RNA editing in trypanosomatid mitochondria.
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Animals↗
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KREPA4, an RNA binding protein essential for editosome integrity and survival of Trypanosoma brucei.
The 20S editosome, a multiprotein complex, catalyzes the editing of most mitochondrial mRNAs in trypanosomatids by uridylate insertion and deletion. RNAi mediated inactivation of expression of KREPA4 (previously TbMP24), a component of the 20S editosome, in procyclic form Trypanosoma brucei resulted in inhibition of cell growth, loss of RNA editing, and disappearance of 20S editosomes. Levels of MRP1 and REAP-1 proteins, which may have roles in editing but are not editosome components, were unaffected. Tagged KREPA4 protein is incorporated into 20S editosomes in vivo with no preference for either insertion or deletion subcomplexes. Consistent with its S1-like motif, recombinant KREPA4 protein binds synthetic gRNA with a preference for the 3' oligo (U) tail. These data suggest that KREPA4 is an RNA binding protein that may be specific for the gRNA Utail and also is important for 20S editosome stability.
Amino Acid Motifs↗
Regulation of bacterial RNase P ribozyme reaction by divalent cation and guide DNA.
The RNA subunit of bacterial ribonuclease P (RNase P) is a ribozyme which can cleave a canonical cloverleaf tRNA precursor and a hairpin RNA with a CCA-3' tag sequence as its substrate. With high concentration of Mg ion, the ribozyme as well as holo enzyme internally cleaves certain tRNAs in vitro. We denoted this unusual reaction as hyperprocessing. By controlling magnesium ion concentration for the reaction and also by forcing the RNA shape with external guide DNAs, we could regulate the hyperprocessing reaction by the bacterial RNase P enzymes. These techniques will lead the RNase P ribozyme to more designable and more applicable RNA-cleaving enzyme.
Bacteria↗
External guide sequences for an RNA enzyme.
Ribonuclease P (RNase P) from Escherichia coli or its catalytic RNA subunit can efficiently cleave small RNA substrates that lack the conserved features of natural substrates of RNase P if an additional small RNA is also present. This additional RNA must contain a sequence complementary to the substrate [external guide sequence (EGS)] and a 3'-proximal CCA sequence to ensure cleavage. The aminoacyl acceptor stem and some additional 5'- and 3'-terminal sequences of a precursor transfer RNA are sufficient to allow efficient cleavage by RNAase P, and the 2'-hydroxyl group at the cleavage site is not absolutely necessary for cleavage. In principle, any RNA could be targeted by a custom-designed EGS RNA for specific cleavage by RNase P in vitro or in vivo.
Base Sequence↗
Tertiary core rearrangements in a tight binding transfer RNA aptamer.
Guided by an in vitro selection experiment designed to obtain tight binding aptamers of Escherichia coli glutamine specific tRNA (tRNAGln) for glutaminyl-tRNA synthetase (GlnRS), we have engineered a tRNA mutant in which the five-nucleotide variable loop sequence 5'-44CAUUC48-3' is replaced by 5'-44AGGU48-3'. This mutant tRNA binds to GlnRS with 30-fold improved affinity compared to the wild type. The 2.7 A cocrystal structure of the RNA aptamer-GlnRS complex reveals major rearrangements in the central tertiary core of the tRNA, while maintaining an RNA-protein interface identical to the wild type. The repacked RNA core features a novel hydrogen bonding arrangement of the trans Levitt pair G15-U48, a new sulfate binding pocket in the major groove, and increased hydrophobic stacking interactions among the bases. These data suggest that enhanced protein binding to a mutant globular RNA can arise from stabilization of RNA tertiary interactions rather than optimization of RNA-protein contacts.
Amino Acyl-tRNA Synthetases↗
Enhancing CRISPR-Cas12a base editing in plants with LbCas12a variants and introns.
Cytosine base editors (CBEs) and adenine base editors (ABEs) are powerful tools for precise genome editing in plants. Conventionally, such base editors are built upon the CRISPR-Cas9 systems where Cas9 nickases are used. To expand the base editing scope and minimize off-target effects, base editors derived from the CRISPR-Cas12a systems are desired. However, the use of deactivated Cas12a (dCas12a) in such base editors constrains the editing activity, preventing the wide use of Cas12a base editors for plant research and trait development. In this study, we demonstrate the use of an ABE based on the efficient LbCas12a-RRV variant to introduce herbicide-resistant mutations in OsACCase in rice. To improve Cas12a CBEs and ABEs, we inserted introns into the coding sequence of dLbCas12a-RRV. This intron-containing Cas12a-CBE shows substantial improvement in editing efficiency in rice, compared to the intron-less counterparts. By contrast, the improvement of ABE with the intron-containing dLbCas12a-RRV is very limited, partly due to the already high baseline editing efficiency of the intron-less dLbCas12a-RRV ABE. Testing of these base editors in poplar shows elevated C-to-T base editing by dLbCas12a-RRV-intron-CBE. For A-to-G editing, ABEs built upon dLbCas12a-RV and dLbCas12a-RRV variants showed significant improvement over ABEs derived from wild-type LbCas12a and the ttLbCas12a variant. The addition of introns to dLbCas12a-RRV does not further improve the base editing efficiency. With whole genome sequencing in rice, we evaluated genome editing specificities with these improved Cas12a base editors. Our analyses show that both intron-containing Cas12a CBE and ABE barely introduce guide RNA-dependent off-target mutations. However, they can generate guide RNA-independent off-target mutations, which are likely attributed to the high enzymatic activities of the deaminases. Collectively, our study demonstrates the successful use of a Cas12a base editor for trait development and reports improved Cas12a CBEs and ABEs for precise base editing in plants.
Oryza↗
Processing of polycistronic guide RNAs is associated with RNA editing complexes in Trypanosoma brucei.
In kinetoplastid mitochondrial mRNA editing, post-transcriptional insertion or deletion of uridines is templated by guide RNAs (gRNAs). Pre-mRNAs are encoded by maxicircles, while gRNAs are encoded by both maxicircles and minicircles. We have investigated minicircle transcription and the processing of gRNAs in Trypanosoma brucei. We find that minicircles are transcribed polycistronically and that transcripts are accurately processed by an approximately 19S complex. This gRNA processing activity co-purifies with RNA editing complexes, and both remain associated in 19S complexes. Furthermore, we show that RNA editing complexes associate preferentially with a polycistronic gRNA over non-processed RNAs. We propose that the approximately 19S complexes initially described as RNA editing complex I are gRNA processing complexes that cleave polycistronic gRNA transcripts into monocistrons.
Animals↗
[Study on the targeting effects of M1-GS RNA on K562 cells].
OBJECTIVE: To determine the effects of M1-GS RNA (M1 RNA) on bcr-abl mRNA and oncoprotein after M1 RNA with guide sequence (M1-GS RNA) targeting the oncogene was transfected into K562 cells. METHODS: pAVGS4 (an eukaryocyte expression vector containing M1-GS RNA sequence) and pNAV-1 (as the control) were transfected into K562 cells by X-tremeGENE Q2. Total RNA was extracted at 24, 48, 72 and 96 hours after transfection. Then RT-PCR was done to compare the products at different time point. After collecting pAVGS4-transfected cells and the control cells at 48 and 96 hours after transfection, total protein was extracted and quantified. Change of P210 was determined by Western blot. Colony formation was analyzed at 96 hours after transfection. RESULTS: RT-PCR based on transfected cells at different time point showed that the amount of bcr-abl mRNA began to decrease at 24 hours and reduced to 9.2% and 2.5% respectively at 48 and 72 hours after transfection. Western blot showed that the expression of P210 in the pAVGS4 group reduced to 10.4% of the control at 48 hours and 6.7% of the control at 96 hours after transfection. The inhibition rate of colony formation was 81.3% after K562 cells were transfected by pAVGS4. CONCLUSION: pAVGS4 can efficiently destroy bcr-abl mRNA in K562 cells. The transcript level of bcr-abl mRNA was reduced with the time after transfection. The expression of P210 was decreased significantly at 48 and 96 hours after transfection. K562 cell colony formation was prominently inhibited.
Escherichia coli Proteins↗
Mapping contacts between gRNA and mRNA in trypanosome RNA editing.
All guide RNAs (gRNAs) identified to date have defined 5' anchor sequences, guiding sequences and a non-encoded 3' uridylate tail. The 5' anchor is required for in vitro editing and is thought to be responsible for selection and binding to the pre-edited mRNA. Little is known, however, about how the gRNAs are used to direct RNA editing. Utilizing the photo-reactive crosslinking agent, azidophenacyl (APA), attached to the 5'- or 3'-terminus of the gRNA, we have begun to map the structural relationships between the different defined regions of the gRNA with the pre-edited mRNA. Analyses of crosslinked conjugates produced with a 5'-terminal APA group confirm that the anchor of the gRNA is correctly positioning the interacting molecules. 3' Crosslinks (X-linker placed at the 3'-end of a U10tail) have also been mapped for three different gRNA/mRNA pairs. In all cases, analyses indicate that the U-tail can interact with a range of nucleotides located upstream of the first edited site. It appears that the U-tail prefers purine-rich sites, close to the first few editing sites. These results suggest that the U-tail may act in concert with the anchor to melt out secondary structure in the mRNA in the immediate editing domain, possibly increasing the accessibility of the editing complex to the proper editing sites.
Animals↗
The involvement of gRNA-binding protein gBP21 in RNA editing-an in vitro and in vivo analysis.
RNA editing in the parasitic organism Trypanosoma brucei is characterised by the insertion and deletion of uridylate residues into otherwise incomplete primary transcripts. The processing reaction is a required pathway for the expression of most mitochondrial genes and proceeds by a cascade of enzyme-catalysed steps. RNA editing involves one or more macromolecular ribonucleoprotein complexes which are likely to interact with additional components as the reaction proceeds. Here we examined the involvement of the gRNA-binding polypeptide gBP21, a protein which has been demonstrated to be associated with active RNA editing complexes. We show that in vitro RNA editing can be suppressed by the addition of a gBP21-specific antibody or by immunodepletion of the protein. By creating a gBP21 knockout mutant we analysed the requirement for the protein in vivo. gBP21(-) trypanosomes are viable as bloodstream stage cells and contain edited mRNAs. However, the knockout mutant is not capable of differentiating from the bloodstream to the insect life cycle stage in vitro. Moreover, mutant cells are characterised by a low mitochondrial transcript abundance. Together, these data establish that gBP21 contributes a non-essential function to the RNA editing reaction and further suggest that the protein is involved in additional mitochondrial processes which impact a larger pool of mitochondrial transcripts.
Animals↗
Unique mitochondrial genome structure in diplonemids, the sister group of kinetoplastids.
Kinetoplastid flagellates are characterized by uniquely massed mitochondrial DNAs (mtDNAs), the kinetoplasts. Kinetoplastids of the trypanosomatid group possess two types of mtDNA molecules: maxicircles bearing protein and mitoribosomal genes and minicircles specifying guide RNAs, which mediate uridine insertion/deletion RNA editing. These circles are interlocked with one another to form dense networks. Whether these peculiar mtDNA features are restricted to kinetoplastids or prevail throughout Euglenozoa (euglenids, diplonemids, and kinetoplastids) is unknown. Here, we describe the mitochondrial genome and the mitochondrial ultrastructure of Diplonema papillatum, a member of the diplonemid flagellates, the sister group of kinetoplastids. Fluorescence and electron microscopy show a single mitochondrion per cell with an ultrastructure atypical for Euglenozoa. In addition, DNA is evenly distributed throughout the organelle rather than compacted. Molecular and electron microscopy studies distinguish numerous 6- and 7-kbp-sized mitochondrial chromosomes of monomeric circular topology and relaxed conformation in vivo. Remarkably, the cox1 gene (and probably other mitochondrial genes) is fragmented, with separate gene pieces encoded on different chromosomes. Generation of the contiguous cox1 mRNA requires trans-splicing, the precise mechanism of which remains to be determined. Taken together, the mitochondrial gene/genome structure of Diplonema is not only different from that of kinetoplastids but unique among eukaryotes as a whole.
Animals↗
DICER-LIKE 4 is required for RNA interference and produces the 21-nucleotide small interfering RNA component of the plant cell-to-cell silencing signal.
In RNA interference, the RNase-III enzyme Dicer processes exogenous double-stranded RNA into small interfering RNAs (siRNAs). siRNAs guide RNA-induced silencing complexes to cleave homologous transcripts, enabling gene-specific knock-down. In plants, double-stranded RNA is processed into siRNA species of 21 nucleotides (nt) and 24 nt (ref. 5), but, unlike in nematodes, the Dicer enzymes involved in this processing have not been identified. Additionally, in both plants and nematodes, systemic signals with RNA components convey the sequence-specific effects of RNA interference between cells. Here, we describe Arabidopsis thaliana mutants with altered silencing cell-to-cell movement beyond the vasculature. At least three SILENCING MOVEMENT DEFICIENT genes (SMD1, SMD2 and SMD3) are required for trafficking, the extent of which correlates with siRNA levels in the veins. Five alleles defective in synthesis of 21-nt, but not 24-nt, siRNAs carry mutations in Dicer-like 4 (DCL4) that are involved in biogenesis of trans-acting siRNAs. We show that the biogenesis and function of trans-acting siRNA can be genetically uncoupled from a bona fide DCL4-dependent pathway that accounts for RNA interference and for production of the 21-nt siRNA component of the plant cell-to-cell silencing signal.
Alleles↗
Genome editing with programmable base editors in human cells.
Genome editing has garnered significant attention over the last decade, resulting in a massive expansion of the genome engineering toolbox. Base editors encompass a class of tools that enable installing single-nucleotide changes in genomic DNA without the use of double-strand breaks. With the ever-increasing development of new and/or improved base editor systems, it is easy to be overwhelmed by the abundance of options. Here, we provide clear guidance to facilitate the selection of a base editor and to design guide RNAs (gRNAs) to suit various needs. Additionally, we describe in detail how to generate gRNA plasmids, transfect various mammalian cell types, and evaluate editing efficiencies. Finally, we give alternative methods and troubleshooting tips for some common pitfalls encountered during base editing.
Humans↗
HCV-RNA detection in ultrasound-guided fine needle biopsies of liver nodules and surrounding tissue.
HCV-RNA was examined in serum and liver tissue obtained from 8 hepatitis B surface antigen (HBsAg) negative patients with liver nodules ranging in size from 2 to 11 cm. Histological examination of ultrasound-guided fine needle biopsies revealed the presence of hepatocellular carcinoma (HCC) in six patients (5 of whom were anti-HCV positive), cholangiocarcinoma in 1 patient (anti-HCV positive) and dysplastic regenerative nodule in 1 patient (anti-HCV negative). The HCCs were surrounded by cirrhosis (3 cases), chronic active hepatitis (CAH) (n = 2) and post hepatitic fibrosis (n = 1), the cholangiocarcinoma by CAH and the regenerative nodule by cirrhotic liver. Total and replicative intermediate HCV-RNA was analyzed by reverse-transcription-nested PCR of the 5'-untranslated region. The five patients with HCC had HCV-RNA in serum, in tumorous and surrounding liver tissues. The viral nucleic acid was also detected in the cirrhotic tissue surrounding the cholangiocarcinoma but not in the tumor. Two out of 5 HCC patients had replicative intermediate RNA (negative strand) in tumorous tissue, 4 in nontumorous tissue and 3 in serum. These results demonstrate that fine needle biopsy can provide sufficient material for both histological examination and HCV-RNA determination and suggest the existence of continuous viral replication during the carcinogenic process.
Aged↗
dCas-Based Tools to Visualize Chromatin or Modify Epigenetic Marks at Specific Plant Genomic Loci.
Development of locus-specific approaches targeting precise regions on chromatin, for locus/transcription visualization or transcription/epigenetic marks editing, is a critical challenge in functional genetics and epigenetics. Systems engineered from the clustered regularly interspaced short palindromic repeats (CRISPR) and its associated endonuclease (Cas) operate through DNA sequence-specific recognition by so-called guide RNAs, which provides high flexibility and modularity for precise chromatin visualization or edition. Here, we provide an overview of the CRISPR/Cas-derived tools developed for visualization of chromatin loci in live imaging or for effective modification of gene expression. These tools make use of effector modules that combine activators, repressors, and epigenetic modifiers with a deactivated Cas protein (dCas). We present how their use in plants brought advances in visualizing or manipulating the expression of loci involved in agronomically interesting traits such as flowering time and response to drought or heat. We also discuss the limitations and future improvements of the dCas-related technologies, such as more compact and combinatorial systems, spatiotemporal targeting for fine-tuning of gene expression, and live visualization of chromatin dynamics.
Chromatin↗
Enhanced cleavage of genomic CCR5 using CASX2Max.
Development of novel CRISPR/Cas systems enhances opportunities for gene editing to treat infectious diseases, cancer, and genetic disorders. CasX2 (PlmCas12e) belongs to the class II CRISPR system derived from Planctomycetes, a non-pathogenic bacterium present in aquatic and terrestrial soils and offers several advantages as a potential therapeutic CRISPR system over Streptococcus pyogenes Cas9 (SpCas9) and Staphylococcus aureus Cas9 (SaCas9). These advantages include its smaller size, distinct protospacer adjacent motif (PAM) requirements, staggered cleavage cuts that promote homology-directed repair, and the absence of pre-existing immunity in humans. We compared the cleavage efficiency and double-stranded break repair characteristics between CasX2 and CasX2Max, a recently generated CasX2 variant with three amino acid substitutions, for targeting CCR5, a gene that encodes the CCR5 receptor important for HIV-1 infection. Two single guide RNAs (sgRNAs) were designed that flank the 32 bases deleted in the natural CCR5 ∆32 mutation. Nanopore sequencing demonstrated that CasX2 using sgRNAs with spacers of 17 nucleotides (nt), 20 nt or 23 nt in length were ineffective at cleaving genomic CCR5. In contrast, CasX2Max using sgRNAs with 20 nt and 23 nt spacer lengths, enabled cleavage of genomic CCR5. Structural modelling indicated that two of the CasX2Max amino acid substitutions enhanced sgRNA-DNA duplex stability, while the third improved DNA strand alignment within the catalytic site. These structural changes likely underlie the increased activity of CasX2Max in cellular gene excision. In sum, CasX2Max consistently outperformed native CasX2 across all assays and represents a superior gene-editing platform for therapeutic applications.
Humans↗
The RNA-induced silencing complex is a Mg2+-dependent endonuclease.
In the Drosophila and mammalian RNA interference (RNAi) pathways, target RNA destruction is catalyzed by the siRNA-guided, RNA-induced silencing complex (RISC). RISC has been proposed to be an siRNA-directed endonuclease, catalyzing cleavage of a single phosphodiester bond on the RNA target. Although 5' cleavage products are readily detected for RNAi in vitro, only 3' cleavage products have been observed in vivo. Proof that RISC acts as an endonuclease requires detection of both 5' and 3' cleavage products in a single experimental system. Here, we show that siRNA-programmed RISC generates both 5' and 3' cleavage products in vitro; cleavage requires Mg(2+), but not Ca(2+), and the cleavage product termini suggest a role for Mg(2+) in catalysis. Moreover, a single phosphorothioate in place of the scissile phosphate blocks cleavage; the phosphorothioate effect can be rescued by the thiophilic cation Mn(2+), but not by Ca(2+) or Mg(2+). We propose that during catalysis, a Mg(2+) ion is bound to the RNA substrate through a nonbridging oxygen of the scissile phosphate. The mechanism of endonucleolytic cleavage is not consistent with the mechanisms of the previously identified RISC nuclease, Tudor-SN. Thus, the RISC-component that mediates endonucleolytic cleavage of the target RNA remains to be identified.
Animals↗
A guide to ions and RNA structure.
RNA folding into stable tertiary structures is remarkably sensitive to the concentrations and types of cations present; an understanding of the physical basis of ion-RNA interactions is therefore a prerequisite for a quantitative accounting of RNA stability. This article summarizes the energetic factors that must be considered when ions interact with two different RNA environments. "Diffuse ions" accumulate near the RNA because of the RNA electrostatic field and remain largely hydrated. A "chelated" ion directly contacts a specific location on the RNA surface and is held in place by electrostatic forces. Energetic costs of ion chelation include displacement of some of the waters of hydration by the RNA surface and repulsion of diffuse ions. Methods are discussed for computing both the free energy of the set of diffuse ions associated with an RNA and the binding free energies of individual chelated ions. Such calculations quantitatively account for the effects of Mg(2+) on RNA stability where experimental data are available. An important conclusion is that diffuse ions are a major factor in the stabilization of RNA tertiary structures.
Animals↗