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An efficient iron-catalyzed hydroarylation of 1,3-dienes with indoles has been developed for the first time.

A palladium-catalyzed regioselective 1,2-alkynyl-carbonalization of conjugated dienes with ethynylbenziodazolone (EBZ) and indoles has been developed for the first time. Various molecules containing alkenyl, alkynyl, and indole groups were readily obtained. Moreover, the resulting products can be applied to various derivatizations. This protocol uses EBZ as an electrophilic alkynylating reagent, avoiding the byproduct of dimerization of alkynes.

Circular RNAs (circRNAs) are covalently enclosed, single-stranded RNAs produced by back-splicing of pre-mRNA exons that have recently emerged as an important class of molecules in gene expression regulation. circRNAs share overlapping sequences with their cognate linear mRNAs except the back-splicing junction (BSJ) sites. This feature makes it difficult to discriminate between the functions of circRNAs and their cognate mRNAs. We previously reported that the programmable RNA-guided, RNA-targeting CRISPR-Cas13 (RfxCas13d) system effectively and specifically discriminates circRNAs from mRNAs by using guide RNAs (gRNAs) targeting sequences across BSJ sites. Here, we describe a detailed protocol based on this RfxCas13d/BSJ-gRNA system for large-scale functional circRNA screening in human cell lines. The protocol includes gRNA library design, construction and transduction, analysis of screening results and validation of functional circRNA candidates. In total, it takes ~3-4 months of collaborative work between a well-trained molecular biologist and a bioinformatic expert. This protocol can be applied both in cells and in vivo to identify highly expressed circRNAs affecting cell growth, either in unperturbed conditions or under environmental stimulation, without disturbing their cognate linear mRNAs.

Background. The tissues and organs of premature infants are immature and easily damaged by external adverse factors, leading to functional development disorders and abnormalities. Besides, the incidence of premature babies in various countries has an increasing trend, with the incidence rate exceeding 10%. Objective. This study aims to investigate the neurodevelopment and the incidence of various developmental delays, cerebral palsy, autism spectrum disorder, and audio-visual impairment in premature infants under 34 weeks of gestation from birth to 2 years of age, so as to provide the basis for early intervention of premature infants in the clinic. Methods A cohort of premature infants was established using 263 premature infants with a gestational age of 28–33 + 6 weeks who were born alive from March 1, 2018, to February 28, 2019, in four tertiary hospitals in Shenzhen. In addition, 263 full-term infants of the same sex who were born in the same period in the four hospitals were randomly selected and paired in a ratio of 1 : 1 as the control group. The subjects were assessed for neurodevelopment using the Gesell test scale at 6, 12, 18, and 24 months after birth (premature infants were corrected for months). We calculated the neurodevelopmental indicators of children in each month of age and the incidence of various developmental delays, cerebral palsy, autism spectrum disorder, and audio-visual impairment in the two groups. Results The results of this study showed that the cohort of premature infants with birth gestational age less than 34 weeks had higher adaptive, fine motor, and personal-social energy domain development quotient (DQ) values from the corrected gestational age of 6 months to the corrected gestational age of 24 months after birth compared with the full-term cohort. And it also achieved catch-up growth in neurological development, but the detection rates of neurodevelopmental abnormalities at the corrected gestational age of 12 and 24 months were higher than those in the full-term cohort. Conclusion It is important to reduce the disability rate and degree of premature infants by strengthening the systematic management, early promotion and supervision, as well as early intervention for preterm infants with developmental abnormalities who were born at gestational age less than 34 weeks after birth.

Long non-coding RNAs (lncRNAs) associate with RNA-binding proteins (RBPs) to form lncRNA-protein complexes that act in a wide range of biological processes. Understanding the molecular mechanism of how a lncRNA-protein complex is assembled and regulated is key for their cellular functions. In this mini-review, we outline molecular methods used to identify lncRNA-protein interactions from large-scale to individual levels using bulk cells as well as those recently developed imaging and single-molecule approaches that are capable of visualizing RNA-protein assemblies in single cells and in real-time. Focusing on the latter group of approaches, we discuss their applications and limitations, which nevertheless have enabled quantification and comprehensive dissection of RNA-protein interactions possible.

Bananas (Musaspp.) are one of the most important tropical fruits and staple food, which are of great significance to human societies. Plantain and Silk are two important banana subgroups, which are both triploid hybrids (AAB) between the wild diploidMusa acuminataandM. balbisiana. In this study, we reported the first haplotype-resolved genome assembly of Plantain and Silk bananas with genome size of approximately 1.4 Gb. We discovered widespread asymmetric evolution in the subgenomes of Plantain and Silk, which could be linked to frequent homologous exchanges (HEs) events. This is the first study to uncover the genetic makeup of triploid banana and verify that subgenome B harbors a rich source of resistance genes. Of the 88,078 and 94,988 annotated genes in Plantain and Silk, only 58.5% and 59.4% were present in all three subgenomes, with >50% genes containing differently expressed alleles in different haplotypes. We also found that Plantain is more resistant to banana Fusarium wilt, exhibiting a much faster defense response after pathogenic fungi infection. Many differentially expressed genes in abscisic acid, ethylene, jasmonic acid and salicylic acid pathways were identified in Plantain. Our analysis revealed that MpMYB36 promotes the biosynthesis of secondary cell wall and deposition of lignin by directly binding to the promoter of MpPAL and MpHCT, which allows Plantain to inhibit the penetration of early infection. Moreover, the insertion of the key carotenoid synthesis gene (CRTISO) may be the potential genetic basis for the richness of carotenoids in Plantain. Our study provides an unprecedented genomic basis for basic research and the development of elite germplasm in cultivated bananas.

Covalently closed circular RNAs (circRNAs) produced by back-splicing of exon(s) are co-expressed with their cognate linear RNAs from the same gene loci. Most circRNAs are fully overlapped with their cognate linear RNAs in sequences except the back-spliced junction (BSJ) site, thus challenging the computational detection, experimental validation and hence functional evaluation of circRNAs. Nevertheless, specific bioinformatic pipelines were developed to identify fragments mapped to circRNA-featured BSJ sites, and circRNAs were pervasively identified from non-polyadenylated RNA-seq datasets in different cell lines/tissues and across species. Precise identification and quantification of circRNAs provide a basis to further understand their functions. Here, we describe detailed computational steps to annotate and quantify circRNAs using a series of CIRCexplorer pipelines.

The human transcriptome contains many types of noncoding RNAs, which rival the number of protein-coding species. From long noncoding RNAs (lncRNAs) that are over 200 nucleotides long to piwi-interacting RNAs (piRNAs) of only 20 nucleotides, noncoding RNAs play important roles in regulating transcription, epigenetic modifications, translation, and cell signaling. Roles for noncoding RNAs in disease mechanisms are also being uncovered, and several species have been identified as potential drug targets. On May 11-14, 2021, the Keystone eSymposium "Noncoding RNAs: Biology and Applications" brought together researchers working in RNA biology, structure, and technologies to accelerate both the understanding of RNA basic biology and the translation of those findings into clinical applications.

Genes specifying long non-coding RNAs (lncRNAs) occupy a large fraction of the genomes of complex organisms. The term 'lncRNAs' encompasses RNA polymerase I (Pol I), Pol II and Pol III transcribed RNAs, and RNAs from processed introns. The various functions of lncRNAs and their many isoforms and interleaved relationships with other genes make lncRNA classification and annotation difficult. Most lncRNAs evolve more rapidly than protein-coding sequences, are cell type specific and regulate many aspects of cell differentiation and development and other physiological processes. Many lncRNAs associate with chromatin-modifying complexes, are transcribed from enhancers and nucleate phase separation of nuclear condensates and domains, indicating an intimate link between lncRNA expression and the spatial control of gene expression during development. lncRNAs also have important roles in the cytoplasm and beyond, including in the regulation of translation, metabolism and signalling. lncRNAs often have a modular structure and are rich in repeats, which are increasingly being shown to be relevant to their function. In this Consensus Statement, we address the definition and nomenclature of lncRNAs and their conservation, expression, phenotypic visibility, structure and functions. We also discuss research challenges and provide recommendations to advance the understanding of the roles of lncRNAs in development, cell biology and disease.

AIM To evaluate the cognitive function in dialysis patients over 60 years old and identify the contributing factors. METHODS A group of elderly dialysis patients in the Department of Nephrology, Pan'an People's Hospital between March 2015 and June 2018 were chosen as the subjects for this study. Patients were divided into two groups, those with cognitive impairment and those with normal cognitive function. Results of their Montreal Cognitive Assessment (MoCA) scores, Controlled Oral Word Association Test (COWAT), Wechsler Adult Intelligence Scale Digit Span subtest (WDMS), and Stanford Diagnostic Math Test (SDMT) were reviewed and analyzed. RESULTS Among the 110 elderly dialysis patients, 75 patients (68.18%) showed different levels of damage to their cognitive function. Their assessment scores on MoCA (total), MoCA subtests: visuospatial/executive, naming, attention, language, delayed recall, abstraction and orientation, COWAT (total), COWAT1, COWAT2, COWAT3, WMDS-Backward, and SDMT are significantly lower than patients with normal cognitive abilities (p

Rice (Oryza sativa), a major staple throughout the world and a model system for plant genomics and breeding, was the first crop genome sequenced almost two decades ago. However, reference genomes for all higher organisms to date contain gaps and missing sequences. Here, we report the assembly and analysis of gap-free reference genome sequences for two elite O. sativa xian/indica rice varieties, Zhenshan 97 and Minghui 63, which are being used as a model system for studying heterosis and yield. Gap-free reference genomes provide the opportunity for a global view of the structure and function of centromeres. We show that all rice centromeric regions share conserved centromere-specific satellite motifs with different copy numbers and structures. In addition, the similarity of CentO repeats in the same chromosome is higher than across chromosomes, supporting a model of local expansion and homogenization. Both genomes have over 395 non-TE genes located in centromere regions, of which ∼41% are actively transcribed. Two large structural variants at the end of chromosome 11 affect the copy number of resistance genes between the two genomes. The availability of the two gap-free genomes lays a solid foundation for further understanding genome structure and function in plants and breeding climate-resilient varieties.

Interpreting the functional impacts of genetic variants (GVs) is an important challenge for functional genomic studies in crops and next-generation breeding. Previous studies in rice (Oryza sativa) have focused mainly on the identification of GVs, whereas systematic functional annotation of GVs has not yet been performed. Here, we present a functional impact map of GVs in rice. We curated haplotype information for 17 397 026 GVs from sequencing data of 4726 rice accessions. We quantitatively evaluated the effects of missense mutations in coding regions in each haplotype based on the conservation of amino acid residues and obtained the effects of 918 848 non-redundant missense GVs. Furthermore, we generated high-quality chromatin accessibility (CA) data from six representative rice tissues and used these data to train deep convolutional neural network models to predict the impacts of 5 067 405 GVs for CA in regulatory regions. We characterized the functional properties and tissue specificity of the GV effects and found that large-effect GVs in coding and regulatory regions may be subject to selection in different directions. Finally, we demonstrated how the functional impact map could be used to prioritize causal variants in mapping populations. This impact map will be a useful resource for accelerating gene cloning and functional studies in rice, and can be freely queried in RiceVarMap V2.0 ( http://ricevarmap.ncpgr.cn ).

Circular intronic RNAs (ciRNAs) escaping from DBR1 debranching of intron lariats are co-transcriptionally produced from pre-mRNA splicing, but their turnover and mechanism of action have remained elusive. We report that RNase H1 degrades a subgroup of ciRNAs in human cells. Many ciRNAs contain high GC% and tend to form DNA:RNA hybrids (R-loops) for RNase H1 cleavage, a process that appears to promote Pol II transcriptional elongation at ciRNA-producing loci. One ciRNA, ciankrd52, shows a stronger ability of R-loop formation than that of its cognate pre-mRNA by maintaining a locally open RNA structure in vitro. This allows the release of pre-mRNA from R-loops by ci-ankrd52 replacement and subsequent ciRNA removal via RNase H1 for efficient transcriptional elongation. We propose that such an R-loop dependent ciRNA degradation likely represents a mechanism that on one hand limits ciRNA accumulation by recruiting RNase H1 and on the other hand resolves R-loops for transcriptional elongation at some GC-rich ciRNA-producing loci.

Leveraging machine learning has been shown to improve the accuracy of structure-based virtual screening. Furthermore, a tremendous amount of empirical data is publicly available, which further enhances the performance of the machine learning approach. In this proof-of-concept study, the 3CLpro enzyme of SARS-CoV-2 was used. Structure-based virtual screening relies heavily on scoring functions. It is widely accepted that target-specific scoring functions may perform more effectively than universal scoring functions in real-world drug research and development processes. It would be beneficial to drug discovery to develop a method that can effectively build target-specific scoring functions. In the current study, the bindingDB database was used to retrieve experimental data. Smina was utilized to generate protein-ligand complexes for the extraction of InteractionFingerPrint (IFP) and SimpleInteractionFingerPrint SIFP fingerprints via the open drug discovery tool (oddt). The present study found that randomforestClassifier and randomforestRegressor performed well when used with the above fingerprints along the Molecular ACCess System (MACCS), Extended Connectivity Fingerprint (ECFP4), and ECFP6. It was found that the area under the precision-recall curve was 0.80, which is considered a satisfactory level of accuracy. In addition, our enrichment factor analysis indicated that our trained scoring function ranked molecules correctly compared to smina’s generic scoring function. Further molecular dynamics simulations indicated that the top-ranked molecules identified by our developed scoring function were highly stable in the active site, supporting the validity of our developed process. This research may provide a template for developing target-specific scoring functions against specific enzyme targets.

Keeping the nucleolus a liquid condensate The nucleolus is a multilayered, membraneless nuclear condensate in which DNA polymerase I (Pol I)–mediated ribosomal DNA (rDNA) transcription and pre-rRNA processing occur in fibrillar center and dense fibrillar component (FC/DFC) units. How its biophysical properties are regulated has remained elusive. Wu et al . found that the RNA helicase DDX21 forms a shell coating each FC/DFC unit in the nucleolus (see the Perspective by Yamazaki and Hirose). The authors found that a long noncoding RNA called SLERT facilitates the transition from the open to the closed configuration of the helicase using a chaperone-like mechanism. DDX21 in the closed conformation forms loose clusters that confer the FC/DFC unit sufficient liquidity and space required for Pol I processivity. In addition, DDX21 within the loose clusters cannot approach and wrap rDNA, thus licensing rDNA for transcription. —DJ

RNA molecules fold into complex structures that enable their diverse functions in cells. Recent revolutionary innovations in transcriptome-wide RNA structural probing of living cells have ushered in a new era in understanding RNA functions. Here, we summarize the latest technological advances for probing RNA secondary structures and discuss striking discoveries that have linked RNA regulation and biological processes through interrogation of RNA structures. In particular, we highlight how different long noncoding RNAs form into distinct secondary structures that determine their modes of interactions with protein partners to realize their unique functions. These dynamic structures mediate RNA regulatory functions through altering interactions with proteins and other RNAs. We also outline current methodological hurdles and speculate about future directions for development of the next generation of RNA structure-probing technologies of higher sensitivity and resolution, which could then be applied in increasingly physiologically relevant studies.

Albicanol is a natural terpenoid derived from Dryopteris fragrans. Herein, we assessed the ability of Albicanol to protect against oxidative stress-induced senescence. Using a murine model of D-galactose (D-gal)-induced aging, we determined that Albicanol treatment can reverse D-gal-mediated learning impairments and behavioral changes, while also remediating brain tissue damage in treated mice. We found that serum SOD, CAT, GSH-Px, and T-AOC levels were significantly decreased in aging mice, and that Albicanol treatment significantly increased the serum levels of these antioxidant enzymes. We additionally evaluated the impact of Albicanol treatment on the Keap1/Nrf2/ARE signaling pathway, and found that it was able to decrease Keap1 expression while increasing the expression of Nrf2, thereby activating this signaling pathway, suppressing oxidative damage, and enhancing the expression of downstream target genes including SOD, GSH, GST, HO-1, and NQO1 in this murine aging model system. Albicanol treatment also inhibited the secretion of inflammatory TNF-a and IL-1b. Together, these data indicated that Albicanol can activate Nrf2 pathway-related genes, thereby inhibition of delayed aging by alleviating oxidative stress-induced damage.

Due to the development of sequencing technology and the great reduction in sequencing costs, an increasing number of plant genomes have been assembled, and numerous genomes have revealed large amounts of variations. However, a single reference genome does not allow the exploration of species diversity, and therefore the concept of pan-genome was developed. A pan-genome is a collection of all sequences available for a species, including a large number of consensus sequences, large structural variations, and small variations including single nucleotide polymorphisms and insertions/deletions. A simple linear pan-genome does not allow these structural variations to be intuitively characterized, so graph-based pan-genomes have been developed. These pan-genomes store sequence and structural variation information in the form of nodes and paths to store and display species variation information in a more intuitive manner. The key role of graph-based pan-genomes is to expand the coordinate system of the linear reference genome to accommodate more regions of genetic diversity. Here, we review the origin and development of graph-based pan-genomes, explore their application in plant research, and further highlight the application of graph-based pan-genomes for future plant breeding.

Covalently closed, single-stranded circular RNAs can be produced from viral RNA genomes as well as from the processing of cellular housekeeping noncoding RNAs and precursor messenger RNAs. Recent transcriptomic studies have surprisingly uncovered that many protein-coding genes can be subjected to backsplicing, leading to widespread expression of a specific type of circular RNAs (circRNAs) in eukaryotic cells. Here, we discuss experimental strategies used to discover and characterize diverse circRNAs at both the genome and individual gene scales. We further highlight the current understanding of how circRNAs are generated and how the mature transcripts function. Some circRNAs act as noncoding RNAs to impact gene regulation by serving as decoys or competitors for microRNAs and proteins. Others form extensive networks of ribonucleoprotein complexes or encode functional peptides that are translated in response to certain cellular stresses. Overall, circRNAs have emerged as an important class of RNAmolecules in gene expression regulation that impact many physiological processes, including early development, immune responses, neurogenesis, and tumorigenesis.

Chen et al. (2021) have identified many internal ribosome entry site-like elements that can potentially drive circRNA translation. Dozens of such element-containing circRNAs-encoded peptides are validated, among which a circFGFR1-encoded protein acts as an antagonist of FGFR1.

Evidence accumulated over the past decade shows that long non-coding RNAs (lncRNAs) are widely expressed and have key roles in gene regulation. Recent studies have begun to unravel how the biogenesis of lncRNAs is distinct from that of mRNAs and is linked with their specific subcellular localizations and functions. Depending on their localization and their specific interactions with DNA, RNA and proteins, lncRNAs can modulate chromatin function, regulate the assembly and function of membraneless nuclear bodies, alter the stability and translation of cytoplasmic mRNAs and interfere with signalling pathways. Many of these functions ultimately affect gene expression in diverse biological and physiopathological contexts, such as in neuronal disorders, immune responses and cancer. Tissue-specific and condition-specific expression patterns suggest that lncRNAs are potential biomarkers and provide a rationale to target them clinically. In this Review, we discuss the mechanisms of lncRNA biogenesis, localization and functions in transcriptional, post-transcriptional and other modes of gene regulation, and their potential therapeutic applications., Recent studies have provided novel insight into the biogenesis of long non-coding RNAs (lncRNAs) and their specific functions. The functions of lncRNAs vary from transcriptional and post-transcriptional gene regulation to the assembly and function of membraneless nuclear bodies, and are relevant to neuronal disorders, immune responses and cancer.

Circular RNAs (circRNAs) produced from back-spliced exons are widely expressed, but individual circRNA functions remain poorly understood owing to the lack of adequate methods for distinguishing circRNAs from cognate messenger RNAs with overlapping exons. Here, we report that CRISPR–RfxCas13d can effectively discriminate circRNAs from mRNAs by using guide RNAs targeting sequences spanning back-splicing junction (BSJ) sites featured in RNA circles. Using a lentiviral library that targets sequences across BSJ sites of highly expressed human circRNAs, we show that a group of circRNAs are important for cell growth mostly in a cell-type-specific manner and that a common oncogenic circRNA, circFAM120A, promotes cell proliferation by preventing the mRNA for family with sequence similarity 120A (FAM120A) from binding the translation inhibitor IGF2BP2. Further application of RfxCas13d–BSJ-gRNA screening has uncovered circMan1a2, which has regulatory potential in mouse embryo preimplantation development. Together, these results establish CRISPR–RfxCas13d as a useful tool for the discovery and functional study of circRNAs at both individual and large-scale levels. This paper describes a CRISPR–Cas13 system to effectively target circRNAs and screen their functions in vitro and in vivo, which enables the study of relevant circRNA phenotypes in human cell proliferation and in mouse embryogenesis.

Paraspeckles are a type of subnuclear bodies built on the long noncoding RNA NEAT1 (nuclear paraspeckle assembly transcript 1, also known as MEN-ε/β or VINC-1). Paraspeckles are involved in many physiological processes including cellular stress responses, cell differentiation, corpus luteum formation and cancer progression. Recently, ultra-resolution microscopy coupled with multicolor-labeling of paraspeckle components (the NEAT1 RNA and paraspeckle proteins) revealed the exquisite details of paraspeckle structure and function. NEAT1 transcripts are radially arranged to form a core–shell spheroidal structure, while paraspeckle proteins (PSPs) localize within different layers. Functional dissection of NEAT1 shows that the subdomains of NEAT1_2 are important for RNA stability, isoform switching and paraspeckle assembly via a liquid–liquid phase separation (LLPS) mechanism. We review recent progress on structure and organization of paraspeckles as well as how paraspeckles spatiotemporally control gene regulation through sequestration of diverse proteins and RNAs in cells.

The papain-like protease (PLpro) is vital for the replication of coronaviruses (CoVs), as well as for escaping innate-immune responses of the host. Hence, it has emerged as an attractive antiviral drug-target. In this study, computational approaches were employed, mainly the structure-based virtual screening coupled with all-atom molecular dynamics (MD) simulations to computationally identify specific inhibitors of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) PLpro, that can be further developed as potential pan-PLpro based broad-spectrum antiviral drugs. The sequence, structure, and functional conserveness of most deadly human CoVs PLpro were exploited, and it was revealed that functionally important catalytic triad residues are well conserved among SARS-CoV, SARS-CoV-2, and middle east respiratory syndrome coronavirus (MERS-CoV). The subsequent screening of a focused protease inhibitors database composed of ∼7000 compounds resulted in the identification of three candidate compounds, ADM_13083841, LMG_15521745, and SYN_15517940. These three compounds established conserved interactions which were further explored through MD simulations, free energy calculations, and residual energy contribution estimated by MM-PB(GB)SA method. All these compounds showed stable conformation and interacted well with the active residues of SARS-CoV-2 PLpro and showed consistent interaction profile with SARS-CoV PLpro and MERS-CoV PLpro as well. Conclusively, the reported SARS-CoV-2 PLpro specific compounds could serve as seeds for developing potent pan-PLpro based broad-spectrum antiviral drugs against deadly human coronaviruses. Moreover, the presented information related to binding site residual energy contribution could lead to further optimization of these compounds., Graphical abstract Image 1, Highlights • An integrated structure-based computational approach identified potential inhibitors of SARS-CoV-2 PLpro. • Quantitative validation of inhibitors’ binding and molecular interaction profiles were elucidated through molecular dynamic simulation and energy decomposition analysis. • The screened inhibitors in present study may lead to develop pan-PLpro based broad spectrum antiviral agents against the diversity of continuedly evolving Coronaviruses.