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Highly structured RNA molecules usually interact with each other, and associate with various RNA-binding proteins, to regulate critical biological processes. However, RNA structures and interactions in intact cells remain largely unknown. Here, by coupling proximity ligation mediated by RNA-binding proteins with deep sequencing, we report an RNA in situ conformation sequencing (RIC-seq) technology for the global profiling of intra- and intermolecular RNA-RNA interactions. This technique not only recapitulates known RNA secondary structures and tertiary interactions, but also facilitates the generation of three-dimensional (3D) interaction maps of RNA in human cells. Using these maps, we identify noncoding RNA targets globally, and discern RNA topological domains and trans-interacting hubs. We reveal that the functional connectivity of enhancers and promoters can be assigned using their pairwise-interacting RNAs. Furthermore, we show that CCAT1-5L--a super-enhancer hub RNA--interacts with the RNA-binding protein hnRNPK, as well as RNA derived from the MYC promoter and enhancer, to boost MYC transcription by modulating chromatin looping. Our study demonstrates the power and applicability of RIC-seq in discovering the 3D structures, interactions and regulatory roles of RNA. RNA in situ conformation sequencing (RIC-seq) enables the generation of three-dimensional interaction maps of RNA in cells, which sheds light on the interactions and regulatory functions of RNA., Author(s): Zhaokui Cai [sup.1] [sup.2] , Changchang Cao [sup.1] , Lei Ji [sup.1] , Rong Ye [sup.1] [sup.2] , Di Wang [sup.1] [sup.2] , Cong Xia [sup.3] , Sui Wang [...]

H2S is an endogenous gas signaling molecule and its multiple biological effects have been demonstrated. The abnormal level of H2S is closely related to the occurrence and development of many diseases, and H2S donors has important pharmacological implications. In recent years, H2S donors represented by ADTOH (5-(4-hydroxyphenyl)-3H-1,2-dithiole-3-thione) are often used to synthesize new 'conjugate' compounds that can release H2S and parent drugs. These hybrids retain the pharmacological activity of the parent drugs and H2S and have a synergistic effect. ADTOH and parent drug hybrids have become one of the important strategies for the development of H2S donor conjugate drugs. This review summarizes molecular hybrids between ADTOH and clinical drugs to provide new ideas for the study of H2S donor drug design. [ABSTRACT FROM AUTHOR]

Chemotherapeutics remain the first choice for advanced gastric cancers (GCs). However, drug resistance and unavoidable severe toxicity lead to chemotherapy failure and poor prognosis. Long noncoding RNAs (lncRNAs) play critical roles in tumor progression in many cancers, including GC. Here, through RNA screening, an apoptotic protease‐activating factor 1 (APAF1)‐binding lncRNA (ABL) that is significantly elevated in cancerous GC tissues and an independent prognostic factor for GC patients is identified. Moreover, ABL overexpression inhibits GC cell apoptosis and promotes GC cell survival and multidrug resistance in GC xenograft and organoid models. Mechanistically, ABL directly binds to the RNA‐binding protein IGF2BP1 via its KH1/2 domain, and then IGF2BP1 further recognizes the METTL3‐mediated m6A modification on ABL, which maintains ABL stability. In addition, ABL can bind to the WD1/WD2 domain of APAF1, which competitively prevent cytochrome c from interacting with APAF1, blocking apoptosome assembly and caspase‐9/3 activation; these events lead to resistance to cell death in GC cells. Intriguingly, targeting ABL using encapsulated liposomal siRNA can significantly enhance the sensitivity of GC cells to chemotherapy. Collectively, the results suggest that ABL can be a potential prognostic biomarker and therapeutic target in GC. [ABSTRACT FROM AUTHOR]

As top predators, falcons possess unique morphological, physiological and behavioral adaptations that allow them to be successful hunters: for example, the peregrine is renowned as the world's fastest animal. To examine the evolutionary basis of predatory adaptations, we sequenced the genomes of both the peregrine (Falco peregrinus) and saker falcon (Falco cherrug), and we present parallel, genome-wide evidence for evolutionary innovation and selection for a predatory lifestyle. The genomes, assembled using Illumina deep sequencing with greater than 100-fold coverage, are both approximately 1.2 Gb in length, with transcriptome-assisted prediction of approximately 16,200 genes for both species. Analysis of 8,424 orthologs in both falcons, chicken, zebra finch and turkey identified consistent evidence for genome-wide rapid evolution in these raptors. SNP-based inference showed contrasting recent demographic trajectories for the two falcons, and gene-based analysis highlighted falcon-specific evolutionary novelties for beak development and olfaction and specifically for homeostasis-related genes in the arid environment-adapted saker. [ABSTRACT FROM AUTHOR]

Background & Aims: The molecular mechanism underlying metabolic dysfunction-associated steatotic liver disease (MASLD) is elusive and whether non-coding RNAs can serve as biomarkers and therapeutic targets of MASLD is less defined., Methods: Exon capture RNA sequencing analysis was used to identify read-through circRNAs (rt-circRNAs) in livers from three MASLD patients and three patients without MASLD. Hepatocyte-specific deletion or overexpression of rt-circRNA RCRIN were utilized to study MASLD pathogenesis., Results: We identified 1126 rt-circRNAs in the liver tissues from MASLD patients. RCRIN was highly expressed in normal livers and was downregulated in MASLD livers. Rcrin deletion in hepatocytes caused lipid accumulation and MASLD development, while Rcrin overexpression suppressed the MASLD progression. Mechanistically, in normal hepatocytes, highly expressed RCRIN bound to RPL8 protein to recruit RNF2 for its degradation, reducing RPL8-contained ribosome numbers and lipid accumulation. In MASLD livers, lowerly expressed RCRIN released RPL8 protein to promote RPL8-contained ribosome numbers and lipid synthesis, leading to higher lipid accumulation and ER stress. We synthesized RCRIN and N-acetylgalactosamine (GalNAc)-Rpl8 siRNA to treat established MASLD in mice, both of which suppressed MASLD pathogenesis., Conclusions: Our findings provide an in vivo function of rt-circRNA RCRIN, show its inhibitory effects in MASLD pathogenesis, indicating RCRIN and RPL8 may be therapeutic targets and candidate nucleic acid drugs for treating MASLD., Impact and Implications: Our finds reveal a novel mechanism connecting a read-through circRNA RCRIN, ribosome heterogeneity and MASLD pathogenesis. In normal hepatocytes, RCRIN exerts its role by reducing liver lipid accumulation and ER stress through promotion of RPL8 degradation. In MASLD patients, lower RCRIN releases RPL8 to form RPL8-contained ribosomes to promote lipid accumulation and ER stress. RCRIN overexpression and RPL8 depletion dramatically suppresses MASLD development and progression. Our findings indicate that RCRIN and RPL8 might be potential therapeutic targets for treatment of MASLD patients., Competing Interests: Declaration of Competing Interest: All of the authors declare no competing financial interests., (Copyright © 2024 Institute of Biophysics, Chinese Academy of Sciences. Published by Elsevier B.V. All rights reserved.)

Postnatal cardiac development requires the orchestrated maturation of diverse cellular components for which unifying control mechanisms are still lacking. Using full-length sequencing, we examined the transcriptomic landscape of the maturating mouse heart (E18.5-P28) at single-cell and transcript isoform resolution. We identified dynamically changing intercellular networks as a molecular basis of the maturing heart and alternative splicing (AS) as a common mechanism that distinguished developmental age. Manipulation of RNA-binding proteins (RBPs) remodeled the AS landscape, cardiac cell maturation, and intercellular communication through direct binding of splice targets, which were enriched for functions related to general, as well as cell-type-specific, maturation. Overexpression of an RBP nuclear cap-binding protein subunit 2 (NCBP2) in neonatal hearts repressed cardiac maturation. Together, our data suggest AS regulation by RBPs as an organ-level control mechanism in mammalian postnatal cardiac development and provide insight into the possibility of manipulating RBPs for therapeutic purposes., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

RNA structure has been increasingly recognized as a critical player in the biogenesis and turnover of many transcripts classes. In eukaryotes, the prediction of RNA structure by thermodynamic modeling meets fundamental limitations due to the large sizes and complex, discontinuous organization of eukaryotic genes. Signatures of functional RNA structures can be found by detecting compensatory substitutions in homologous sequences, but a comparative approach is applicable only within conserved sequence blocks. Here, we developed a computational pipeline called PHRIC, which is not limited to conserved regions and relies on RNA contacts derived from RNA in situ conformation sequencing (RIC-seq) experiments. It extracts pairs of short RNA fragments surrounded by nested clusters of RNA contacts and predicts long, nearly perfect complementary base pairings formed between these fragments. In application to a panel of RIC-seq experiments in seven human cell lines, PHRIC predicted ~12,000 stable long-range RNA structures with equilibrium free energy below -15 kcal/mol, the vast majority of which fall outside of regions annotated as conserved among vertebrates. These structures, nevertheless, show some level of sequence conservation and remarkable compensatory substitution patterns in other clades. Furthermore, we found that introns have a higher propensity to form stable long-range RNA structures between each other, and moreover that RNA structures tend to concentrate within the same intron rather than connect adjacent introns. These results for the first time extend the application of proximity ligation assays to RNA structure prediction beyond conserved regions., Competing Interests: The authors declare that they have no competing interests., (© 2023 Margasyuk et al.)

Over recent years, long-range RNA structure has emerged as a factor that is fundamental to alternative splicing regulation. An increasing number of human disorders are now being associated with splicing defects; hence it is essential to develop methods that assess long-range RNA structure experimentally. RNA in situ conformation sequencing (RIC-seq) is a method that recapitulates RNA structure within physiological RNA-protein complexes. In this work, we juxtapose pairs of conserved complementary regions (PCCRs) that were predicted in silico with the results of RIC-seq experiments conducted in seven human cell lines. We show statistically that RIC-seq support of PCCRs correlates with their properties, such as equilibrium free energy, presence of compensatory substitutions, and occurrence of A-to-I RNA editing sites and forked eCLIP peaks. Exons enclosed in PCCRs that are supported by RIC-seq tend to have weaker splice sites and lower inclusion rates, which is indicative of post-transcriptional splicing regulation mediated by RNA structure. Based on these findings, we prioritize PCCRs according to their RIC-seq support and show, using antisense nucleotides and minigene mutagenesis, that PCCRs in two disease-associated human genes, PHF20L1 and CASK , and also PCCRs in their murine orthologs, impact alternative splicing. In sum, we demonstrate how RIC-seq experiments can be used to discover functional long-range RNA structures, and particularly those that regulate alternative splicing., (© 2023 Margasyuk et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

H 2 S is an endogenous gas signaling molecule and its multiple biological effects have been demonstrated. The abnormal level of H 2 S is closely related to the occurrence and development of many diseases, and H 2 S donors has important pharmacological implications. In recent years, H 2 S donors represented by ADTOH (5-(4-hydroxyphenyl)-3H-1,2-dithiole-3-thione) are often used to synthesize new 'conjugate' compounds that can release H 2 S and parent drugs. These hybrids retain the pharmacological activity of the parent drugs and H 2 S and have a synergistic effect. ADTOH and parent drug hybrids have become one of the important strategies for the development of H 2 S donor conjugate drugs. This review summarizes molecular hybrids between ADTOH and clinical drugs to provide new ideas for the study of H 2 S donor drug design.

Enhancers are noncoding DNA elements that are present upstream or downstream of a gene to control its spatial and temporal expression. Specific histone modifications, such as monomethylation on histone H3 lysine 4 (H3K4me1) and H3K27ac, have been widely used to assign enhancer regions in mammalian genomes. In recent years, emerging evidence suggests that active enhancers are bidirectionally transcribed to produce enhancer RNAs (eRNAs). This finding not only adds a new reliable feature to define enhancers but also raises a fundamental question of how eRNAs function to activate transcription. Although some believe that eRNAs are merely transcriptional byproducts, many studies have demonstrated that eRNAs execute crucial tasks in regulating chromatin conformation and transcription activation. In this review, we summarize the current understanding of eRNAs from their biogenesis, functions, and regulation to their pathological significance. Additionally, we discuss the challenges and possible mechanisms of eRNAs in regulated transcription., (© 2020 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Nanowires that transfer electrons to extracellular acceptors are important in organic matter degradation and nutrient cycling in the environment. Geobacter pili of the group of Type IV pilus are regarded as nanowire-like biological structures. However, determination of the structure of pili remains challenging due to the insolubility of monomers, presence of surface appendages, heterogeneity of the assembly, and low-resolution of electron microscopy techniques. Our previous study provided a method to predict structures for Type IV pili. In this work, we improved on our previous method using molecular dynamics simulations to optimize structures of Neisseria gonorrhoeae (GC), Neisseria meningitidis and Geobacter uraniireducens pilus. Comparison between the predicted structures for GC and Neisseria meningitidis pilus and their native structures revealed that proposed method could predict Type IV pilus successfully. According to the predicted structures, the structural basis for conductivity in G.uraniireducens pili was attributed to the three N-terminal aromatic amino acids. The aromatics were interspersed within the regions of charged amino acids, which may influence the configuration of the aromatic contacts and the rate of electron transfer. These results will supplement experimental research into the mechanism of long-rang electron transport along pili of electricigens.

Chromosome-long haplotyping of human genomes is important to identify genetic variants with differing gene expression, in human evolution studies, clinical diagnosis, and other biological and medical fields. Although several methods have realized haplotyping based on sequencing technologies or population statistics, accuracy and cost are factors that prohibit their wide use. Borrowing ideas from group testing theories, we proposed a clone-based haplotyping method by overlapping pool sequencing. The clones from a single individual were pooled combinatorially and then sequenced. According to the distinct pooling pattern for each clone in the overlapping pool sequencing, alleles for the recovered variants could be assigned to their original clones precisely. Subsequently, the clone sequences could be reconstructed by linking these alleles accordingly and assembling them into haplotypes with high accuracy. To verify the utility of our method, we constructed 130 110 clones in silico for the individual NA12878 and simulated the pooling and sequencing process. Ultimately, 99.9% of variants on chromosome 1 that were covered by clones from both parental chromosomes were recovered correctly, and 112 haplotype contigs were assembled with an N50 length of 3.4 Mb and no switch errors. A comparison with current clone-based haplotyping methods indicated our method was more accurate., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: Continual progress in next-generation sequencing allows for generating increasingly large metagenomes which are over time or space. Comparing and classifying the metagenomes with different microbial communities is critical. Alignment-free supervised classification is important for discriminating between the multifarious components of metagenomic samples, because it can be accomplished independently of known microbial genomes., Results: We propose an alignment-free supervised metagenomic classification method called DectICO. The intrinsic correlation of oligonucleotides provides the feature set, which is selected dynamically using a kernel partial least squares algorithm, and the feature matrices extracted with this set are sequentially employed to train classifiers by support vector machine (SVM). We evaluated the classification performance of DectICO on three actual metagenomic sequencing datasets, two containing deep sequencing metagenomes and one of low coverage. Validation results show that DectICO is powerful, performs well based on long oligonucleotides (i.e., 6-mer to 8-mer), and is more stable and generalized than a sequence-composition-based method. The classifiers trained by our method are more accurate than non-dynamic feature selection methods and a recently published recursive-SVM-based classification approach., Conclusions: The alignment-free supervised classification method DectICO can accurately classify metagenomic samples without dependence on known microbial genomes. Selecting the ICO dynamically offers better stability and generality compared with sequence-composition-based classification algorithms. Our proposed method provides new insights in metagenomic sample classification.

Domestic yaks (Bos grunniens) provide meat and other necessities for Tibetans living at high altitude on the Qinghai-Tibetan Plateau and in adjacent regions. Comparison between yak and the closely related low-altitude cattle (Bos taurus) is informative in studying animal adaptation to high altitude. Here, we present the draft genome sequence of a female domestic yak generated using Illumina-based technology at 65-fold coverage. Genomic comparisons between yak and cattle identify an expansion in yak of gene families related to sensory perception and energy metabolism, as well as an enrichment of protein domains involved in sensing the extracellular environment and hypoxic stress. Positively selected and rapidly evolving genes in the yak lineage are also found to be significantly enriched in functional categories and pathways related to hypoxia and nutrition metabolism. These findings may have important implications for understanding adaptation to high altitude in other animal species and for hypoxia-related diseases in humans.