[Progress of research on circular RNA encoding proteins in disease].

With the advance of research, non-coding RNA has been found to surpass the traditional definition to directly code functional proteins by coding sequence elements and binding with ribosomes. Among the non-coding RNAs, the function of circRNA encoded proteins has been most extensively studied. This study has used "circRNA", "encoded", and "translation" as the key words to search the PubMed and Web of Science databases. The retrieved literature was screened and traced, with the translation mechanism, related research methods, and correlation with diseases of circRNA reviewed. CircRNA can translate proteins through a non-cap-dependent pathway. Multiple molecular techniques, in particular mass spectrometry analysis, have important value in identifying unique peptide segments of circRNA encoded proteins for confirming their existence. The proteins encoded by the circRNA are involved in the pathogenesis of diseases of the digestive, neurological, urinary systems and the breast, and have the potential to serve as novel targets for disease diagnosis and treatment. This article has provided a comprehensive review for the basic theory, experimental methods, and disease-related research in the field of circRNA translation, which may provide clues for the identification of new diagnostic and therapeutic targets.

Analysis of culture and RNA isolation methods for precision-cut liver slices from cirrhotic rats.

Precision-cut liver slices (PCLS) are increasingly used as a model to investigate anti-fibrotic therapies. However, many studies use PCLS from healthy animals treated with pro-fibrotic stimuli in culture, which reflects only the early stages of fibrosis. The effects of different culture conditions on PCLS from cirrhotic animals has not been well characterized and there is no consensus on optimal methods. In this study, we report a method for the collection and culture of cirrhotic PCLS and compare the effect of common culture conditions on viability, function, and gene expression. Additionally, we compared three methods of RNA isolation and identified a protocol with high yield and purity. We observed significantly increased albumin production when cultured with insulin-transferrin-selenium and dexamethasone, and when incubated on a rocking platform. Culturing with insulin-transferrin-selenium and dexamethasone maintained gene expression closer to the levels in fresh slices. However, despite stable viability and function up to 4 days, we found significant changes in expression of key genes by day 2. Interestingly, we also observed that cirrhotic PCLS maintain viability in culture longer than slices from healthy animals. Due to the influence of matrix stiffness on fibrosis and hepatocellular function, it is important to evaluate prospective anti-fibrotic therapies in a platform that preserves tissue biomechanics. PCLS from cirrhotic animals represent a promising tool for the development of treatments for chronic liver disease.

ImmRNA: a database of RNAs associated with tumor immunity.

The relationship between different ribonucleic acids (RNAs) and tumor immunity has been widely investigated. However, a systematic description of tumor immune-related RNAs in different tumors is still lacking. We collected the relationship of tumor immune-related RNAs from the published literature and presented them in a user-friendly interface, "ImmRNA" (http://www.immrna.cn/), to provide a resource to study immune-RNA-cancer regulatory relations. The ImmRNA contains 49 996 curated entries. Each entry includes gene symbols, gene types, target genes, downstream effects, functions, immune cells, and other information. By rearranging and reanalyzing the data, our dataset contains the following key points: (i) providing the links between RNAs and the immune in cancers, (ii) displaying the downstream effects and functions of RNAs, (iii) listing immune cells and immune pathways related to RNA function, (iv) showing the relationship between RNAs and prognostic outcomes, and (v) exhibiting the experimental methods described in the article. ImmRNA provides a valuable resource for understanding the functions of tumor immune-related RNAs. Database URL:  http://www.immrna.cn/.

[Advances in mapping analysis of ribonucleic acid modifications through sequencing].

Over 170 chemical modifications have been discovered in various types of ribonucleic acids (RNAs), including messenger RNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), and small nuclear RNA (snRNA). These RNA modifications play crucial roles in a wide range of biological processes such as gene expression regulation, RNA stability maintenance, and protein translation. RNA modifications represent a new dimension of gene expression regulation known as the "epitranscriptome". The discovery of RNA modifications and the relevant writers, erasers, and readers provides an important basis for studies on the dynamic regulation and physiological functions of RNA modifications. Owing to the development of detection technologies for RNA modifications, studies on RNA epitranscriptomes have progressed to the single-base resolution, multilayer, and full-coverage stage. Transcriptome-wide methods help discover new RNA modification sites and are of great importance for elucidating the molecular regulatory mechanisms of epitranscriptomics, exploring the disease associations of RNA modifications, and understanding their clinical applications. The existing RNA modification sequencing technologies can be categorized according to the pretreatment approach and sequencing principle as direct high-throughput sequencing, antibody-enrichment sequencing, enzyme-assisted sequencing, chemical labeling-assisted sequencing, metabolic labeling sequencing, and nanopore sequencing technologies. These methods, as well as studies on the functions of RNA modifications, have greatly expanded our understanding of epitranscriptomics. In this review, we summarize the recent progress in RNA modification detection technologies, focusing on the basic principles, advantages, and limitations of different methods. Direct high-throughput sequencing methods do not require complex RNA pretreatment and allow for the mapping of RNA modifications using conventional RNA sequencing methods. However, only a few RNA modifications can be analyzed by high-throughput sequencing. Antibody enrichment followed by high-throughput sequencing has emerged as a crucial approach for mapping RNA modifications, significantly advancing the understanding of RNA modifications and their regulatory functions in different species. However, the resolution of antibody-enrichment sequencing is limited to approximately 100-200 bp. Although chemical crosslinking techniques can achieve single-base resolution, these methods are often complex, and the specificity of the antibodies used in these methods has raised concerns. In particular, the issue of off-target binding by the antibodies requires urgent attention. Enzyme-assisted sequencing has improved the accuracy of the localization analysis of RNA modifications and enables stoichiometric detection with single-base resolution. However, the enzymes used in this technique show poor reactivity, specificity, and sequence preference. Chemical labeling sequencing has become a widely used approach for profiling RNA modifications, particularly by altering reverse transcription (RT) signatures such as RT stops, misincorporations, and deletions. Chemical-assisted sequencing provides a sequence-independent RNA modification detection strategy that enables the localization of multiple RNA modifications. Additionally, when combined with the biotin-streptavidin affinity method, low-abundance RNA modifications can be enriched and detected. Nevertheless, the specificity of many chemical reactions remains problematic, and the development of specific reaction probes for particular modifications should continue in the future to achieve the precise localization of RNA modifications. As an indirect localization method, metabolic labeling sequencing specifically localizes the sites at which modifying enzymes act, which is of great significance in the study of RNA modification functions. However, this method is limited by the intracellular labeling of RNA and cannot be applied to biological samples such as clinical tissues and blood samples. Nanopore sequencing is a direct RNA-sequencing method that does not require RT or the polymerase chain reaction (PCR). However, challenges in analyzing the data obtained from nanopore sequencing, such as the high rate of false positives, must be resolved. Discussing sequencing analysis methods for various types of RNA modifications is instructive for the future development of novel RNA modification mapping technologies, and will aid studies on the functions of RNA modifications across the entire transcriptome.

A point cloud segmentation framework for image-based spatial transcriptomics.

Recent progress in image-based spatial RNA profiling enables to spatially resolve tens to hundreds of distinct RNA species with high spatial resolution. It presents new avenues for comprehending tissue organization. In this context, the ability to assign detected RNA transcripts to individual cells is crucial for downstream analyses, such as in-situ cell type calling. Yet, accurate cell segmentation can be challenging in tissue data, in particular in the absence of a high-quality membrane marker. To address this issue, we introduce ComSeg, a segmentation algorithm that operates directly on single RNA positions and that does not come with implicit or explicit priors on cell shape. ComSeg is applicable in complex tissues with arbitrary cell shapes. Through comprehensive evaluations on simulated and experimental datasets, we show that ComSeg outperforms existing state-of-the-art methods for in-situ single-cell RNA profiling and in-situ cell type calling. ComSeg is available as a documented and open source pip package at https://github.com/fish-quant/ComSeg .

Thermodynamic coupling of the tandem RRM domains of hnRNP A1 underlie its pleiotropic RNA binding functions.

The functional properties of RNA binding proteins (RBPs) require allosteric regulation through interdomain communication. Despite the importance of allostery to biological regulation, only a few studies have been conducted to describe the biophysical nature by which interdomain communication manifests in RBPs. Here, we show for hnRNP A1 that interdomain communication is vital for the unique stability of its amino-terminal domain, which consists of two RNA recognition motifs (RRMs). These RRMs exhibit drastically different stability under pressure. RRM2 unfolds as an individual domain but remains stable when appended to RRM1. Variants that disrupt interdomain communication between the tandem RRMs show a significant decrease in stability. Carrying these mutations over to the full-length protein for in vivo experiments revealed that the mutations affected the ability of the disordered carboxyl-terminal domain to engage in protein-protein interactions and influenced the protein's RNA binding capacity. Collectively, this work reveals that thermodynamic coupling between the tandem RRMs of hnRNP A1 accounts for its allosteric regulatory functions.

TracrRNA reprogramming enables direct PAM-independent detection of RNA with diverse DNA-targeting Cas12 nucleases.

Many CRISPR-Cas immune systems generate guide (g)RNAs using trans-activating CRISPR RNAs (tracrRNAs). Recent work revealed that Cas9 tracrRNAs could be reprogrammed to convert any RNA-of-interest into a gRNA, linking the RNA's presence to Cas9-mediated cleavage of double-stranded (ds)DNA. Here, we reprogram tracrRNAs from diverse Cas12 nucleases, linking the presence of an RNA-of-interest to dsDNA cleavage and subsequent collateral single-stranded DNA cleavage-all without the RNA necessarily encoding a protospacer-adjacent motif (PAM). After elucidating nuclease-specific design rules, we demonstrate PAM-independent RNA detection with Cas12b, Cas12e, and Cas12f nucleases. Furthermore, rationally truncating the dsDNA target boosts collateral cleavage activity, while the absence of a gRNA reduces background collateral activity and enhances sensitivity. Finally, we apply this platform to detect 16 S rRNA sequences from five different bacterial pathogens using a universal reprogrammed tracrRNA. These findings extend tracrRNA reprogramming to diverse dsDNA-targeting Cas12 nucleases, expanding the flexibility and versatility of CRISPR-based RNA detection.

Self-supervised learning for characterising histomorphological diversity and spatial RNA expression prediction across 23 human tissue types.

As vast histological archives are digitised, there is a pressing need to be able to associate specific tissue substructures and incident pathology to disease outcomes without arduous annotation. Here, we learn self-supervised representations using a Vision Transformer, trained on 1.7 M histology images across 23 healthy tissues in 838 donors from the Genotype Tissue Expression consortium (GTEx). Using these representations, we can automatically segment tissues into their constituent tissue substructures and pathology proportions across thousands of whole slide images, outperforming other self-supervised methods (43% increase in silhouette score). Additionally, we can detect and quantify histological pathologies present, such as arterial calcification (AUROC = 0.93) and identify missing calcification diagnoses. Finally, to link gene expression to tissue morphology, we introduce RNAPath, a set of models trained on 23 tissue types that can predict and spatially localise individual RNA expression levels directly from H&E histology (mean genes significantly regressed = 5156, FDR 1%). We validate RNAPath spatial predictions with matched ground truth immunohistochemistry for several well characterised control genes, recapitulating their known spatial specificity. Together, these results demonstrate how self-supervised machine learning when applied to vast histological archives allows researchers to answer questions about tissue pathology, its spatial organisation and the interplay between morphological tissue variability and gene expression.

Circular at the very beginning: on the initial genomes in the RNA world.

It is likely that an RNA world existed in early life, when RNA played both the roles of the genome and functional molecules, thereby undergoing Darwinian evolution. However, even with only one type of polymer, it seems quite necessary to introduce a labour division concerning these two roles because folding is required for functional molecules (ribozymes) but unfavourable for the genome (as a template in replication). Notably, while ribozymes tend to have adopted a linear form for folding without constraints, a circular form, which might have been topologically hindered in folding, seems more suitable for an RNA template. Another advantage of involving a circular genome could have been to resist RNA's end-degradation. Here, we explore the scenario of a circular RNA genome plus linear ribozyme(s) at the precellular stage of the RNA world through computer modelling. The results suggest that a one-gene scene could have been 'maintained', albeit with rather a low efficiency for the circular genome to produce the ribozyme, which required precise chain-break or chain-synthesis. This strict requirement may have been relieved by introducing a 'noncoding' sequence into the genome, which had the potential to derive a second gene through mutation. A two-gene scene may have 'run well' with the two corresponding ribozymes promoting the replication of the circular genome from different respects. Circular genomes with more genes might have arisen later in RNA-based protocells. Therefore, circular genomes, which are common in the modern living world, may have had their 'root' at the very beginning of life.

RNA scaffolds the Golgi ribbon by forming condensates with GM130.

The mammalian Golgi is composed of stacks that are laterally connected into a continuous ribbon-like structure. The integrity and function of the ribbon is disrupted under stress conditions, but the molecular mechanisms remain unclear. Here we show that the ribbon is maintained by biomolecular condensates of RNA and the Golgi matrix protein GM130 (GOLGA2). We identify GM130 as a membrane-bound RNA-binding protein, which directly recruits RNA and associated RNA-binding proteins to the Golgi membrane. Acute degradation of RNA or GM130 in cells disrupts the ribbon. Under stress conditions, RNA dissociates from GM130 and the ribbon is disjointed, but after the cells recover from stress the ribbon is restored. When overexpressed in cells, GM130 forms RNA-dependent liquid-like condensates. GM130 contains an intrinsically disordered domain at its amino terminus, which binds RNA to induce liquid-liquid phase separation. These co-condensates are sufficient to link purified Golgi membranes, reconstructing lateral linking of stacks into a ribbon-like structure. Together, these studies show that RNA acts as a structural biopolymer that together with GM130 maintains the integrity of the Golgi ribbon.

RNA Adduction Resulting from the Metabolic Activation of Myristicin by P450 Enzymes and Sulfotransferases.

Myristicin (MYR) mainly occurs in nutmeg and belongs to alkoxy-substituted allylbenzenes, a class of potentially toxic natural chemicals. RNA interaction with MYR metabolites in vitro and in vivo has been investigated in order to gain a better understanding of MYR toxicities. We detected two guanosine adducts (GA1 and GA2), two adenosine adducts (AA1 and AA2), and two cytosine adducts (CA1 and CA2) by LC-MS/MS analysis of total RNA extracts from cultured primary mouse hepatocytes and liver tissues of mice after exposure to MYR. An order of nucleoside adductions was found to be GAs > AAs > CAs, and the result of density functional theory calculations was in agreement with that detected by the LC-MS/MS-based approach. In vitro and in vivo studies have shown that MYR was oxidized by cytochrome P450 enzymes to 1'-hydroxyl and 3'-hydroxyl metabolites, which were then sulfated by sulfotransferases (SULTs) to form sulfate esters. The resulting sulfates would react with the nucleosides by SN1 and/or SN2 reactions, resulting in RNA adduction. The modification may alter the biochemical properties of RNA and disrupt RNA functions, perhaps partially contributing to the toxicities of MYR.

The glycine-rich domain of GRP7 plays a crucial role in binding long RNAs and facilitating phase separation.

Microscale thermophoresis (MST) is a well-established method to quantify protein-RNA interactions. In this study, we employed MST to analyze the RNA binding properties of glycine-rich RNA binding protein 7 (GRP7), which is known to have multiple biological functions related to its ability to bind different types of RNA. However, the exact mechanism of GRP7's RNA binding is not fully understood. While the RNA-recognition motif of GRP7 is known to be involved in RNA binding, the glycine-rich region (known as arginine-glycine-glycine-domain or RGG-domain) also influences this interaction. To investigate to which extend the RGG-domain of GRP7 is involved in RNA binding, mutation studies on putative RNA interacting or modulating sites were performed. In addition to MST experiments, we examined liquid-liquid phase separation of GRP7 and its mutants, both with and without RNA. Furthermore, we systemically investigated factors that might affect RNA binding selectivity of GRP7 by testing RNAs of different sizes, structures, and modifications. Consequently, our study revealed that GRP7 exhibits a high affinity for a variety of RNAs, indicating a lack of pronounced selectivity. Moreover, we established that the RGG-domain plays a crucial role in binding longer RNAs and promoting phase separation.

Adeno-associated-virus-mediated delivery of CRISPR-CasRx induces efficient RNA knockdown in the mouse testis.

Rationale: In male mammals, many developmental-stage-specific RNA transcripts (both coding and noncoding) are preferentially or exclusively expressed in the testis, where they play important roles in spermatogenesis and male fertility. However, a reliable platform for efficiently depleting various types of RNA transcripts to study their biological functions during spermatogenesis in vivo has not been developed. Methods: We used an adeno-associated virus serotype nine (AAV9)-mediated CRISPR-CasRx system to knock down the expression of exogenous and endogenous RNA transcripts in the testis. Virus particles were injected into the seminiferous tubules via the efferent duct. Using an autophagy inhibitor, 3-methyladenine (3-MA), we optimized the AAV9 transduction efficiency in germ cells in vivo. Results: AAV9-mediated delivery of CRISPR-CasRx effectively and specifically induces RNA transcripts (both coding and noncoding) knockdown in the testis in vivo. In addition, we showed that the co-microinjection of AAV9 and 3-MA into the seminiferous tubules enabled long-term transgene expression in the testis. Finally, we found that a promoter of Sycp1 gene induced CRISPR-CasRx-mediated RNA transcript knockdown in a germ-cell-type-specific manner. Conclusion: Our results demonstrate the efficacy and versatility of the AAV9-mediated CRISPR-CasRx system as a flexible knockdown platform for studying gene function during spermatogenesis in vivo. This approach may advance the development of RNA-targeting therapies for conditions affecting reproductive health.

Mudskipper detects combinatorial RNA binding protein interactions in multiplexed CLIP data.

The uncovering of protein-RNA interactions enables a deeper understanding of RNA processing. Recent multiplexed crosslinking and immunoprecipitation (CLIP) technologies such as antibody-barcoded eCLIP (ABC) dramatically increase the throughput of mapping RNA binding protein (RBP) binding sites. However, multiplex CLIP datasets are multivariate, and each RBP suffers non-uniform signal-to-noise ratio. To address this, we developed Mudskipper, a versatile computational suite comprising two components: a Dirichlet multinomial mixture model to account for the multivariate nature of ABC datasets and a softmasking approach that identifies and removes non-specific protein-RNA interactions in RBPs with low signal-to-noise ratio. Mudskipper demonstrates superior precision and recall over existing tools on multiplex datasets and supports analysis of repetitive elements and small non-coding RNAs. Our findings unravel splicing outcomes and variant-associated disruptions, enabling higher-throughput investigations into diseases and regulation mediated by RBPs.

Tradeoffs in the design of RNA thermometers.

The synthesis of RNA thermometers is aimed at achieving temperature responses with desired thresholds and sensitivities. Although previous works have generated thermometers with a variety of thresholds and sensitivities as well as guidelines for design, possible constraints in the achievable thresholds and sensitivities remain unclear. We addressed this issue using a two-state model and its variants, as well as melt profiles generated from thermodynamic computations. In the two-state model, we found that the threshold was inversely proportional to the sensitivity, in the case of a fixed energy difference between the two states. Notably, this constraint could persist in variations of the two-state model with sequentially unfolding states and branched parallel pathways. Furthermore, the melt profiles generated from a library of thermometers exhibited a similar constraint. These results should inform the design of RNA thermometers as well as other responses that are mediated in a similar fashion.

Atomistic insights into the reentrant phase-transitions in polyuracil and polylysine mixtures.

The phase separation of protein and RNA mixtures underpins the assembly and regulation of numerous membraneless organelles in cells. The ubiquity of protein-RNA condensates in cellular regulatory processes is in part due to their sensitivity to RNA concentration, which affects their physical properties and stability. Recent experiments with poly-cationic peptide-RNA mixtures have revealed closed-loop phase diagrams featuring lower and upper critical solution temperatures. These diagrams indicate reentrant phase transitions shaped by biomolecular interactions and entropic forces such as solvent and ion reorganization. We employed atomistic simulations to study mixtures with various RNA-polylysine stoichiometries and temperatures to elucidate the microscopic driving forces behind reentrant phase transitions in protein-RNA mixtures. Our findings reveal an intricate interplay between hydration, ion condensation, and specific RNA-polylysine hydrogen bonding, resulting in distinct stoichiometry-dependent phase equilibria governing stabilities and structures of the condensate phase. Our simulations show that reentrant transitions are accompanied by desolvation around the phosphate groups of RNA, with increased contacts between phosphate and lysine side chains. In RNA-rich systems at lower temperatures, RNA molecules can form an extensive pi-stacking and hydrogen bond network, leading to percolation. In protein-rich systems, no such percolation-induced transitions are observed. Furthermore, we assessed the performance of three prominent water force fields-Optimal Point Charge (OPC), TIP4P-2005, and TIP4P-D-in capturing reentrant phase transitions. OPC provided a superior balance of interactions, enabling effective capture of reentrant transitions and accurate characterization of changes in solvent reorganization. This study offers atomistic insights into the nature of reentrant phase transitions using simple model peptide and nucleotide mixtures. We believe that our results are broadly applicable to larger classes of peptide-RNA mixtures exhibiting reentrant phase transitions.

Characterization of m6A Modifiers and RNA Modifications in Uterine Fibroids.

Uterine leiomyoma or fibroids are prevalent noncancerous tumors of the uterine muscle layer, yet their origin and development remain poorly understood. We analyzed RNA expression profiles of 15 epigenetic mediators in uterine fibroids compared to myometrium using publicly available RNA sequencing (RNA-seq) data. To validate our findings, we performed RT-qPCR on a separate cohort of uterine fibroids targeting these modifiers confirming our RNA-seq data. We then examined protein profiles of key N6-methyladenosine (m6A) modifiers in fibroids and their matched myometrium, showing no significant differences in concordance with our RNA expression profiles. To determine RNA modification abundance, mRNA and small RNA from fibroids and matched myometrium were analyzed by ultra-high performance liquid chromatography-mass spectrometry identifying prevalent m6A and 11 other known modifiers. However, no aberrant expression in fibroids was detected. We then mined a previously published dataset and identified differential expression of m6A modifiers that were specific to fibroid genetic subtype. Our analysis also identified m6A consensus motifs on genes previously identified to be dysregulated in uterine fibroids. Overall, using state-of-the-art mass spectrometry, RNA expression, and protein profiles, we characterized and identified differentially expressed m6A modifiers in relation to driver mutations. Despite the use of several different approaches, we identified limited differential expression of RNA modifiers and associated modifications in uterine fibroids. However, considering the highly heterogenous genomic and cellular nature of fibroids, and the possible contribution of single molecule m6A modifications to fibroid pathology, there is a need for greater in-depth characterization of m6A marks and modifiers in a larger and diverse patient cohort.

Lupus IgA1 autoantibodies synergize with IgG to enhance plasmacytoid dendritic cell responses to RNA-containing immune complexes.

Autoantibodies to nuclear antigens are hallmarks of systemic lupus erythematosus (SLE) where they contribute to pathogenesis. However, there remains a gap in our knowledge regarding how different isotypes of autoantibodies contribute to this autoimmune disease, including the production of the critical type I interferon (IFN) cytokines by plasmacytoid dendritic cells (pDCs) in response to immune complexes (ICs). We focused on IgA, which is the second-most prevalent isotype in serum and, along with IgG, is deposited in glomeruli in individuals with lupus nephritis. We show that individuals with SLE have serum IgA autoantibodies against most nuclear antigens, correlating with IgG against the same antigen. We investigated whether IgA autoantibodies against a major SLE autoantigen, Smith ribonucleoprotein (Sm/RNP), played a role in IC activation of pDCs. We found that pDCs expressed the IgA-specific Fc receptor, FcαR, and IgA1 autoantibodies synergized with IgG in RNA-containing ICs to generate robust primary blood pDC IFN-α responses in vitro. pDC responses to these ICs required both FcαR and FcγRIIa, showing synergy between these Fc receptors. Sm/RNP IC binding to and internalization by pDCs were greater when ICs contained both IgA1 and IgG. Circulating pDCs from individuals with SLE had higher binding of IgA1-containing ICs and higher expression of FcαR than pDCs from healthy control individuals. Although pDC FcαR expression correlated with the blood IFN-stimulated gene signature in SLE, Toll-like receptor 7 agonists, but not IFN-α, up-regulated pDC FcαR expression in vitro. Together, we show a mechanism by which IgA1 autoantibodies contribute to SLE pathogenesis.

Ratiometric sandwich-type assays for RNAs with a point mutation using benzo[a]pyrene-modified probes.

Benzo[a]pyrene-modified oligonucleotides were developed for the detection of RNAs with a point mutation. The probes produced two distinct fluorescence signals in response to single nucleotide differences in the RNA sequences, allowing for discrimination between the matched and single base mismatched RNA sequences in colorimetric and ratiometric manners.