Editing Function Research Articles

Role of a DEF/Y motif in histone H2A-H2B recognition and nucleosome editing

The SWR complex edits the histone composition of nucleosomes at promoters to facilitate transcription by replacing the two nucleosomal H2A-H2B (A-B) dimers with H2A.Z-H2B (Z-B) dimers. Swc5, a subunit of SWR, binds to A-B dimers, but its role in the histone replacement reaction was unclear. In this study, we showed that Swc5 uses a tandem DEF/Y motif within an intrinsically disordered region to engage the A-B dimer. A 2.37-Å X-ray crystal structure of the histone binding domain of Swc5 in complex with an A-B dimer showed that consecutive acidic residues and flanking hydrophobic residues of Swc5 form a cap over the histones, excluding histone-DNA interaction. Mutations in Swc5 DEF/Y inhibited the nucleosome editing function of SWR in vitro. Swc5 DEF/Y interacts with histones in vivo, and the extent of this interaction is dependent on the remodeling ATPase of SWR, supporting a model in which Swc5 acts as a wedge to promote A-B dimer eviction. Given that DEF/Y motifs are found in other evolutionary unrelated chromatin regulators, this work provides the molecular basis for a general strategy used repeatedly during eukaryotic evolution to mobilize histones in various genomic functions.

The Function and Genomic Landscape of RNA Editing in HhESCs and Neural Lineage Differentiation

The Function and Genomic Landscape of RNA Editing in HhESCs and Neural Lineage Differentiation

Trans-editing by aminoacyl-tRNA synthetase-like editing domains.

Aminoacyl-tRNA synthetases (aaRS) are ubiquitous enzymes responsible for aminoacyl-tRNA (aa-tRNA) synthesis. Correctly formed aa-tRNAs are necessary for proper decoding of mRNA and accurate protein synthesis. tRNAs possess specific nucleobases that promote selective recognition by cognate aaRSs. Selecting the cognate amino acid can be more challenging because all amino acids share the same peptide backbone and several are isosteric or have similar side chains. Thus, aaRSs can misactivate non-cognate amino acids and produce mischarged aa-tRNAs. If left uncorrected, mischarged aa-tRNAs deliver their non-cognate amino acid to the ribosome resulting in misincorporation into the nascent polypeptide chain. This changes the primary protein sequence and potentially causes misfolding or formation of non-functional proteins that impair cell survival. A variety of proofreading or editing pathways exist to prevent and correct mistakes in aa-tRNA formation. Editing may occur before the amino acid transfer step of aminoacylation via hydrolysis of the aminoacyl-adenylate. Alternatively, post-transfer editing, which occurs after the mischarged aa-tRNA is formed, may be carried out via a distinct editing site on the aaRS where the mischarged aa-tRNA is deacylated. In recent years, it has become clear that most organisms also encode factors that lack aminoacylation activity but resemble aaRS editing domains and function to clear mischarged aa-tRNAs in trans. This review focuses on these trans-editing factors, which are encoded in all three domains of life and function together with editing domains present within aaRSs to ensure that the accuracy of protein synthesis is sufficient for cell survival.

CRISPR/Cas9 high-throughput screening in cancer research

In recent years, CRISPR/Cas9 technology has developed rapidly. With its accurate, fast, and simple editing functions that can achieve gene activation, interference, knockout, and knock-in, it has become a powerful genetic screening tool that is widely used in various models, including cell lines of mice and zebrafish. The use of CRISPR system to construct a genomic library for high-throughput screening is the main strategy for research of disease, especially tumor target gene research. This article reviews the basic principles and latest developments of CRISPR/Cas9 library screening technology strategies to improve its off-target effect, the basic workflow of library screening, and its application in tumor research.

An Integrated Development Environment for the Prototype Verification System

The steep learning curve of formal technologies is a well-known barrier to the adoption of formal verification tools in industry. This paper presents VSCode-PVS, a modern integrated development environment for the Prototype Verification System (PVS). This new environment integrates the editing and proof management functionalities of PVS in Visual Studio Code, a popular code editor widely used by software developers. VSCode-PVS provides functionalities that developers expect to find in modern verification tools, but are not available in the standard Emacs front-end of PVS, such as auto-completion, point-and-click navigation of definitions, live diagnostics for errors, and literate programming. The main features and architecture of the environment are presented, along with a comparison with other similar tools.

The mechanism of β-N-methylamino-l-alanine inhibition of tRNA aminoacylation and its impact on misincorporation.

β-N-methylamino-l-alanine (BMAA) is a nonproteinogenic amino acid that has been associated with neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and Alzheimer's disease (AD). BMAA has been found in human protein extracts; however, the mechanism by which it enters the proteome is still unclear. It has been suggested that BMAA is misincorporated at serine codons during protein synthesis, but direct evidence of its cotranslational incorporation is currently lacking. Here, using LC-MS-purified BMAA and several biochemical assays, we sought to determine whether any aminoacyl-tRNA synthetase (aaRS) utilizes BMAA as a substrate for aminoacylation. Despite BMAA's previously predicted misincorporation at serine codons, following a screen for amino acid activation in ATP/PPi exchange assays, we observed that BMAA is not a substrate for human seryl-tRNA synthetase (SerRS). Instead, we observed that BMAA is a substrate for human alanyl-tRNA synthetase (AlaRS) and can form BMAA-tRNAAla by escaping from the intrinsic AlaRS proofreading activity. Furthermore, we found that BMAA inhibits both the cognate amino acid activation and the editing functions of AlaRS. Our results reveal that, in addition to being misincorporated during translation, BMAA may be able to disrupt the integrity of protein synthesis through multiple different mechanisms.

CRISPR System: A High-throughput Toolbox for Research and Treatment of Parkinson's Disease.

In recent years, the innovation of gene-editing tools such as the CRISPR/Cas9 system improves the translational gap of treatments mediated by gene therapy. The privileges of CRISPR/Cas9 such as working in living cells and organs candidate this technology for using in research and treatment of the central nervous system (CNS) disorders. Parkinson's disease (PD) is a common, debilitating, neurodegenerative disorder which occurs due to loss of dopaminergic neurons and is associated with progressive motor dysfunction. Knowledge about the pathophysiological basis of PD has altered the classification system of PD, which manifests in familial and sporadic forms. The first genetic linkage studies in PD demonstrated the involvement of Synuclein alpha (SNCA) mutations and SNCA genomic duplications in the pathogenesis of PD familial forms. Subsequent studies have also insinuated mutations in leucine repeat kinase-2 (LRRK2), Parkin, PTEN-induced putative kinase 1 (PINK1), as well as DJ-1 causing familial forms of PD. This review will attempt to discuss the structure, function, and development in genome editing mediated by CRISP/Cas9 system. Further, it describes the genes involved in the pathogenesis of PD and the pertinent alterations to them. We will pursue this line by delineating the PD linkage studies in which CRISPR system was employed. Finally, we will discuss the pros and cons of CRISPR employment vis-à-vis the process of genome editing in PD patients' iPSCs.

КЛАСИФІКАЦІЯ ТИПІВ ТА ПРИЙОМІВ МОНТАЖУ ЗА ПРИНЦИПОМ МОТИВАЦІЇ ЗМІНИ КАДРУ

Going back to history of film editing we can see quite a number of systematic attempts to classify the editing types. Bela Balazs, Vsevolod Pudovkin, Semen Timoshenko, Sergei Eisenstein, Raymond Spottiswoode were doing their the best at the time of 1920s-1930s to construct the classification of film editing types. Being at that time at the edge of the “film practice”, nowadays these attempts become aware of the lack of conjunction in “film practice” and “film science” that have been developed up to now. Moreover, the mentioned attempts were accented mainly on emotional component of audience perception. The real functionality of the cut, at this very moment, just at this very frame happened to be out of the look. In addition, all classifications were built on linear principle – i.e. the simple list of types. It is clear now that the simple listing cannot cover all the variety of existing editing types, and that it is not possible to mix the editing types and editing techniques in one simple listing.The scope of the article is within the multi-level hierarchical classification that is based on the motivation principle of the cut. That means the functional causes for film director or film editor to cut at a frame to achieve the desired impact on audiovisual perception. This makes it easy to construct the conjunction of editing types and editing techniques, the latter being the consequently subordinated to functions of editing.There are three main functions of editing in terms of primitive audiovisual perception of shot sequences that apply on content perception. These are:continuity (or continuity editing), meaning just to tell the story without the redundant details (i.e. shots);intellectual (or intellectual comparison of the shots), meaning the effect of any one shot differs sharply from that of its precursor and successor, resulting in an impact of the sensation and concept deriving from contiguous shots; and from this impact may arise a third concept different from that of either of the components which produced it;disjunctive (or provocative editing), meaning the effect of combining the shots together that are against the editing rules, with the deliberate aim to attract the audience attention, sometimes even to shock (the best samples could be found with the almost every music video, however, it happened to be the distinctive practice for many feature films, as well).

RR-APET - Heart rate variability analysis software

Background and ObjectivesHeart rate variability (HRV) has increasingly been linked to medical phenomena and several HRV metrics have been found to be good indicators of patient health. This has enabled generalised treatment plans to be developed in order to respond to subtle personal differences that are reflected in HRV metrics. There are several established HRV analysis platforms and methods available within the literature; some of which provide command line operation across databases but do not offer extensive graphical user interface (GUI) and editing functionality, while others offer extensive ECG editing but are not feasible over large datasets without considerable manual effort. The aim of this work is to provide a comprehensive open-source package, in a well known and multi-platform language, that offers considerable graphical signal editing features, flexibility within the algorithms used for R-peak detection and HRV quantification, and includes graphical functionality for batch processing. Thereby, providing a platform suited to either physician or researcher. MethodsRR-APET’s software was developed in the Python language and is modular in format, providing a range of different modules for established R-peak detection algorithms, as well as an embedded template for alternate algorithms. These modules also include several easily adjustable features, allowing the user to optimise any of the algorithms for different ECG signals or databases. Additionally, the software’s user-friendly GUI platform can be operated by both researchers or medical professionals to accomplish different tasks, such as: the in-depth visual analysis of a single ECG, or the analysis multiple signals in a single iteration using batch processing. RR-APET also supports several popular data formats, including text, HDF5, Matlab, and Waveform Database (WFDB) files. ResultsThe RR-APET platform presents multiple metrics that quantify the heart rate variability features of an R-to-R interval series, including time-domain, frequency-domain, and nonlinear metrics. When known R-peak annotations are available, positive predictability, sensitivity, detection error rate, and accuracy measures are also provided to assess the validity of the implemented R-peak detection algorithm. RR-APET scored an overall usability rating of 4.16 out of a possible 5, when released on a trial basis for user evaluation. ConclusionsWith its unique ability to both create and operate on large databases, this software provides a strong platform from which to conduct further research in the field of HRV analytics and its correlation to patient healthcare outcomes. This software is available free of charge at https://gitlab.com/MegMcC/rr-apet-hrv-analysis-software and can be operated as an executable file within Windows, Mac and Linux systems.

Opacity Enforcement Using Nondeterministic Publicly Known Edit Functions

This note investigates enforcement of opacity by nondeterministic edit functions. The edit functions alter the system's output by inserting fictitious events or erasing observed events. The edit decisions are randomly made while not known by the outside environment a priori . There is an intruder characterized as a passive observer with malicious goals to infer the secrets of the system. We require that opacity be enforced when the intruder may or may not know the implementation of edit functions. This requirement is termed as private and public safety. We also restrict the operation of edit functions by defining edit constraints. Then, the opacity enforcement problem is transformed to a three-player game among the edit function, the environment, and a dummy player, which helps to determine edit decisions. A game structure called the all edit structure (AES) is introduced to characterize the interaction among those players. It embeds all privately safe edit functions and may also embed publicly safe edit functions. Based on the AES, we present an algorithm that provably synthesizes nondeterministic edit functions that satisfy both private safety and public safety.

Design of a Lightweight Autonomous Learning System for the Course of Software Testing Based on Android

A lightweight autonomous learning system for the course of software testing based on Android is developed according to the local teaching environment, and the system is fully applied on autonomous learning process outside class on campus. The system is designed on the architecture of SSH (Struts-Spring-Hibernate) which is based on J2EE for web server, and the functions of Course Information, Resource Management, Exercise, User Management and Online Discussion can be provided to client users by Android platform. Finally, further work such as how to add the function of online exam for subjective exam items in the system, how to add powerful function of micro video content editing for the management of course resource, and how to integrate this system into the local informational learning platform to complete the blended learning mode on SPOC (Small Private Online Course) are expected in the paper.

Identifying the growth associating genes of Nannochloropsis oceanica by bulked mutant analysis (BMA) and RNA sequencing (BMR-seq)

Microalgal strains (lines), either economically or ecologically important or both, are usually clones derived from a single cell. The functions of their genes are verified usually through either loss of function or gain of function approaches, for example, overexpression, knockout, silencing, and editing. In comparison with plants and animals, the genetic studies of microalgae are scarce, and less genes were de novo cloned. Bulked segregant analysis (BSA) and bulked segregant RNA sequencing (BSR-seq) are widely used to identify the linking markers or control genes of sexually reproducing organisms. In order to avoid genotyping, a large amount of individuals in genome-wide association study (GWAS), we modified BSA and BSR-seq in this study to bulked mutant analysis (BMR) and bulked mutant RNA sequencing (BMR-seq). In order to generate stable variations, we used Zeocin to cause the double-strand breaks of DNA (DSBs), and generate rich InDel variations during DNA repairing. We identified a set of growth performance associating SNPs and InDels among the bulked DNA and bulked RNA. We found that the function-known genes either containing these variations or locating upstream or downstream of these variations are highly related to the complex growth performance trait of Nannochloropsis oceanica. Our findings verified that Zeocin is effective to create rich InDels in monoploid and asexual N. oceanica genome, and BMA and BMR-seq are applicable for identifying the associating SNPs and InDels of the growth performance trait. We prefer to use BMA to identify trait associating genes and variations; microalgae are fast growing with a short generation time, and mRNAs are the transcripts of genes and share the variations with DNA. We believe that the methods we developed in this study will facilitate the genetic studies of the traits of diverse microalgae.

CRISPR/Cas9-based epigenome editing: An overview of dCas9-based tools with special emphasis on off-target activity.

Molecular tools for gene regulation and epigenome editing consist of two main parts: the targeting moiety binding a specific genomic locus and the effector domain performing the editing or regulatory function. The advent of CRISPR-Cas9 technology enabled easy and flexible targeting of almost any locus by co-expression of a small sgRNA molecule, which is complementary to the target sequence and forms a complex with Cas9, directing it to that particular target. Here, we review strategies for recruitment of effector domains, used in gene regulation and epigenome editing, to the dCas9 DNA-targeting protein. To date, the most important CRISPR-Cas9 applications in gene regulation are CRISPR activation or interference, while epigenome editing focuses on targeted changes in DNA methylation and histone modifications. Several strategies for signal amplification by recruitment of multiple effector domains deserve special focus. While some approaches rely on altering the sgRNA molecule and extending it with aptamers for effector domain recruitment, others use modifications to the Cas9 protein by direct fusions with effector domains or by addition of an epitope tag, which also has the ability to bind multiple effector domains. A major barrier to the widespread use of CRISPR-Cas9 technology for therapeutic purposes is its off-target effect. We review efforts to enhance CRISPR-Cas9 specificity by selection of Cas9 orthologs from various bacterial species and their further refinement by introduction of beneficial mutations. The molecular tools available today enable a researcher to choose the best balance of targeting flexibility, activity amplification, delivery method and specificity.

JViz.RNA 4.0-Visualizing pseudoknots and RNA editing employing compressed tree graphs.

Previously, we have introduced an improved version of jViz.RNA which enabled faster and more stable RNA visualization by employing compressed tree graphs. However, the new RNA representation and visualization method required a sophisticated mechanism of pseudoknot visualization. In this work, we present our novel pseudoknot classification and implementation of pseudoknot visualization in the context of the new RNA graph model. We then compare our approach with other RNA visualization software, and demonstrate jViz.RNA 4.0’s benefits compared to other software. Additionally, we introduce interactive editing functionality into jViz.RNA and demonstrate its benefits in exploring and building RNA structures. The results presented highlight the new high degree of utility jViz.RNA 4.0 now offers. Users are now able to visualize pseudoknotted RNA, manipulate the resulting automatic layouts to suit their individual needs, and change both positioning and connectivity of the RNA molecules examined. Care was taken to limit overlap between structural elements, particularly in the case of pseudoknots to ensure an intuitive and informative layout of the final RNA structure.Availability: The software is freely available at: https://jviz.cs.sfu.ca/.

Getting in the groove: Editing of MHC-I antigen repertoires by molecular chaperones is governed by a network of protein dynamics

Abstract Molecular chaperones TAPBPR (TAP-binding protein related) and tapasin associate with class-I major histocompatibility complex (MHC-I) molecules to promote optimization (editing) of peptide cargo. Using solution NMR, we investigate the molecular mechanism of peptide exchange performed by the 90 kDa chaperone protein complex. We identify TAPBPR-induced conformational changes on conserved MHC-I surfaces, consistent with our independently determined X-ray structure of the empty complex. Conformational dynamics present in the empty MHC-I are stabilized by TAPBPR in a peptide-deficient complex, and become progressively dampened with increasing peptide occupancy. Incoming peptides are recognized by the chaperoned groove according to the global stability of the final pMHC-I product, and anneal in a native-like conformation. Our results demonstrate an inverse relationship between MHC-I occupancy by peptide and the affinity of TAPBPR for such pMHC-I molecules, where the lifetime of transiently bound peptides controls the dynamic regulation of a conformational switch, located near the TAPBPR binding site, which triggers TAPBPR release. We further discuss the role of protein dynamics in shaping chaperone specificity towards different human and murine class-I MHC allotypes, and present the high-resolution cryoEM structure of a human A*02/TAPBPR complex with novel insights into the antigen editing mechanism. These results suggest a similar mechanism for the editing function of tapasin in the peptide-loading complex, and provide a molecular blueprint for the design of novel chaperones with tailored antigen editing functions.

Studying Lysine Acetylation of Aminoacyl‐tRNA Synthetases in Escherichia coli

Aminoacyl‐tRNA synthetases (AARSs) generate aminoacylated tRNAs for ribosomal protein synthesis, and play key roles in maintaining high fidelity of translation. A series of acetylome studies have shown that the family of AARSs is one of the protein families which have both the most acetylation sites and the highest acetylation levels. However, those putative acetylation targets have been less studied, mostly because of the challenge to produce homogeneously acetylated proteins at specific positions which needs the knowledge of specific acetyltransferases for target lysine residues. To overcome this issue, we applied the genetic code expansion strategy to co‐translationally incorporate acetyllysine at the target positions directly. We totally generated 25 site‐specifically acetylated variants of four AARSs. Kinetic analyses were performed to biochemically characterize the impact of site‐specific acetylation on AARS functions, including both tRNA aminoacylation and editing activities. The results showed that impacts of acetylation were different for class I and class II AARSs, and also varied among the same class of AARSs. Combined with quantitative proteomic studies, we proposed that the impact of acetylation on AARS editing activities could be more significant than that on tRNA aminoacylation activities. To the best of our knowledge, this is the first work to show the association of protein acetylation with the AARS editing function. Furthermore, using threonyl‐tRNA synthetase (ThrRS) as a representative, both in vivo and in vitro studies were performed to characterize the acetylation and deacetylation processes of AARSs. Although nonenzymatic acetylation and CobB‐dependent deacetylation were concluded, our results also indicated existence of additional acetyltransferases and deacetylases for AARSs.Support or Funding InformationThis work was supported by Oak Ridge Associated Universities, Arkansas Biosciences Institute, and the University of Arkansas.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A Novel Direct Manipulation Technique for Motion-editing using a Timeline-based Interface

Recently, motion data is becoming increasingly available for creating computer animation. Motion capture is one of the systems that can generate such motion data. However, it is not suitable to capture a lot of motion due to the cost of motion capture technique, and the diculty of its postprocessing. This paper presents a timeline-based motion-editing system that enables users to perform motion-editing tasks easily and quickly. A motion sequence is summarized and displayed in the 3D environment as a set of editable icons. Users can edit the motion data by performing a sequence of operations on a single key frame or over an interval. The recorded sequence is then propagated automatically to a set of target key frames or intervals, which can be either user dened or system dened. In addition, we provide a simple interaction method for manipulating the duration of specic intervals in the motion data. Methods for combining and synchronizing two dierent motions are also provided in this system. In contrast with the previous work that allows only temporal editing, the proposed system provides editing functions for both geometry and temporal editing. We describe a user study that demonstrated the eciency of the proposed system.

Structure, dynamics and roX2-lncRNA binding of tandem double-stranded RNA binding domains dsRBD1,2 of Drosophila helicase Maleless

Maleless (MLE) is an evolutionary conserved member of the DExH family of helicases in Drosophila. Besides its function in RNA editing and presumably siRNA processing, MLE is best known for its role in remodelling non-coding roX RNA in the context of X chromosome dosage compensation in male flies. MLE and its human orthologue, DHX9 contain two tandem double-stranded RNA binding domains (dsRBDs) located at the N-terminal region. The two dsRBDs are essential for localization of MLE at the X-territory and it is presumed that this involves binding roX secondary structures. However, for dsRBD1 roX RNA binding has so far not been described. Here, we determined the solution NMR structure of dsRBD1 and dsRBD2 of MLE in tandem and investigated its role in double-stranded RNA (dsRNA) binding. Our NMR and SAXS data show that both dsRBDs act as independent structural modules in solution and are canonical, non-sequence-specific dsRBDs featuring non-canonical KKxAXK RNA binding motifs. NMR titrations combined with filter binding experiments and isothermal titration calorimetry (ITC) document the contribution of dsRBD1 to dsRNA binding in vitro. Curiously, dsRBD1 mutants in which dsRNA binding in vitro is strongly compromised do not affect roX2 RNA binding and MLE localization in cells. These data suggest alternative functions for dsRBD1 in vivo.

Evolutionarily significant A-to-I RNA editing events originated through G-to-A mutations in primates

BackgroundRecent studies have revealed thousands of A-to-I RNA editing events in primates, but the origination and general functions of these events are not well addressed.ResultsHere, we perform a comparative editome study in human and rhesus macaque and uncover a substantial proportion of macaque A-to-I editing sites that are genomically polymorphic in some animals or encoded as non-editable nucleotides in human. The occurrence of these recent gain and loss of RNA editing through DNA point mutation is significantly more prevalent than that expected for the nearby regions. Ancestral state analyses further demonstrate that an increase in recent gain of editing events contribute to the over-representation, with G-to-A mutation site as a favorable location for the origination of robust A-to-I editing events. Population genetics analyses of the focal editing sites further reveal that a portion of these young editing events are evolutionarily significant, indicating general functional relevance for at least a fraction of these sites.ConclusionsOverall, we report a list of A-to-I editing events that recently originated through G-to-A mutations in primates, representing a valuable resource to investigate the features and evolutionary significance of A-to-I editing events at the population and species levels. The unique subset of primate editome also illuminates the general functions of RNA editing by connecting it to particular gene regulatory processes, based on the characterized outcome of a gene regulatory level in different individuals or primate species with or without these editing events.

An Approach of Extending Net Disks’ Collaborative Document Editing Functionality

Net disks are playing an increasingly important role in our daily works. Currently, net disks have given us an ideal ecosystem to store and share files; however, they generally cannot support collaborative document editing. To overcome the shortage, after analyzing the model of cloud storage and net disks, this paper proposes a solution to extend net disks’ collaborative editing functionality via API. Based on the proposed method, a net disk system coupled with collaborative document editing functionality is built. The experiment result shows that the extending approach is feasible and effective. This approach can not only make net disks support document collaborative editing, but also be easy to be implemented.