A strategy for developing a hammerhead ribozyme for selective RNA cleavage depending on substitutional RNA editing

Substitutional RNA editing plays a crucial role in the regulation of biological processes. Cleavage of target RNA that depends on the specific site of substitutional RNA editing is a useful tool for analyzing and regulating intracellular processes related to RNA editing. Hammerhead ribozymes have been utilized as small catalytic RNAs for cleaving target RNA at a specific site and may be used for RNA-editing-specific RNA cleavage. Here we reveal a design strategy for a hammerhead ribozyme that specifically recognizes adenosine to inosine (A-to-I) and cytosine to uracil (C-to-U) substitutional RNA-editing sites and cleaves target RNA. Because the hammerhead ribozyme cleaves one base upstream of the target-editing site, the base that pairs with the target-editing site was utilized for recognition. RNA-editing-specific ribozymes were designed such that the recognition base paired only with the edited base. These ribozymes showed A-to-I and C-to-U editing-specific cleavage activity against synthetic serotonin receptor 2C and apolipoprotein B mRNA fragments in vitro, respectively. Additionally, the ribozyme designed for recognizing A-to-I RNA editing at the Q/R site on filamin A (FLNA) showed editing-specific cleavage activity against physiologically edited FLNA mRNA extracted from cells. We demonstrated that our strategy is effective for cleaving target RNA in an editing-dependent manner. The data in this study provided an experimental basis for the RNA-editing-dependent degradation of specific target RNA in vivo.     

Determinants for association and guide RNA-directed endonuclease cleavage by purified RNA editing complexes from Trypanosoma brucei

U-insertion/deletion RNA editing in the single mitochondrion of kinetoplastids, an ancient lineage of eukaryotes, is a unique mRNA maturation process needed for translation. Multisubunit editing complexes recognize many pre-edited mRNA sites and modify them via cycles of three catalytic steps: guide RNA (gRNA)-directed cleavage, insertion or deletion of uridylates at the 3′-terminus of the upstream cleaved piece, and ligation of the two mRNA pieces. While catalytic and many structural protein subunits of these complexes have been identified, the mechanisms and basic determinants of substrate recognition are still poorly understood. This study defined relatively simple single- and double-stranded determinants for association and gRNA-directed cleavage. To this end, we used an electrophoretic mobility shift assay to directly score the association of purified editing complexes with RNA ligands, in parallel with UV photocrosslinking and functional studies. The cleaved strand required a minimal 5′ overhang of 12 nt and an ∼ 15-bp duplex with gRNA to direct the cleavage site. A second protruding element in either the cleaved or the guide strand was required unless longer duplexes were used. Importantly, the single-stranded RNA requirement for association can be upstream or downstream of the duplex, and the binding and cleavage activities of purified editing complexes could be uncoupled. The current observations together with our previous reports in the context of purified native editing complexes show that the determinants for association, cleavage and full-round editing gradually increase in complexity as these stages progress. The native complexes in these studies contained most, if not all, known core subunits in addition to components of the MRP complex. Finally, we found that the endonuclease KREN1 in purified complexes photocrosslinks with a targeted editing site. A model is proposed whereby one or more RNase III-type endonucleases mediate the initial binding and scrutiny of potential ligands and subsequent catalytic selectivity triggers either insertion or deletion editing enzymes.     

Improved design of hammerhead ribozyme for selective digestion of target RNA through recognition of site-specific adenosine-to-inosine RNA editing

Adenosine-to-inosine (A-to-I) RNA editing is an endogenous regulatory mechanism involved in various biological processes. Site-specific, editing-state–dependent degradation of target RNA may be a powerful tool both for analyzing the mechanism of RNA editing and for regulating biological processes. Previously, we designed an artificial hammerhead ribozyme (HHR) for selective, site-specific RNA cleavage dependent on the A-to-I RNA editing state. In the present work, we developed an improved strategy for constructing a trans-acting HHR that specifically cleaves target editing sites in the adenosine but not the inosine state. Specificity for unedited sites was achieved by utilizing a sequence encoding the intrinsic cleavage specificity of a natural HHR. We used in vitro selection methods in an HHR library to select for an extended HHR containing a tertiary stabilization motif that facilitates HHR folding into an active conformation. By using this method, we successfully constructed highly active HHRs with unedited-specific cleavage. Moreover, using HHR cleavage followed by direct sequencing, we demonstrated that this ribozyme could cleave serotonin 2C receptor (HTR2C) mRNA extracted from mouse brain, depending on the site-specific editing state. This unedited-specific cleavage also enabled us to analyze the effect of editing state at the E and C sites on editing at other sites by using direct sequencing for the simultaneous quantification of the editing ratio at multiple sites. Our approach has the potential to elucidate the mechanism underlying the interdependencies of different editing states in substrate RNA with multiple editing sites.     

N6-Methyladenosines Modulate A-to-I RNA Editing

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基于机器学习的RNA编辑位点预测方法综述

RNA编辑是一个十分重要的生物细胞分子机制。作为转录后修饰的一步,它可以增加蛋白质组学多样性,改变转录产物的稳定性,调节基因表达等。RNA编辑失调会导致各种疾病,包括神经疾病和癌症。在动物中,腺苷到肌苷(A-to-I)的编辑是最普遍的。高通量测序技术的进步大大提高了在全局范围内检测和量化RNA编辑的能力,使得RNA编辑的大规模全基因组分析变得可行,产生了一系列基于高通量测序技术的RNA编辑位点预测方法。通过对这些方法进行介绍、总结和分析,为RNA编辑的进一步研究提供一些思路。     

Evidence for a mitochondrial chromosome in Xenopus laevis oocytes

When Xenopus laevis mitochondria are gently lysed at physiologic ionic strength, mitochondrial DNA (mitDNA) is extracted associated with proteins. Sedimentation and buoyant density studies indicate that proteins are bound to mitDNA at a ratio of about 1/1. This DNA-protein complex visualized by electron microscopy after fixation with glutaraldehyde appears as a relaxed circular molecule consisting of an average of 48 globular particles interconnected by a thin filament.     

Dramatic Increase of the RNA Editing for Glutamate Receptor Subunits During Terminal Differentiation of Clonal Human Neurons

Abstract: RNA editing plays an important role in determining physiological characteristics of certain glutamate-gated receptor (GluR) channels such as Ca²⁺ permeability and desensitization kinetics. In one case, the editing changes a gene-encoded glutamine (Q) to an arginine (R) codon located in the channel-forming domain of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor subunit GluR-B and also the kainate receptor subunits GluR5 and GluR6. Another case of RNA editing alters an arginine (R) to a glycine (G) codon at a position termed the "R/G" site of AMPA subunits GluR-B, C, and D. Double-stranded RNA-specific adenosine deaminases (DRADA) have been implicated as agents involved in the editing. By using a human teratocarcinoma cell line, NT2, we investigated the change of the RNA editing of GluR subunits in conjunction with the expression of two DRADA members, DRADA1 and DRADA2 genes, during neuronal differentiation. Whereas Q/R and R/G site RNA editing both become progressively activated in differentiating NT2 cells, the expression of the two DRADA genes can already be detected even in the undifferentiated NT2 cells. Development of the editing machinery appears to require, in addition to DRADA enzymes, a currently unidentified mechanism(s) that may become activated during neuronal differentiation.     

Sexually distinct development of vocal pathways in Xenopus laevis

Deterministic rules, rather than experience, are thought to regulate the development of simple behaviors in vertebrates and invertebrates. We revisited this issue through examination of the sexually distinct vocalizations of African clawed frogs (Xenopus laevis), a reproductive behavior used by sexually mature males and females. We discovered that, as expected for simple behavior, female vocalizations develop through deterministic rules. The rare calls of juvenile females are indistinguishable from those of adult females. The vocal pathways of juvenile females, as measured by the contractile properties of the laryngeal muscles (the vocal muscles) and the laryngeal motoneuron somata (vocal motoneurons) size, are the developmental default and do not differentiate as they mature. Male Xenopus, in contrast, produce extensive vocalizations with rudimentary acoustic structure before reaching sexual maturity. Moreover, the functional properties of the vocal central pattern generator mature before muscle fibers and motoneuron size are fully masculinized. The results suggest that neuronal activity during development may be important in organizing the contractile properties of the muscle fibers in male, but not in female Xenopus. © 2010 Wiley Periodicals, Inc. Develop Neurobiol 70: 862–874, 2010     

Distribution of prosome proteins and their relationship with the cytoskeleton in oogenesis of Xenopus laevis

The presence of prosome proteins (p25K and p27K) was shown and their distribution was studied in oogenesis of Xenopus laevis using immunoblotting and immunofluorescence. These proteins form numerous granular clusters of variable size all over the cell. At previteilogenic stages, the prosome antibodies homogeneously stain the oocyte nucleus and the evenly distributed relatively large clusters in the cytoplasm. As the oocyte grows, the pattern of distribution of the prosome proteins undergoes changes: animal-vegetal and cortical gradients appear in the cytoplasm. In the course of oocyte maturation the size of clusters diminishes. Artificial activation of the egg leads to a dorso-ventral gradient in distribution of the prosome proteins. In this way, specific localization of prosome proteins is first visualized during formation of the dorso-ventral polarity. Co-localization of prosome proteins and actin and myosin was found in the oocyte by double staining. Small clusters of prosomes dispersed in the cytoplasm acquire capability of movement (after artificial activation) due, in all likelihood, to persisting connection with the acto-myosin complex of the egg. © 1994 Wiley-Liss, Inc.     

Neural-inducing activity of nuclei and nuclear fractions from Xenopus laevis embryos

Summary From embryos (Xenopus laevis) of different developmental stages nuclei were isolated which exert neural inducing activity in the biological test. The active material could partly be extracted from the nuclei. Experiments for the isolation of nuclear ribonucleoprotein (RNP) particles have shown that the activity is localized at least in part in these particles. On the other hand, some neural inducer is not detached from chromatin and the nuclear matrix even with ionic detergents. Inducing activity was found in germinal vesicles and to a higher degree in the cytoplasm of oocytes, but in a masked, biologically inactive state.     

Evidence for intracellular sodium pumps in permeabilized Xenopus laevis oocytes.

Ouabain binding was studied in Xenopus laevis oocytes permeabilized by detergents. The behaviour of markers showed that 10 microM-digitonin selectively disrupts the plasma membrane. In the presence of ATP, oocytes permeabilized at 10 microM-digitonin bound no more ouabain molecules than were required to abolish active 86Rb+ uptake in the intact cells. However, the ouabain binding capacity increased approx. 2-fold when inner membranes were disrupted by SDS or excess digitonin, as judged from the accompanying release of the lysosomal marker beta-hexosaminidase. The results suggest that oocytes have a large internal pool of functional sodium pumps.     

Evidence for polarizing zone in the limb buds of Xenopus laevis

To test for the presence of polarizing mesoderm in an amphibian, Xenopus laevis hindlimb bud tips were rotated 180° on the proximodistal axis and returned to the stump. Supernumerary outgrowths were induced in the preaxial stump and preaxial tip tissues, and the most postaxial digit always formed next to the grafted postaxial tissue. The occurrence of polarized supernumerary outgrowths indicated that the posterior limb border contained a polarizing zone. When the limb tip was cut at varying known lengths from the body wall, rotated, and grafted to the limb stump, the incidence of twinning along the proximodistal axis permitted insight into the distribution of the polarizing zone along the posterior border. The location of polarizing tissues was found to be similar to that in the chick wing bud at comparable stages. To confirm the posterior border stump influence on the rotated preaxial limb tip tissues, 180° tip rotations were made at the proximodistal level with the highest incidence of twinning. In these cases, the adjacent stump posterior border tissues (polarizing zone) were removed, leaving a substantial amount of the deeper postaxial stump tissue, however. The frequency of twinning from tip tissues was greatly reduced in these larvae compared to those with rotated limb tips on intact stumps. Cytological examination of supernumerary outgrowths resulting from grafts of two-nucleolate tips onto one-nucleolate stumps confirmed the preaxial source of the supernumerary outgrowths.     

人A-to-I RNA编辑事件对外显子剪接增强子潜在影响的生物信息学分析

目的:研究人A-to-I RNA编辑事件对外显子剪接增强子(ESE)的潜在影响。方法:搜集文献报道的人A-to-I RNA编辑位点,并筛选包含有A-to-I RNA编辑位点的ESE,分析人A-to-I RNA编辑前后单碱基变化对ESE的潜在影响。结果:3640个A-to-I RNA编辑位点可能使其所在的ESE功能发生潜在改变;A-to-I RNA编辑事件对不同类型ESE的潜在影响不同。结论:A-to-I RNA编辑事件可能潜在影响ESE的功能,对ESE的潜在影响为量的调节,而非质的改变。     

Isolation and characterization of a 7 S RNP particle from mature Xenopus laevis oocytes

Mature oocytes of Xenopus laevis contain a 7 S RNP particle consisting of two components, ribosomal 5 S RNA and a protein of Mr approximately 45000. The structure of the free 5 S rRNA and the 7 S RNP complex has been studied by diethylpyrocarbonate modification of adenines. A74, A77, A90, A100, A101 and A103 of the 5 S rRNA are protected upon association of the protein.