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 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 Guide RNA-mRNA chimeras, which are potential RNA editing intermediates, are formed by endonuclease and RNA ligase in a trypanosome mitochondrial extract.

1995 ◽  
Vol 15 (6) ◽  
pp. 2933-2941 ◽  
Author(s):  
L N Rusché ◽  
K J Piller ◽  
B Sollner-Webb
Keyword(s):  
Rna Editing ◽  
Mitochondrial Rna ◽  
Site Specific ◽  
Rna Ligase ◽  
Guide Rna ◽  
Guide Rnas ◽  
Editing Domain ◽  
Mitochondrial Transcripts ◽  
Mitochondrial Extract

RNA editing in kinetoplast mitochondrial transcripts involves the insertion and/or deletion of uridine residues and is directed by guide RNAs (gRNAs). It is thought to occur through a chimeric intermediate in which the 3' oligo(U) tail of the gRNA is covalently joined to the 3' portion of the mRNA at the site being edited. Chimeras have been proposed to be formed by a transesterification reaction but could also be formed by the known mitochondrial site-specific nuclease and RNA ligase. To distinguish between these models, we studied chimera formation in vitro directed by a trypanosome mitochondrial extract. This reaction was found to occur in two steps. First, the mRNA is cleaved in the 3' portion of the editing domain, and then the 3' fragment derived from this cleavage is ligated to the gRNA. The isolated mRNA 3' cleavage product is a more efficient substrate for chimera formation than is the intact mRNA, inconsistent with a transesterification mechanism but supporting a nuclease-ligase mechanism. Also, when normal mRNA cleavage is inhibited by the presence of a phosphorothioate, normal chimera formation no longer occurs. Rather, this phosphorothioate induces both cleavage and chimera formation at a novel site within the editing domain. Finally, levels of chimera-forming activity correlate with levels of mitochondrial RNA ligase activity when reactions are conducted under conditions which inhibit the ligase, including the lack of ATP containing a cleavable alpha-beta bond. These data show that chimera formation in the mitochondrial extract occurs by a nuclease-ligase mechanism rather than by transesterification.

scholarly journals Uridine insertion into preedited mRNA by a mitochondrial extract from Leishmania tarentolae: stereochemical evidence for the enzyme cascade model.

1996 ◽  
Vol 16 (8) ◽  
pp. 4584-4589 ◽  
Author(s):  
G C Frech ◽  
L Simpson
Keyword(s):  
Rna Editing ◽  
Cascade Model ◽  
Transfer Reactions ◽  
Leishmania Tarentolae ◽  
Rna Ligase ◽  
Guide Rnas ◽  
Enzyme Cascade ◽  
Mitochondrial Extract

An RNA editing-like internal uridine (U) incorporation activity (G. C. Frech, N. Bakalara, L Simpson, and A. M. Simpson, EMBO J. 14:178-187, 1995) and a 3'-terminal U addition activity (N. Bakalara, A. M. Simpson, and L. Simpson, J. Biol. Chem. 264:18679-18686, 1989) have been previously described by using a mitochondrial extract from Leishmania tarentolae. Chiral phosphorothioates were used to investigate the stereoconfiguration requirements and the stereochemical course of these nucleotidyl transfer reactions. The extract utilizes (SP)-alpha-S-UTP for both 3' and internal U incorporation into substrate RNA. The internal as well as the 3' incorporation of (SP)-alpha-S-UTP proceeds via inversion of the stereoconfiguration. Furthermore, internal U incorporation does not occur at sites containing thiophosphodiesters of the RP configuration. Our results are compatible with an enzyme cascade model for this in vitro U insertion activity involving sequential endonuclease and uridylyl transferase directly from UTP and RNA ligase steps and are incompatible with models involving the transfer of U residues from the 3' ends of guide RNAs.

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 Complexes from Trypanosoma brucei that exhibit deletion editing and other editing-associated properties.

1996 ◽  
Vol 16 (4) ◽  
pp. 1410-1418 ◽  
Author(s):  
R A Corell ◽  
L K Read ◽  
G R Riley ◽  
J K Nellissery ◽  
T E Allen ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Rna Editing ◽  
Uv Treatment ◽  
Rna Ligase ◽  
Guide Rna ◽  
Ribonucleoprotein Complexes ◽  
Multicomponent Complex ◽  
Editing Activity ◽  
Atpase 6

Transcripts from many mitochondrial genes in kinetoplastids undergo RNA editing, a posttranscriptional process which inserts and deletes uridines. By assaying for deletion editing in vitro, we found that the editing activity from Trypanosoma brucei mitochondrial lysates (S.D. Seiwert and K.D. Stuart), Science 266:114-117,1994) sediments with a peak of approximately 20S. RNA helicase, terminal uridylyl transferase, RNA ligase, and adenylation activities, which may have a role in editing, cosediment in a broad distribution, with most of each activity at 35 to 40S. Most ATPase 6 (A6) guide RNA and unedited A6 mRNA sediments at 20 to 30S, with some sedimenting further into the gradient, while most edited A6 mRNA sediments at >35S. Several mitochondrial proteins which cross-link specifically with guide RNA upon UV treatment also sediment in glycerol gradients. Notably, a 65-kDa protein sediments primarily at approximately 20S, a 90-kDa protein sediments at 35 to 40S, and a 25-kDa protein is present at <10S. Most ribonucleoprotein complexes that form with gRNA in vitro sediment at 10 to 20S, except for one, which sediments at 30 to 45S. These results suggest that RNA editing takes place within a multicomponent complex. The potential functions of and relationships between the 20S and 35 to 40S complexes are discussed.

scholarly journals Guide RNA requirement for editing-site-specific endonucleolytic cleavage of preedited mRNA by mitochondrial ribonucleoprotein particles in Trypanosoma brucei.

1997 ◽  
Vol 17 (9) ◽  
pp. 5377-5385 ◽  
Author(s):  
B K Adler ◽  
S L Hajduk
Keyword(s):  
Cytochrome B ◽  
Editing Site ◽  
Mitochondrial Rna ◽  
Ample Evidence ◽  
Site Specific ◽  
Rna Ligase ◽  
Guide Rna ◽  
Guide Rnas ◽  
Endonucleolytic Cleavage

RNA editing in trypanosome mitochondria entails the posttranscriptional internal addition and occasional deletion of uridines from precursor mRNAs. Ample evidence exists to show that the information specifying the site and number of uridines added or deleted comes from small, mitochondrially encoded guide RNAs (gRNAs). More recent work indicates that the process involves an enzymatic cascade, initiating with an endonucleolytic cleavage of the pre-mRNA at an editing site. The cleaved editing site can undergo uridine (U) addition to or deletion from the 3' end of the 5' fragment via a mitochondrial terminal uridylyl transferase (TUTase) or terminal uridylyl exonuclease, respectively. Mitochondrial RNA ligase subsequently rejoins the mRNA. Activities to carry out these processes have been found in trypanosome mitochondria, including an editing-site-specific endonuclease activity which cleaves preedited but not edited mRNAs. We have found that this enzymatic activity cosediments with the same 19S ribonucleoprotein particle previously shown to contain TUTase, RNA ligase, and gRNAs and remains stable after salt treatment. Depletion of endogenous cytochrome b gRNAs by the addition of complementary oligonucleotides in vitro completely inhibits editing-site cleavage of synthetic preedited cytochrome b mRNA. The addition of synthetic cognate gRNA for cytochrome b but not unrelated small RNA restores editing-site cleavage. These studies show that in addition to specifying the site and number of uridines added or deleted, gRNAs provide the necessary information for cleavage by the editing-site-specific endonuclease.

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 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.

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