Combining Insertion and Deletion in RNA-editing Preserves Regularity

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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ON THE AMBIGUITY OF INSERTION SYSTEMS

International Journal of Foundations of Computer Science ◽

10.1142/s0129054111009008 ◽

2011 ◽

Vol 22 (07) ◽

pp. 1747-1758

Author(s):

LAKSHMANAN KUPPUSAMY ◽

ANAND MAHENDRAN ◽

KAMALA KRITHIVASAN

Keyword(s):

Rna Editing ◽

Formal Language ◽

Formal Language Theory ◽

Language Theory ◽

Gene Insertion ◽

Computing Model ◽

Insertion And Deletion ◽

Dna Processing

Gene insertion and deletion are the operations that occur commonly in DNA processing and RNA editing. Based on these evolutionary transformations, a computing model has been formulated in formal language theory known as insertion-deletion systems. In this paper, we study about the ambiguity issues of insertion systems. First, we define six levels of ambiguity for insertion systems based on the components used in the derivation such as axiom, contexts and strings. Next, we show that there are inherently i-ambiguous insertion languages which are j-unambiguous for the combinations (i, j) ∈ {(5,0), (5,4), (4,3), (4,2), (3,1),(2,1), (1,0), (0,1)}. Finally, we prove an important result that the ambiguity problem of insertion systems is undecidable.

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

Molecular Cell ◽

10.1016/s1097-2765(03)00286-7 ◽

2003 ◽

Vol 12 (2) ◽

pp. 307-319 ◽

Cited By ~ 86

Author(s):

Achim Schnaufer ◽

Nancy Lewis Ernst ◽

Setareh S Palazzo ◽

Jeff O'Rear ◽

Reza Salavati ◽

...

Keyword(s):

Trypanosoma Brucei ◽

Rna Editing ◽

Insertion And Deletion

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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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Assembly and Function of the RNA Editing Complex in Trypanosoma brucei Requires Band III Protein

Molecular and Cellular Biology ◽

10.1128/mcb.22.9.3194-3203.2002 ◽

2002 ◽

Vol 22 (9) ◽

pp. 3194-3203 ◽

Cited By ~ 36

Author(s):

Catherine E. Huang ◽

Sean F. O'Hearn ◽

Barbara Sollner-Webb

Keyword(s):

Rna Editing ◽

Guide Rna ◽

Insertion And Deletion ◽

Cell Extracts ◽

Zinc Finger Motifs ◽

Acid Protein ◽

Mitochondrial Transcripts ◽

Insertion Sites ◽

And Function ◽

Amino Acid Protein

ABSTRACT Trypanosome RNA editing, the posttranscriptional insertion and deletion of U residues in mitochondrial transcripts, is catalyzed by a protein complex containing seven distinct proteins. In this study, we cloned the gene for band III, a 555-amino-acid protein with two separate zinc finger motifs. We prepared antibodies that showed band III protein cofractionates with the previously characterized band IV protein throughout the purification of the editing complex and is not found free or in other protein associations; therefore, it is a true constituent of the editing complex. Double-stranded RNA interference efficiently depleted band III protein and demonstrated that band III expression is essential for growth of procyclic trypanosomes and for RNA editing. These depleted cell extracts were deficient specifically in guide RNA-directed endonuclease cleavage at both U deletion and U insertion sites and in the activity of the band IV ligase, but they retained the 3′-U-exonuclease and terminal-U-transferase activities as well as band V ligase of the editing complex. Loss of band III protein also resulted in almost complete loss of the band IV ligase protein and altered sedimentation of the band V ligase. These data indicate that band III is either the RNA editing endonuclease or a factor critical for cleavage activity in the editing complex. They also demonstrate that band III is required for proper assembly of the editing complex.

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