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

2008 ◽  
Vol 28 (22) ◽  
pp. 6939-6953 ◽  
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.

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 A UDP-GlcNAc : βGal β1-6 GlcNAc transferase involved in bloodstream form N-glycan and procyclic form GPI anchor elaboration in Trypanosoma brucei.

2021 ◽  
Author(s):  
Samuel Martin Duncan ◽  
Rupa Nagar ◽  
Manuela Damerow ◽  
Dmitry V. Yashunsky ◽  
Benedetta Buzzi ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Bloodstream Form ◽  
Wild Type ◽  
Dual Roles ◽  
Gpi Anchor ◽  
Procyclic Form ◽  
Life Cycle Stages ◽  
Glcnac Transferase

Trypanosoma brucei has large carbohydrate extensions on its N-linked glycans and glycosylphosphatidylinositol (GPI) anchors in its bloodstream form (BSF) and procyclic form (PCF), respectively. The parasites glycoconjugate repertoire suggests at least 38 glycosyltransferase (GT) activities, 16 of which are unknown. Here, we probe the function(s) of a putative β3GT gene, TbGT10. The BSF null mutant is viable in vitro and in vivo and can differentiate into PCF, demonstrating non-essentiality. However, the absence of TbGT10 led to impaired elaboration of N-glycans and GPI anchor sidechains in BSF and PCF parasites, respectively. Glycosylation defects include reduced BSF glycoprotein binding to ricin and to monoclonal antibodies mAb139 and mAbCB1. The latter bind a carbohydrate epitope of lysosomal glycoprotein p67 that we show here, using synthetic glycans, consists of (-6Gal1-4GlcNAc1-)≥4 poly-N-acetyllactosamine repeats. Methylation linkage analysis of Pronase glycopeptides isolated from BSF wild-type and TbGT10 null parasites show a reduction in 6-O-substituted- and 3,6-di-O-substituted-Gal residues. Together, these data suggest that TbGT10 encodes a UDP-GlcNAc : βGal β1-6 GlcNAc-transferase active in both BSF and PCF life-cycle stages elaborating complex N-glycans and GPI sidechains, respectively. The β1-6 specificity of this β3GT gene product and its dual roles in N-glycan and GPI glycan elaboration are notable.

scholarly journals An Arginine-Glycine-Rich RNA Binding Protein Impacts the Abundance of Specific mRNAs in the Mitochondria of Trypanosoma brucei

2014 ◽  
Vol 14 (2) ◽  
pp. 149-157 ◽  
Author(s):  
Natalie M. McAdams ◽  
Michelle L. Ammerman ◽  
Julee Nanduri ◽  
Kaylen Lott ◽  
John C. Fisk ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Rna Editing ◽  
Rna Binding ◽  
Mitochondrial Gene ◽  
Mitochondrial Rna ◽  
Insect Vector ◽  
Sequence Specificity ◽  
Content Type

ABSTRACT In kinetoplastid parasites, regulation of mitochondrial gene expression occurs posttranscriptionally via RNA stability and RNA editing. In addition to the 20S editosome that contains the enzymes required for RNA editing, a dynamic complex called the mitochondrial RNA binding 1 (MRB1) complex is also essential for editing. Trypanosoma brucei RGG3 (TbRGG3) was originally identified through its interaction with the guide RNA-associated proteins 1 and 2 (GAP1/2), components of the MRB1 complex. Both the arginine-glycine-rich character of TbRGG3, which suggests a function in RNA binding, and its interaction with MRB1 implicate TbRGG3 in mitochondrial gene regulation. Here, we report an in vitro and in vivo characterization of TbRGG3 function in T. brucei mitochondria. We show that in vitro TbRGG3 binds RNA with broad sequence specificity and has the capacity to modulate RNA-RNA interactions. In vivo , inducible RNA interference (RNAi) studies demonstrate that TbRGG3 is essential for proliferation of insect vector stage T. brucei . TbRGG3 ablation does not cause a defect in RNA editing but, rather, specifically affects the abundance of two preedited transcripts as well as their edited counterparts. Protein-protein interaction studies show that TbRGG3 associates with GAP1/2 apart from the remainder of the MRB1 complex, as well as with several non-MRB1 proteins that are required for mitochondrial RNA editing and/or stability. Together, these studies demonstrate that TbRGG3 is an essential mitochondrial gene regulatory factor that impacts the stabilities of specific RNAs.

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 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 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 Trypanosoma brucei RNA Editing Complex

2003 ◽  
Vol 23 (21) ◽  
pp. 7909-7919 ◽  
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.

scholarly journals Functional dissection of the catalytic mechanism of mammalian RNA polymerase II

2005 ◽  
Vol 83 (4) ◽  
pp. 497-504 ◽  
Author(s):  
Benoit Coulombe ◽  
Marie-France Langelier
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Catalytic Mechanism ◽  
Mutational Analysis ◽  
Structural Elements ◽  
Catalytic Mechanisms ◽  
Rnap Ii ◽  
Affinity Peptide

High resolution X-ray crystal structures of multisubunit RNA polymerases (RNAP) have contributed to our understanding of transcriptional mechanisms. They also provided a powerful guide for the design of experiments aimed at further characterizing the molecular stages of the transcription reaction. Our laboratory used tandem-affinity peptide purification in native conditions to isolate human RNAP II variants that had site-specific mutations in structural elements located strategically within the enzyme's catalytic center. Both in vitro and in vivo analyses of these mutants revealed novel features of the catalytic mechanisms involving this enzyme.Key words: RNA polymerase II, transcriptional mechanisms, mutational analysis, mRNA synthesis.

scholarly journals Important Role for Phylogenetically Invariant PP2Acα Active Site and C-Terminal Residues Revealed by Mutational Analysis in Saccharomyces cerevisiae

Genetics ◽  
2000 ◽  
Vol 156 (1) ◽  
pp. 21-29 ◽  
Author(s):  
David R H Evans ◽  
Brian A Hemmings
Keyword(s):  
Protein Interactions ◽  
Active Site ◽  
Protein Function ◽  
Mutational Analysis ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Active Site Residues ◽  
Catalytic Function

Abstract PP2A is a central regulator of eukaryotic signal transduction. The human catalytic subunit PP2Acα functionally replaces the endogenous yeast enzyme, Pph22p, indicating a conservation of function in vivo. Therefore, yeast cells were employed to explore the role of invariant PP2Ac residues. The PP2Acα Y127N substitution abolished essential PP2Ac function in vivo and impaired catalysis severely in vitro, consistent with the prediction from structural studies that Tyr-127 mediates substrate binding and its side chain interacts with the key active site residues His-118 and Asp-88. The V159E substitution similarly impaired PP2Acα catalysis profoundly and may cause global disruption of the active site. Two conditional mutations in the yeast Pph22p protein, F232S and P240H, were found to cause temperature-sensitive impairment of PP2Ac catalytic function in vitro. Thus, the mitotic and cell lysis defects conferred by these mutations result from a loss of PP2Ac enzyme activity. Substitution of the PP2Acα C-terminal Tyr-307 residue by phenylalanine impaired protein function, whereas the Y307D and T304D substitutions abolished essential function in vivo. Nevertheless, Y307D did not reduce PP2Acα catalytic activity significantly in vitro, consistent with an important role for the C terminus in mediating essential protein-protein interactions. Our results identify key residues important for PP2Ac function and characterize new reagents for the study of PP2A in vivo.

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