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.

scholarly journals 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.

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 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 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 Trypanosome RNA Editing: Simple Guide RNA Features Enhance U Deletion 100-Fold

2001 ◽  
Vol 21 (3) ◽  
pp. 884-892 ◽  
Author(s):  
Jorge Cruz-Reyes ◽  
Alevtina Zhelonkina ◽  
Laura Rusche ◽  
Barbara Sollner-Webb
Keyword(s):  
Rna Editing ◽  
Central Region ◽  
Marked Improvement ◽  
Base Pairing ◽  
Guide Rna ◽  
Guide Rnas ◽  
Evolutionary Forces

ABSTRACT Trypanosome RNA editing is a massive processing of mRNA by U deletion and U insertion, directed by trans-acting guide RNAs (gRNAs). A U deletion cycle and a U insertion cycle have been reproduced in vitro using synthetic ATPase (A6) pre-mRNA and gRNA. Here we examine which gRNA features are important for this U deletion. We find that, foremost, this editing depends critically on the single-stranded character of a few gRNA and a few mRNA residues abutting the anchor duplex, a feature not previously appreciated. That plus any base-pairing sequence to tether the upstream mRNA are all the gRNA needs to direct unexpectedly efficient in vitro U deletion, using either the purified editing complex or whole extract. In fact, our optimized gRNA constructs support faithful U deletion up to 100 times more efficiently than the natural gRNA, and they can edit the majority of mRNA molecules. This is a marked improvement of in vitro U deletion, in which previous artificial gRNAs were no more active than natural gRNA and the editing efficiencies were at most a few percent. Furthermore, this editing is not stimulated by most other previously noted gRNA features, including its potential ligation bridge, 3′ OH moiety, any U residues in the tether, the conserved structure of the central region, or proteins that normally bind these regions. Our data also have implications about evolutionary forces active in RNA editing.

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.

DEVELOPMENT OF APPROACHES FOR DETECTION OF GENOME-INTEGRATED PROVIRAL DNA OF THE HUMAN IMMUNODEFICIENCY VIRUS (HIV-1) IN ULTRA LOW CONCENTRATIONS USING THE CRISPR/CAS SYSTEM

2020 ◽  
Author(s):  
M.A. Tyumentseva ◽  
◽  
A.I. Tyumentsev ◽  
V.G. Akimkin ◽  
◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Health Monitoring ◽  
Biological Samples ◽  
Proviral Dna ◽  
Guide Rnas ◽  
Ribonucleoprotein Complexes ◽  
Low Concentrations ◽  
Immunodeficiency Virus ◽  
Hiv 1

For the effective functioning of supervisory and health monitoring services, it is necessary to introduce modern molecular technologies into their practice. Therefore, the task of developing new effective methods for detecting pathogen, for example HIV, based on CRISPR/CAS genome editing systems, remains urgent. In the present work, guide RNAs and specific oligonucleotides were developed for preliminary amplification of highly conserved regions of the HIV-1 genome. The developed guide RNAs make it possible to detect single copies of HIV-1 proviral DNA in vitro as part of CRISPR/CAS ribonucleoprotein complexes in biological samples after preliminary amplification.

ASSESSMENT OF EFFICIENCY AND OFF-TARGET ACTIVITY OF CRISPR/CAS RIBONUCLEOPROTEIN COMPLEXES

2020 ◽  
Author(s):  
Y.V. Mikhaylova ◽  
◽  
M.A. Tyumentseva ◽  
A.A. Shelenkov ◽  
Y.G. Yanushevich ◽  
...  
Keyword(s):  
High Sensitivity ◽  
Correct Choice ◽  
Target Sequence ◽  
Dna Breaks ◽  
Gene Encoding ◽  
Guide Rna ◽  
Guide Rnas ◽  
Ribonucleoprotein Complexes ◽  
Chemokine Receptor Ccr5 ◽  
Target Activity

In this study, we assessed the efficiency and off-target activity of the CRISPR/CAS complex with one of the selected guide RNAs using the CIRCLE-seq technology. The gene encoding the human chemokine receptor CCR5 was used as a target sequence for genome editing. The results of this experiment indicate the correct choice of the guide RNA and efficient work of the CRISPR- CAS ribonucleoprotein complex used. CIRCLE-seq technology has shown high sensitivity compared to bioinformatic methods for predicting off-target activity of CRISPR/CAS complexes. We plan to evaluate the efficiency and off-target activity of CRISPR/CAS ribonucleoprotein complexes with other guide RNAs by slightly adjusting the CIRCLE-seq-technology protocol in order to reduce nonspecific DNA breaks and increase the number of reliable reads.

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.

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