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 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 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 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.

Assembly of mitochondrial ribonucleoprotein complexes involves specific guide RNA (gRNA)-binding proteins and gRNA domains but does not require preedited mRNA

1994 ◽  
Vol 14 (4) ◽  
pp. 2629-2639
Author(s):  
L K Read ◽  
H U Göringer ◽  
K Stuart
Keyword(s):  
Complex Formation ◽  
Rna Editing ◽  
Binding Proteins ◽  
Macromolecular Complex ◽  
Differential Distribution ◽  
Guide Rna ◽  
Guide Rnas ◽  
Ribonucleoprotein Complexes ◽  
Recognition Elements

RNA editing in kinetoplastids probably employs a macromolecular complex, the editosome, that is likely to include the guide RNAs (gRNAs) which specify the edited sequence. Specific ribonucleoprotein (RNP) complexes which form in vitro with gRNAs (H. U. Göringer, D. J. Koslowsky, T. H. Morales, and K. D. Stuart, Proc. Natl. Acad. Sci. USA, in press) are potential editosomes or their precursors. We find that several factors are important for in vitro formation of these RNP complexes and identify specific gRNA-binding proteins present in the complexes. Preedited mRNA promotes the in vitro formation of the four major gRNA-containing RNP complexes under some conditions but is required for the formation of only a subcomponent of one complex. The 5' gRNA sequence encompassing the RYAYA and anchor regions and the 3' gRNA oligo(U) tail are both important in complex formation, since their deletion results in a dramatic decrease of some complexes and the absence of others. UV cross-linking experiments identify several proteins which are in contact with gRNA and preedited mRNA in mitochondrial extracts. Proteins of 25 and 90 kDa are highly specific for gRNAs, and the 90-kDa protein binds specifically to gRNA oligo(U) tails. The gRNA-binding proteins exhibit a differential distribution between the four in vitro-formed complexes. These experiments reveal several proteins potentially involved in RNA editing and indicate that multiple recognition elements in gRNAs are used for complex formation.

Modification of Trypanosoma brucei mitochondrial rRNA by posttranscriptional 3' polyuridine tail formation

1991 ◽  
Vol 11 (12) ◽  
pp. 5878-5884
Author(s):  
B K Adler ◽  
M E Harris ◽  
K I Bertrand ◽  
S L Hajduk
Keyword(s):  
Trypanosoma Brucei ◽  
Rna Editing ◽  
Total Cell ◽  
Metabolic Labeling ◽  
Mechanism Of Formation ◽  
Guide Rnas ◽  
Mitochondrial Rrna ◽  
Mitochondrial Transcripts ◽  
Reading Frames

Trypanosoma brucei mitochondrial transcripts can be posttranscriptionally processed by uridine addition or deletion. With editing of mRNAs, uridine addition and deletion create precisely altered reading frames. The addition of nonencoded uridines to mitochondrial guide RNAs results in a less precise modification. Although uridines are specifically added to the 3' termini, their number varies, which results in heterogeneous oligo(U) tails on guide RNAs. In this paper, we show that the mitochondrial 9S and 12S rRNAs are also modified by uridine addition. These modifications appear to have aspects in common with both RNA editing and oligo(U) tail formation. Metabolic labeling studies with intact mitochondria and [alpha-32P]UTP, in the absence of transcription, demonstrated the posttranscriptional timing of the event. T1 RNase comparison analyses of cytidine 3',5'-[5'-32P]biphosphate 3'-end-labeled and [alpha-32P]UTP metabolically labeled rRNAs, along with direct RNA sequencing of the 3' termini, identified the site of uridine addition and revealed the creation of an oligo(U) tail for both rRNAs. 12S and 9S rRNAs hybrid selected from total cell RNA exhibited the same modification, demonstrating the presence of this processing in vivo. Moreover, only 3'-poly(U)-tailed 9S and 12S rRNAs were detected in total cellular and mitochondrial RNAs, which suggests that they are the most abundant and probable mature forms. The 12S and 9S rRNA oligo(U) tails differed significantly from each other, with the 12S having a heterogeneous tail of 2 to 17 uridines and the 9S having a tail of precisely 11 uridines. The mechanism of formation and the function of the rRNA poly(U) tails remain to be determined.

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 Association of Guide RNA Binding Protein gBP21 with Active RNA Editing Complexes in Trypanosoma brucei

1998 ◽  
Vol 18 (10) ◽  
pp. 6014-6022 ◽  
Author(s):  
Thomas E. Allen ◽  
Stefan Heidmann ◽  
RoseMary Reed ◽  
Peter J. Myler ◽  
H. Ulrich Göringer ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Rna Editing ◽  
Rna Binding ◽  
Binding Protein ◽  
Protein A ◽  
Mitochondrial Fraction ◽  
Macromolecular Complex ◽  
Guide Rna ◽  
Ribonucleoprotein Complexes

ABSTRACT RNA editing in Trypanosoma brucei mitochondria produces mature mRNAs by a series of enzyme-catalyzed reactions that specifically insert or delete uridylates in association with a macromolecular complex. Using a mitochondrial fraction enriched for in vitro RNA editing activity, we produced several monoclonal antibodies that are specific for a 21-kDa guide RNA (gRNA) binding protein initially identified by UV cross-linking. Immunofluorescence studies localize the protein to the mitochondrion, with a preference for the kinetoplast. The antibodies cause a supershift of previously identified gRNA-specific ribonucleoprotein complexes and immunoprecipitate in vitro RNA editing activities that insert and delete uridylates. The immunoprecipitated material also contains gRNA-specific endoribonuclease, terminal uridylyltransferase, and RNA ligase activities as well as gRNA and both edited and unedited mRNA. The immunoprecipitate contains numerous proteins, of which the 21-kDa protein, a 90-kDa protein, and novel 55- and 16-kDa proteins can be UV cross-linked to gRNA. These studies indicate that the 21-kDa protein associates with the ribonucleoprotein complex (or complexes) that catalyze RNA editing.

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 Robust RNA editing via recruitment of endogenous ADARs using circular guide RNAs

2021 ◽  
Author(s):  
Dhruva Katrekar ◽  
James Yen ◽  
Yichen Xiang ◽  
Anushka Saha ◽  
Dario Meluzzi ◽  
...  
Keyword(s):  
Rna Editing ◽  
Transcript Level ◽  
Rnase H ◽  
Type I ◽  
Rna Molecules ◽  
Guide Rnas ◽  
Cellular Machinery ◽  
Dna Vectors

ABSTRACTAkin to short-hairpin RNAs and antisense oligonucleotides which efficaciously recruit endogenous cellular machinery such as Argonaute and RNase H to enable targeted RNA knockdown, simple long antisense guide RNAs (1) can recruit endogenous adenosine deaminases acting on RNA (ADARs) to enable programmable A-to-I RNA editing, without requiring co-delivery of any exogenous proteins. This approach is highly specific, however the efficiency is typically lower than observed with enzyme overexpression. Conjecturing this was due in part to the short half-life and residence times of guide RNAs, here we engineer highly stable circular ADAR recruiting guide RNAs (cadRNAs), which can be delivered not only by genetically encoding on DNA vectors, but also via transfection of RNA molecules transcribed in vitro. Using these cadRNAs, we observed robust RNA editing across multiple sites and cell lines, in both untranslated and coding regions of RNAs, vastly improved efficiency and durability of RNA editing, and high transcriptome-wide specificity. High transcript-level specificity was achieved by further engineering to reduce bystander editing. Additionally, in vivo delivery of cadRNAs via adeno-associated viruses (AAVs) enabled robust 38% RNA editing of the mPCSK9 transcript in C57BL/6J mice livers, and 12% UAG-to-UGG RNA correction of the amber nonsense mutation in the IDUA-W392X mouse model of mucopolysaccharidosis type I-Hurler (MPS I-H) syndrome. Taken together, cadRNAs enable efficacious programmable RNA editing with application across diverse protein modulation and gene therapeutic settings.

scholarly journals Specific cleavage of pre-edited mRNAs in trypanosome mitochondrial extracts

1992 ◽  
Vol 12 (6) ◽  
pp. 2591-2598
Author(s):  
M Harris ◽  
C Decker ◽  
B Sollner-Webb ◽  
S Hajduk
Keyword(s):  
Cytochrome Oxidase ◽  
Rna Editing ◽  
Mitochondrial Gene ◽  
Cytochrome Oxidase Subunit ◽  
Oxidase Subunit ◽  
Editing Domain ◽  
Posttranscriptional Processing ◽  
Processing Event

RNA editing in Trypanosoma brucei is a posttranscriptional processing event that results in the addition and deletion of uridine residues within several mitochondrial mRNAs. We have examined reactions involving pre-edited precursor RNAs in vitro. In this study, we report specific cleavage of pre-edited cytochrome b (CYb), cytochrome oxidase subunit II (COII), and cytochrome oxidase subunit III (COIII) mRNAs when incubated with T. brucei mitochondrial extracts. The pre-edited CYb RNA was cleaved near the 3'-most uridine addition sites, within the region where editing would be expected to commence. Pre-edited COII mRNA was similarly cleaved adjacent to its small editing domain, while pre-edited COIII RNA was cleaved at multiple sites in the region where uridine addition and deletion occurs in vivo. In contrast, edited versions of CYb, COII, and COIII RNAs were not cleaved within the editing domains. Such differential cleavage of the edited and pre-edited forms of these mRNAs suggests either a direct involvement in RNA editing or involvement in another aspect of mitochondrial gene expression requiring cleavage of pre-edited RNAs.

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