal Structure ◽

Cell Extract ◽

Insertion And Deletion ◽

Vitro Analysis ◽

Enzymatic Complex ◽

In Vitro Analysis

ABSTRACT Maturation of Trypanosoma brucei mitochondrial mRNA involves massive posttranscriptional insertion and deletion of uridine residues. This RNA editing utilizes an enzymatic complex with seven major proteins, band I through band VII. We here use RNA interference (RNAi) to examine the band II and band V proteins. Band II is found essential for viability; it is needed to maintain the normal structure of the editing complex and to retain the band V ligase protein. Previously, band III was found essential for certain activities, including maintenance of the editing complex and retention of the band IV ligase protein. Thus, band II and band V form a protein pair with features analogous to the band III/band IV ligase pair. Since band V is specific for U insertion and since band IV is needed for U deletion, their parallel organization suggests that the editing complex has a pseudosymmetry. However, unlike the essential band IV ligase, RNAi to band V has only a morphological but no growth rate effect, suggesting that it is stimulatory but nonessential. Indeed, in vitro analysis of band V RNAi cell extract demonstrates that band IV can seal U insertion when band V is lacking. Thus, band IV ligase is the first activity of the basic editing complex shown able to serve in both forms of editing. Our studies also indicate that the U insertional portion may be less central in the editing complex than the corresponding U deletional portion.

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Cell-line specific RNA editing patterns in Trypanosoma brucei suggest a unique mechanism to generate protein variation in a system intolerant to genetic mutations

Nucleic Acids Research ◽

10.1093/nar/gkz1131 ◽

2019 ◽

Vol 48 (3) ◽

pp. 1479-1493

Author(s):

Laura E Kirby ◽

Donna Koslowsky

Keyword(s):

Trypanosoma Brucei ◽

Rna Editing ◽

Dual Coding ◽

Reading Frame ◽

Guide Rnas ◽

Insertion And Deletion ◽

Editing Domain ◽

Ribosomal Protein S12 ◽

Reading Frames ◽

Unique Mechanism

Abstract Trypanosoma brucei possesses a highly complex RNA editing system that uses guide RNAs to direct the insertion and deletion of uridines in mitochondrial mRNAs. These changes extensively alter the target mRNAs and can more than double them in length. Recently, analyses showed that several of the edited genes possess the capacity to encode two different protein products. The overlapped reading frames can be accessed through alternative RNA editing that shifts the translated reading frame. In this study, we analyzed the editing patterns of three putative dual-coding genes, ribosomal protein S12 (RPS12), the 5′ editing domain of NADH dehydrogenase subunit 7 (ND7 5′), and C-rich region 3 (CR3). We found evidence that alternatively 5′-edited ND7 5′ and CR3 transcripts are present in the transcriptome, providing evidence for the use of dual ORFs in these transcripts. Moreover, we found that CR3 has a complex set of editing pathways that vary substantially between cell lines. These findings suggest that alternative editing can work to introduce genetic variation in a system that selects against nucleotide mutations.

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In Trypanosoma brucei RNA Editing, TbMP18 (Band VII) Is Critical for Editosome Integrity and for both Insertional and Deletional Cleavages

Molecular and Cellular Biology ◽

10.1128/mcb.01460-06 ◽

2006 ◽

Vol 27 (2) ◽

pp. 777-787 ◽

Cited By ~ 17

Author(s):

Julie A. Law ◽

Sean O'Hearn ◽

Barbara Sollner-Webb

Keyword(s):

Rna Interference ◽

Trypanosoma Brucei ◽

Rna Editing ◽

Protein Complex ◽

Substrate Recognition ◽

The Other ◽

Major Protein

ABSTRACT In trypanosome RNA editing, uridylate (U) residues are inserted and deleted at numerous sites within mitochondrial pre-mRNAs by an ∼20S protein complex that catalyzes cycles of cleavage, U addition/U removal, and ligation. We used RNA interference to deplete TbMP18 (band VII), the last unexamined major protein of our purified editing complex, showing it is essential. TbMP18 is critical for the U-deletional and U-insertional cleavages and for integrity of the ∼20S editing complex, whose other major components, TbMP99, TbMP81, TbMP63, TbMP52, TbMP48, TbMP42 (bands I through VI), and TbMP57, instead sediment as ∼10S associations. Additionally, TbMP18 augments editing substrate recognition by the TbMP57 terminal U transferase, possibly aiding the recognition component, TbMP81. The other editing activities and their coordination in precleaved editing remain active in the absence of TbMP18. These data are reminiscent of the data on editing subcomplexes reported by A. Schnaufer et al. (Mol. Cell 12:307-319, 2003) and suggest that these subcomplexes are held together in the ∼20S complex by TbMP18, as was proposed previously. Our data additionally imply that the proteins are less long-lived in these subcomplexes than they are when held in the complete editing complex. The editing endonucleolytic cleavages being lost when the editing complex becomes fragmented, as upon TbMP18 depletion, should be advantageous to the trypanosome, minimizing broken mRNAs.

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Combining Insertion and Deletion in RNA-editing Preserves Regularity

Electronic Proceedings in Theoretical Computer Science ◽

10.4204/eptcs.100.4 ◽

2012 ◽

Vol 100 ◽

pp. 48-62

Author(s):

E.P. de Vink ◽

H. Zantema ◽

D. Bošnački

Keyword(s):

Rna Editing ◽

Insertion And Deletion

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