scholarly journals Pentatricopeptide repeat poly(A) binding protein from mitochondria of trypanosomes

2018 ◽  
Author(s):  
Mikhail V. Mesitov ◽  
Tian Yu ◽  
Takuma Suematsu ◽  
Francois M. Sement ◽  
Liye Zhang ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Rna Editing ◽  
Mechanism Of Action ◽  
Binding Protein ◽  
Check Point ◽  
Pentatricopeptide Repeat ◽  
Transcript Stability ◽  
Internal Sequence ◽  
Quality Check ◽  
Editing Process

AbstractIn Trypanosoma brucei, most mitochondrial mRNAs undergo U-insertion/deletion editing, and 3′ adenylation and uridylation. The internal sequence changes and terminal extensions are coordinated: Pre-editing addition of the short (A) tail protects the edited transcript against 3′-5′ degradation, while post-editing A/U-tailing renders mRNA competent for ribosome recruitment. Participation of a poly(A) binding protein (PABP) in coupling of editing and 3′ modification processes has been inferred, but its identity and mechanism of action remained elusive. We report identification of KPAF4, a pentatricopeptide repeat-containing PABP which sequesters the A-tail and impedes exonucleolytic degradation. Conversely, KPAF4 inhibits uridylation of A-tailed transcripts and, therefore, premature A/U-tailing of partially-edited mRNAs. This quality check point prevents translation of incompletely edited mRNAs. Our findings also implicate the RNA editing substrate binding complex (RESC) in mediating the interaction between the 5′ end bound pyrophosphohydrolase MERS1 and 3′ end associated KPAF4 to enable mRNA circularization. This event is critical for transcript stability during the editing process.

scholarly journals Poly(A) binding KPAF4/5 complex stabilizes kinetoplast mRNAs in Trypanosoma brucei

2020 ◽  
Vol 48 (15) ◽  
pp. 8645-8662
Author(s):  
Inna Aphasizheva ◽  
Tian Yu ◽  
Takuma Suematsu ◽  
Qiushi Liu ◽  
Mikhail V Mesitov ◽  
...  
Keyword(s):  
Quality Control ◽  
Trypanosoma Brucei ◽  
Mrna Stability ◽  
Critical Element ◽  
Pentatricopeptide Repeat ◽  
Present Evidence ◽  
Mrna Stabilization ◽  
Translational Activation ◽  
Pyrophosphate Hydrolysis ◽  
Editing Process

Abstract In Trypanosoma brucei, mitochondrial pre-mRNAs undergo 3′-5′ exonucleolytic processing, 3′ adenylation and uridylation, 5′ pyrophosphate removal, and, often, U-insertion/deletion editing. The 3′ modifications are modulated by pentatricopeptide repeat (PPR) Kinetoplast Polyadenylation Factors (KPAFs). We have shown that KPAF3 binding to the 3′ region stabilizes properly trimmed transcripts and stimulates their A-tailing by KPAP1 poly(A) polymerase. Conversely, poly(A) binding KPAF4 shields the nascent A-tail from uridylation and decay thereby protecting pre-mRNA upon KPAF3 displacement by editing. While editing concludes in the 5′ region, KPAF1/2 dimer induces A/U-tailing to activate translation. Remarkably, 5′ end recognition and pyrophosphate hydrolysis by the PPsome complex also contribute to mRNA stabilization. Here, we demonstrate that KPAF4 functions as a heterodimer with KPAF5, a protein lacking discernable motifs. We show that KPAF5 stabilizes KPAF4 to enable poly(A) tail recognition, which likely leads to mRNA stabilization during the editing process and impedes spontaneous translational activation of partially-edited transcripts. Thus, KPAF4/5 represents a poly(A) binding element of the mitochondrial polyadenylation complex. We present evidence that RNA editing substrate binding complex bridges the 5′ end-bound PPsome and 3′ end-bound polyadenylation complexes. This interaction may enable mRNA circularization, an apparently critical element of mitochondrial mRNA stability and quality control.

scholarly journals Disruption of a gene encoding a novel mitochondrial DEAD-box protein in Trypanosoma brucei affects edited mRNAs.

1997 ◽  
Vol 17 (9) ◽  
pp. 4895-4903 ◽  
Author(s):  
A Missel ◽  
A E Souza ◽  
G Nörskau ◽  
H U Göringer
Keyword(s):  
Trypanosoma Brucei ◽  
Rna Editing ◽  
Molecular Level ◽  
Mitochondrial Proteins ◽  
Null Mutant ◽  
Gene Encoding ◽  
Guide Rnas ◽  
Dead Box ◽  
Posttranscriptional Processing ◽  
Editing Process

The majority of mitochondrial pre-mRNAs in kinetoplastid protozoa such as Trypanosoma, Leishmania, and Crithidia are substrates of a posttranscriptional processing reaction referred to as RNA editing. The process results in the insertion and, to a lesser extent, deletion of uridylates, thereby completing the informational content of the mRNAs. The specificity of the RNA editing reaction is provided by guide RNAs (gRNAs), which serve as templates for the editing apparatus. In addition, the process relies on mitochondrial proteins, presumably acting within a high-molecular-mass ribonucleoprotein complex. Although several enzymatic activities have been implicated in the editing process, no protein has been identified to date. Here we report the identification of a novel mitochondrial DEAD-box protein, which we termed mHel61p. Disruption of the mHEL61 alleles in insect-stage Trypanosoma brucei cells resulted in a reduced growth rate phenotype. On a molecular level, the null mutant showed significantly reduced amounts of edited mRNAs, whereas never-edited and nuclear mRNAs were unaffected. Reexpression of mHel61p in the knockout cell line restored the ability to efficiently synthesize edited mRNAs. The results suggest an involvement of mHel61p in the control of the abundance of edited mRNAs and thus reveal a novel function for DEAD-box proteins.

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 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 The 27 kDa Trypanosoma brucei Pentatricopeptide Repeat Protein is a G-tract Specific RNA Binding Protein

2018 ◽  
Vol 8 (1) ◽  
Author(s):  
Pakoyo F. Kamba ◽  
David A. Dickson ◽  
Neil A. White ◽  
Jennifer L. Ekstrom ◽  
Donna J. Koslowsky ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Pentatricopeptide Repeat ◽  
Repeat Protein ◽  
Pentatricopeptide Repeat Protein

scholarly journals Pentatricopeptide repeat poly(A) binding protein KPAF4 stabilizes mitochondrial mRNAs in Trypanosoma brucei

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Mikhail V. Mesitov ◽  
Tian Yu ◽  
Takuma Suematsu ◽  
Francois M. Sement ◽  
Liye Zhang ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Binding Protein ◽  
Pentatricopeptide Repeat

The RNA editing process in Trypanosoma brucei

1993 ◽  
Vol 4 (4) ◽  
pp. 251-260 ◽  
Author(s):  
Kenneth Stuart
Keyword(s):  
Trypanosoma Brucei ◽  
Rna Editing ◽  
Editing Process

scholarly journals RiceMPR25encodes a pentatricopeptide repeat protein and is essential for RNA editing ofnad5transcripts in mitochondria

2012 ◽  
Vol 72 (3) ◽  
pp. 450-460 ◽  
Author(s):  
Takushi Toda ◽  
Sota Fujii ◽  
Ko Noguchi ◽  
Tomohiko Kazama ◽  
Kinya Toriyama
Keyword(s):  
Rna Editing ◽  
Pentatricopeptide Repeat ◽  
Repeat Protein ◽  
Pentatricopeptide Repeat Protein

scholarly journals TbUNC119 and Its Binding Protein Complex Are Essential for Propagation, Motility, and Morphogenesis of Trypanosoma brucei Procyclic Form Cells

PLoS ONE ◽  
2010 ◽  
Vol 5 (12) ◽  
pp. e15577 ◽  
Author(s):  
Shigeru Ohshima ◽  
Mitsuko Ohashi-Suzuki ◽  
Yutaka Miura ◽  
Yoshisada Yabu ◽  
Noriko Okada ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Protein Complex ◽  
Binding Protein ◽  
Procyclic Form
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