The RNA editing core complex (RECC) catalyzes mitochondrial U-insertion/deletion mRNA editing in trypanosomatid flagellates. Some naphthalene-based sulfonated compounds, such as C35 and MrB, competitively inhibit the auto-adenylylation activity of an essential RECC enzyme, kinetoplastid RNA editing ligase 1 (KREL1), required for the final step in editing. Previous studies revealed the ability of these compounds to interfere with the interaction between the editosome and its RNA substrates, consequently affecting all catalytic activities that comprise RNA editing. This observation implicates a critical function for the affected RNA binding proteins in RNA editing. In this study, using the inhibitory compounds, we analyzed the composition and editing activities of functional editosomes and identified the mitochondrial RNA binding proteins 1 and 2 (MRP1/2) as their preferred targets. While the MRP1/2 heterotetramer complex is known to bind guide RNA and promote annealing to its cognate pre-edited mRNA, its role in RNA editing remained enigmatic. We show that the compounds affect the association between the RECC and MRP1/2 heterotetramer. Furthermore, RECC purified post-treatment with these compounds exhibit compromised in vitro RNA editing activity that, remarkably, recovers upon the addition of recombinant MRP1/2 proteins. This work provides experimental evidence that the MRP1/2 heterotetramer is required for in vitro RNA editing activity and substantiates the hypothesized role of these proteins in presenting the RNA duplex to the catalytic complex in the initial steps of RNA editing.
- MeSH
- RNA Editing drug effects genetics MeSH
- RNA, Guide, Kinetoplastida drug effects MeSH
- Ligases antagonists & inhibitors MeSH
- RNA, Messenger genetics MeSH
- Mitochondrial Proteins genetics MeSH
- Mitochondria drug effects genetics MeSH
- RNA-Binding Proteins genetics MeSH
- Protozoan Proteins genetics MeSH
- Recombinant Proteins genetics MeSH
- RNA, Mitochondrial genetics MeSH
- RNA, Protozoan genetics MeSH
- Trypanosoma brucei brucei drug effects MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't 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
- RNA Editing * MeSH
- RNA, Guide, Kinetoplastida metabolism MeSH
- Kinetoplastida genetics metabolism MeSH
- RNA, Messenger metabolism MeSH
- Protozoan Proteins metabolism MeSH
- RNA, Protozoan genetics metabolism MeSH
- Trypanosoma brucei brucei genetics metabolism MeSH
- Uridine metabolism MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Review MeSH
- Research Support, N.I.H., Extramural MeSH
CRISPR is a prokaryotic defence system that was adapted as a tool for genome editing and has become one of the most important discoveries of this century. CRISPR-associated endonucleases cleave DNA at precise sites, which are marked by complementary short-guided RNA. The recently developed versions of endonucleases are compatible with a broad range of PAM motifs, have a higher specificity and enable a specific nucleotide to be replaced.
- MeSH
- DNA genetics MeSH
- Gene Editing * MeSH
- Endonucleases genetics metabolism MeSH
- RNA, Guide, Kinetoplastida genetics MeSH
- Humans MeSH
- Clustered Regularly Interspaced Short Palindromic Repeats genetics MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
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
- RNA Editing * MeSH
- Phylogeny MeSH
- Genome, Protozoan * MeSH
- RNA, Guide, Kinetoplastida chemistry metabolism MeSH
- RNA, Messenger metabolism MeSH
- RNA, Mitochondrial metabolism MeSH
- Transcriptome MeSH
- Trypanosomatina genetics metabolism MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Kinetoplastid flagellates are known for several unusual features, one of which is their complex mitochondrial genome, known as kinetoplast (k) DNA, composed of mutually catenated maxi- and minicircles. Trypanosoma lewisi is a member of the Stercorarian group of trypanosomes which is, based on human infections and experimental data, now considered a zoonotic pathogen. By assembling a total of 58 minicircle classes, which fall into two distinct categories, we describe a novel type of kDNA organization in T. lewisi. RNA-seq approaches allowed us to map the details of uridine insertion and deletion editing events upon the kDNA transcriptome. Moreover, sequencing of small RNA molecules enabled the identification of 169 unique guide (g) RNA genes, with two differently organized minicircle categories both encoding essential gRNAs. The unprecedented organization of minicircles and gRNAs in T. lewisi broadens our knowledge of the structure and expression of the mitochondrial genomes of these human and animal pathogens. Finally, a scenario describing the evolution of minicircles is presented.
- MeSH
- Adenosine Triphosphatases genetics MeSH
- RNA Editing MeSH
- Phylogeny MeSH
- Genome, Mitochondrial MeSH
- RNA, Guide, Kinetoplastida genetics MeSH
- Mitochondria genetics MeSH
- Protein Subunits genetics MeSH
- DNA, Protozoan genetics MeSH
- RNA, Protozoan genetics MeSH
- Trypanosoma lewisi genetics MeSH
- High-Throughput Nucleotide Sequencing MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
317 s. : il.
- Keywords
- Laboratorní technika, RNA,
- MeSH
- Clinical Laboratory Techniques MeSH
- RNA MeSH
- Publication type
- Monograph MeSH
- Conspectus
- Biochemie. Molekulární biologie. Biofyzika
- NML Fields
- molekulární biologie, molekulární medicína
[1st ed.] xix, 375 s. : il.
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
- Chromatography MeSH
- RNA Editing MeSH
- Microscopy, Electron MeSH
- Financing, Organized MeSH
- RNA, Guide, Kinetoplastida metabolism ultrastructure MeSH
- Humans MeSH
- Mitochondrial Proteins physiology metabolism ultrastructure MeSH
- RNA Precursors metabolism ultrastructure MeSH
- Multidrug Resistance-Associated Proteins physiology chemistry MeSH
- RNA-Binding Proteins metabolism ultrastructure MeSH
- Recombinant Proteins pharmacology MeSH
- RNA, Protozoan genetics MeSH
- Trypanosoma brucei brucei genetics metabolism MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Animals MeSH
BACKGROUND: Current SARS-CoV-2 detection platforms lack the ability to differentiate among variants of concern (VOCs) in an efficient manner. CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated) based detection systems have the potential to transform the landscape of COVID-19 diagnostics due to their programmability; however, most of these methods are reliant on either a multi-step process involving amplification or elaborate guide RNA designs. METHODS: Three Cas12b proteins from Alicyclobacillus acidoterrestris (AacCas12b), Alicyclobacillus acidiphilus (AapCas12b), and Brevibacillus sp. SYP-B805 (BrCas12b) were expressed and purified, and their thermostability was characterised by differential scanning fluorimetry, cis-, and trans-cleavage activities over a range of temperatures. The BrCas12b was then incorporated into a reverse transcription loop-mediated isothermal amplification (RT-LAMP)-based one-pot reaction system, coined CRISPR-SPADE (CRISPR Single Pot Assay for Detecting Emerging VOCs). FINDINGS: Here we describe a complete one-pot detection reaction using a thermostable Cas12b effector endonuclease from Brevibacillus sp. to overcome these challenges detecting and discriminating SARS-CoV-2 VOCs in clinical samples. CRISPR-SPADE was then applied for discriminating SARS-CoV-2 VOCs, including Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), and Omicron (B.1.1.529) and validated in 208 clinical samples. CRISPR-SPADE achieved 92·8% sensitivity, 99·4% specificity, and 96·7% accuracy within 10-30 min for discriminating the SARS-CoV-2 VOCs, in agreement with S gene sequencing, achieving a positive and negative predictive value of 99·1% and 95·1%, respectively. Interestingly, for samples with high viral load (Ct value ≤ 30), 100% accuracy and sensitivity were attained. To facilitate dissemination and global implementation of the assay, a lyophilised version of one-pot CRISPR-SPADE reagents was developed and combined with an in-house portable multiplexing device capable of interpreting two orthogonal fluorescence signals. INTERPRETATION: This technology enables real-time monitoring of RT-LAMP-mediated amplification and CRISPR-based reactions at a fraction of the cost of a qPCR system. The thermostable Brevibacillus sp. Cas12b offers relaxed primer design for accurately detecting SARS-CoV-2 VOCs in a simple and robust one-pot assay. The lyophilised reagents and simple instrumentation further enable rapid deployable point-of-care diagnostics that can be easily expanded beyond COVID-19. FUNDING: This project was funded in part by the United States-India Science & Technology Endowment Fund- COVIDI/247/2020 (P.K.J.), Florida Breast Cancer Foundation- AGR00018466 (P.K.J.), National Institutes of Health- NIAID 1R21AI156321-01 (P.K.J.), Centers for Disease Control and Prevention- U01GH002338 (R.R.D., J.A.L., & P.K.J.), University of Florida, Herbert Wertheim College of Engineering (P.K.J.), University of Florida Vice President Office of Research and CTSI seed funds (M.S.), and University of Florida College of Veterinary Medicine and Emerging Pathogens Institute (R.R.D.).
- MeSH
- Brevibacillus * genetics MeSH
- COVID-19 * diagnosis MeSH
- RNA, Guide, Kinetoplastida MeSH
- Humans MeSH
- SARS-CoV-2 genetics MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
The canonical stop codons of the nuclear genome of the trypanosomatid Blastocrithidia nonstop are recoded. Here, we investigated the effect of this recoding on the mitochondrial genome and gene expression. Trypanosomatids possess a single mitochondrion and protein-coding transcripts of this genome require RNA editing in order to generate open reading frames of many transcripts encoded as 'cryptogenes'. Small RNAs that can number in the hundreds direct editing and produce a mitochondrial transcriptome of unusual complexity. We find B. nonstop to have a typical trypanosomatid mitochondrial genetic code, which presumably requires the mitochondrion to disable utilization of the two nucleus-encoded suppressor tRNAs, which appear to be imported into the organelle. Alterations of the protein factors responsible for mRNA editing were also documented, but they have likely originated from sources other than B. nonstop nuclear genome recoding. The population of guide RNAs directing editing is minimal, yet virtually all genes for the plethora of known editing factors are still present. Most intriguingly, despite lacking complex I cryptogene guide RNAs, these cryptogene transcripts are stochastically edited to high levels.
- MeSH
- Cell Nucleus * genetics metabolism MeSH
- RNA Editing * MeSH
- Genetic Code MeSH
- Genome, Mitochondrial * MeSH
- RNA, Guide, Kinetoplastida genetics metabolism MeSH
- Codon genetics MeSH
- RNA, Messenger genetics metabolism MeSH
- Mitochondria genetics metabolism MeSH
- Open Reading Frames genetics MeSH
- Protozoan Proteins genetics metabolism MeSH
- RNA, Transfer * genetics metabolism MeSH
- Codon, Terminator genetics MeSH
- Trypanosomatina genetics metabolism MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
