Uridine insertion/deletion (U-indel) editing of mitochondrial mRNA, unique to the protistan class Kinetoplastea, generates canonical as well as potentially non-productive editing events. While the molecular machinery and the role of the guide (g) RNAs that provide required information for U-indel editing are well understood, little is known about the forces underlying its apparently error-prone nature. Analysis of a gRNA:mRNA pair allows the dissection of editing events in a given position of a given mitochondrial transcript. A complete gRNA dataset, paired with a fully characterized mRNA population that includes non-canonically edited transcripts, would allow such an analysis to be performed globally across the mitochondrial transcriptome. To achieve this, we have assembled 67 minicircles of the insect parasite Leptomonas pyrrhocoris, with each minicircle typically encoding one gRNA located in one of two similar-sized units of different origin. From this relatively narrow set of annotated gRNAs, we have dissected all identified mitochondrial editing events in L. pyrrhocoris, the strains of which dramatically differ in the abundance of individual minicircle classes. Our results support a model in which a multitude of editing events are driven by a limited set of gRNAs, with individual gRNAs possessing an inherent ability to guide canonical and non-canonical editing.
- MeSH
- editace RNA * MeSH
- fylogeneze MeSH
- genom protozoální * MeSH
- guide RNA, Kinetoplastida chemie metabolismus MeSH
- messenger RNA metabolismus MeSH
- RNA mitochondriální metabolismus MeSH
- transkriptom MeSH
- Trypanosomatina genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Small interfering RNAs (siRNAs) that silence genes of infectious diseases are potentially potent drugs. A continuing obstacle for siRNA-based drugs is how to improve their efficacy for adequate dosage. To overcome this obstacle, the interactions of antiviral siRNAs, tested in vivo, were computationally examined within the RNA-induced silencing complex (RISC). Thermodynamics data show that a persistent RISC cofactor is significantly more exothermic for effective antiviral siRNAs than their ineffective counterparts. Detailed inspection of viral RNA secondary structures reveals that effective antiviral siRNAs target hairpin or pseudoknot loops. These structures are critical for initial RISC interactions since they partially lack intramolecular complementary base pairing. Importing two temporary RISC cofactors from magnesium-rich hairpins and/or pseudoknots then kickstarts full RNA hybridization and hydrolysis. Current siRNA design guidelines are based on RNA primary sequence data. Herein, the thermodynamics of RISC cofactors and targeting magnesium-rich RNA secondary structures provide additional guidelines for improving siRNA design.
- MeSH
- Argonaut proteiny chemie metabolismus MeSH
- guide RNA, Kinetoplastida chemie metabolismus MeSH
- hořčík MeSH
- hybridizace nukleových kyselin MeSH
- hydrolýza MeSH
- komplex RISC MeSH
- konformace nukleové kyseliny MeSH
- krystalografie rentgenová MeSH
- lidé MeSH
- malá interferující RNA chemie genetika metabolismus MeSH
- messenger RNA chemie metabolismus MeSH
- metoda Monte Carlo MeSH
- párování bází MeSH
- racionální návrh léčiv MeSH
- RNA interference * MeSH
- RNA virová antagonisté a inhibitory chemie MeSH
- simulace molekulového dockingu MeSH
- termodynamika MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Lactococcus lactis is a food-grade lactic acid bacterium that is used in the dairy industry as a cell factory and as a host for recombinant protein expression. The nisin-controlled inducible expression (NICE) system is frequently applied in L. lactis; however new tools for its genetic modification are highly desirable. In this work NICE was adapted for dual protein expression. Plasmid pNZDual, that contains two nisin promoters and multiple cloning sites (MCSs), and pNZPolycist, that contains a single nisin promoter and two MCSs separated by the ribosome binding site, were constructed. Genes for the infrared fluorescent protein and for the human IgG-binding DARPin were cloned in all possible combinations to assess the protein yield. The dual promoter plasmid pNZDual enabled balanced expression of the two model proteins. It was exploited for the development of a single-plasmid inducible CRISPR-Cas9 system (pNZCRISPR) by using a nisin promoter, first to drive Cas9 expression and, secondly, to drive single guide RNA transcription. sgRNAs against htrA and ermR directed Cas9 against genomic or plasmid DNA and caused changes in bacterial growth and survival. Replacing Cas9 by dCas9 enabled CRISPR interference-mediated silencing of the upp gene. The present study introduces a new series of plasmids for advanced genetic modification of lactic acid bacterium L. lactis.
- MeSH
- antibakteriální látky farmakologie MeSH
- CRISPR-Cas systémy MeSH
- editace genu metody MeSH
- fermentace MeSH
- genetické inženýrství metody MeSH
- genom bakteriální * MeSH
- guide RNA, Kinetoplastida genetika metabolismus MeSH
- imunoglobulin G genetika metabolismus MeSH
- klonování DNA MeSH
- Lactococcus lactis genetika metabolismus MeSH
- lidé MeSH
- methyltransferasy genetika metabolismus MeSH
- nisin farmakologie MeSH
- plazmidy chemie metabolismus MeSH
- promotorové oblasti (genetika) účinky léků MeSH
- proteiny tepelného šoku genetika metabolismus MeSH
- regulace genové exprese u bakterií * MeSH
- rekombinantní fúzní proteiny genetika metabolismus MeSH
- transgeny * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Our understanding of kinetoplastid RNA (kRNA) editing has centered on this paradigm: guide RNAs (gRNAs) provide a blueprint for uridine insertion/deletion into mitochondrial mRNAs by the RNA editing core complex (RECC). The characterization of constituent subunits of the mitochondrial RNA-binding complex 1 (MRB1) implies that it too is vital to the editing process. The recently elucidated MRB1 architecture will be instrumental in putting functional data from individual subunits into context. Our model depicts two functions for MRB1: mediating multi-round kRNA editing by coordinating the exchange of multiple gRNAs required by RECC to edit lengthy regions of mRNAs, and then linking kRNA editing with other RNA processing events.
- MeSH
- editace RNA * MeSH
- guide RNA, Kinetoplastida metabolismus MeSH
- Kinetoplastida genetika metabolismus MeSH
- messenger RNA metabolismus MeSH
- protozoální proteiny metabolismus MeSH
- RNA protozoální genetika metabolismus MeSH
- Trypanosoma brucei brucei genetika metabolismus MeSH
- uridin metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
- Research Support, N.I.H., Extramural MeSH
Trypanosoma brucei brucei has two distinct developmental stages, the procyclic stage in the insect and the bloodstream stage in the mammalian host. The significance of each developmental stage is punctuated by specific changes in metabolism. In the insect, T. b. brucei is strictly dependent on mitochondrial function and thus respiration to generate the bulk of its ATP, whereas in the mammalian host it relies heavily on glycolysis. These observations have raised questions about the importance of mitochondrial function in the bloodstream stage. Peculiarly, akinetoplastic strains of Trypanosoma brucei evansi that lack mitochondrial DNA do exist in the wild and are developmentally locked in the glycolysis-dependent bloodstream stage. Using RNAi we show that two mitochondrion-imported proteins, mitochondrial RNA polymerase and guide RNA associated protein 1, are still imported into the nucleic acids-lacking organelle of T. b. evansi, making the need for these proteins futile. We also show that, like in the T. b. brucei procyclic stage, the mitochondria of both bloodstream stage of T. b. brucei and T. b. evansi import various tRNAs, including those that undergo thiolation. However, we were unable to detect mitochondrial thiolation in the akinetoplastic organelle. Taken together, these data suggest a lack of connection between nuclear and mitochondrial communication in strains of T. b. evansi that lost mitochondrial genome and that do not required an insect vector for survival.
- MeSH
- adenosintrifosfát metabolismus MeSH
- buněčné jádro genetika metabolismus MeSH
- DNA řízené RNA-polymerasy genetika metabolismus MeSH
- geneticky modifikované organismy MeSH
- glykolýza fyziologie MeSH
- guide RNA, Kinetoplastida metabolismus MeSH
- kinetoplastová DNA metabolismus MeSH
- mezibuněčná komunikace MeSH
- mitochondrie genetika metabolismus MeSH
- oxidativní fosforylace MeSH
- proteiny genetika metabolismus MeSH
- RNA interference MeSH
- RNA transferová genetika metabolismus MeSH
- transport proteinů MeSH
- transport RNA MeSH
- Trypanosoma fyziologie MeSH
- trypanozomiáza parazitologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
The mitochondrial RNA-binding proteins (MRP) 1 and 2 play a regulatory role in RNA editing and putative role(s) in RNA processing in Trypanosoma brucei. Here, we report the purification of a high molecular weight protein complex consisting solely of the MRP1 and MRP2 proteins from the mitochondrion of T. brucei. The MRP1/MRP2 complex natively purified from T. brucei and the one reconstituted in Escherichia coli in vivo bind guide (g) RNAs and pre-mRNAs with dissociation constants in the nanomolar range, and efficiently promote annealing of pre-mRNAs with their cognate gRNAs. In addition, the MRP1/MRP2 complex stimulates annealing between two non-cognate RNA molecules suggesting that along with the cognate duplexes, spuriously mismatched RNA hybrids may be formed at some rate in vivo. A mechanism of catalysed annealing of gRNA/pre-mRNA by the MRP1/MRP2 complex is proposed.
- MeSH
- chromatografie MeSH
- editace RNA MeSH
- elektronová mikroskopie MeSH
- financování organizované MeSH
- guide RNA, Kinetoplastida metabolismus ultrastruktura MeSH
- lidé MeSH
- mitochondriální proteiny fyziologie metabolismus ultrastruktura MeSH
- prekurzory RNA metabolismus ultrastruktura MeSH
- proteiny spojené s mnohočetnou rezistencí k lékům fyziologie chemie MeSH
- proteiny vázající RNA metabolismus ultrastruktura MeSH
- rekombinantní proteiny farmakologie MeSH
- RNA protozoální genetika MeSH
- Trypanosoma brucei brucei genetika metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
