Distinct Processing of lncRNAs Contributes to Non-conserved Functions in Stem Cells.

Long noncoding RNAs (lncRNAs) evolve more rapidly than mRNAs. Whether conserved lncRNAs undergo conserved processing, localization, and function remains unexplored. We report differing subcellular localization of lncRNAs in human and mouse embryonic stem cells (ESCs). A significantly higher fraction of lncRNAs is localized in the cytoplasm of hESCs than in mESCs. This turns out to be important for hESC pluripotency. FAST is a positionally conserved lncRNA but is not conserved in its processing and localization. In hESCs, cytoplasm-localized hFAST binds to the WD40 domain of the E3 ubiquitin ligase ß-TrCP and blocks its interaction with phosphorylated ß-catenin to prevent degradation, leading to activated WNT signaling, required for pluripotency. In contrast, mFast is nuclear retained in mESCs, and its processing is suppressed by the splicing factor PPIE, which is highly expressed in mESCs but not hESCs. These findings reveal that lncRNA processing and localization are previously under-appreciated contributors to the rapid evolution of function.

IntS6 and the Integrator phosphatase module tune the efficiency of select premature transcription termination events.

The metazoan-specific Integrator complex catalyzes 3' end processing of small nuclear RNAs (snRNAs) and premature termination that attenuates the transcription of many protein-coding genes. Integrator has RNA endonuclease and protein phosphatase activities, but it remains unclear if both are required for complex function. Here, we show IntS6 (Integrator subunit 6) over-expression blocks Integrator function at a subset of Drosophila protein-coding genes, although having no effect on snRNAs or attenuation of other loci. Over-expressed IntS6 titrates protein phosphatase 2A (PP2A) subunits, thereby only affecting gene loci where phosphatase activity is necessary for Integrator function. IntS6 functions analogous to a PP2A regulatory B subunit as over-expression of canonical B subunits, which do not bind Integrator, is also sufficient to inhibit Integrator activity. These results show that the phosphatase module is critical at only a subset of Integrator-regulated genes and point to PP2A recruitment as a tunable step that modulates transcription termination efficiency.

Approaches and challenges in genome-wide circular RNA identification and quantification.

Numerous circular RNAs (circRNAs) produced from back-splicing of exon(s) have been recently revealed on a genome-wide scale across species. Although generally expressed at a low level, some relatively abundant circRNAs can play regulatory roles in various biological processes, prompting continuous profiling of circRNA in broader conditions. Over the past decade, distinct strategies have been applied in both transcriptome enrichment and bioinformatic tools for detecting and quantifying circRNAs. Understanding the scope and limitations of these strategies is crucial for the subsequent annotation and characterization of circRNAs, especially those with functional potential. Here, we provide an overview of different transcriptome enrichment, deep sequencing and computational approaches for genome-wide circRNA identification, and discuss strategies for accurate quantification and characterization of circRNA.

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OBJECTIVES: To study the correlation between 25-hydroxyvitamin D [25-(OH)D] and nephroblastoma in children and its value in assessing the prognosis of the disease. METHODS: A total of 50 children with nephroblastoma who were admitted from January 2018 to December 2022 were included as the nephroblastoma group, and according to the postoperative pathological type, they were divided into a good prognosis group with 38 children and a poor prognosis group with 12 children. A total of 50 healthy children who underwent physical examination during the same period of time served as the healthy control group. The above groups were compared in terms of serum creatinine and 25-(OH)D level. A Spearman correlation analysis was used to investigate the correlation between serum 25-(OH)D level and therapeutic effect reaction. A multivariate logistic regression analysis was used to identify the risk factors affecting the prognosis of nephroblastoma in children. RESULTS: The nephroblastoma group had significantly lower levels of serum creatinine and 25-(OH)D than the healthy control group (P<0.05). Compared with the good prognosis group, the poor prognosis group had a significantly larger tumor diameter, a significantly higher proportion of children with stage III-IV tumors, a significantly higher rate of tumor metastasis, and significantly lower serum levels of creatinine and 25-(OH)D (P<0.05). The Spearman correlation analysis showed that serum 25-(OH)D level was negatively correlated with therapeutic effect reaction (rs=-0.685, P<0.001). The multivariate logistic regression analysis showed that tumor diameter ≥10 cm, stage III-IV tumors, presence of tumor metastasis, and 25-(OH)D <19 ng/mL were closely associated with the poor prognosis of nephroblastoma in children (P<0.05). Serum 25-(OH)D level had an area under the curve of 0.805 (95%CI: 0.706-0.903, P<0.001) in evaluating the prognosis of nephroblastoma in children, with a Youden index of 0.512, a sensitivity of 0.938, and a specificity of 0.575 at the optimal cut-off value of 1.764 ng/mL. CONCLUSIONS: There is a significant correlation between 25-(OH)D level and the prognosis of nephroblastoma in children, and 25-(OH)D can be used for prognosis prediction.

CIRCexplorer pipelines for circRNA annotation and quantification from non-polyadenylated RNA-seq datasets.

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.

Increased complexity of circRNA expression during species evolution.

Circular RNAs (circRNAs) are broadly identified from precursor mRNA (pre-mRNA) back-splicing across various species. Recent studies have suggested a cell-/tissue- specific manner of circRNA expression. However, the distinct expression pattern of circRNAs among species and its underlying mechanism still remain to be explored. Here, we systematically compared circRNA expression from human and mouse, and found that only a small portion of human circRNAs could be determined in parallel mouse samples. The conserved circRNA expression between human and mouse is correlated with the existence of orientation-opposite complementary sequences in introns that flank back-spliced exons in both species, but not the circRNA sequences themselves. Quantification of RNA pairing capacity of orientation-opposite complementary sequences across circRNA-flanking introns by Complementary Sequence Index (CSI) identifies that among all types of complementary sequences, SINEs, especially Alu elements in human, contribute the most for circRNA formation and that their diverse distribution across species leads to the increased complexity of circRNA expression during species evolution. Together, our integrated and comparative reference catalog of circRNAs in different species reveals a species-specific pattern of circRNA expression and suggests a previously under-appreciated impact of fast-evolved SINEs on the regulation of (circRNA) gene expression.

Base-Editor-Mediated circRNA Knockout by Targeting Predominantly Back-Splice Sites.

Back-splicing of eukaryotic exon(s) leads to the production of covalently closed circular RNAs (circRNAs). Generally, most circRNAs contain overlapping sequences to their cognate linear RNAs from the same gene loci, leading to difficulties in distinguishing them from each other. A recent study has shown that some circRNAs can be specifically depleted by using base editing systems to target their predominantly back-splice sites for circularization, suggesting an efficient approach for circRNA knockout (KO). Here, we describe the detailed protocol for applying base editors to disrupt back-splice sites of predominantly circularized exons for circRNA KO at the genomic DNA level.

CRISPR-dCas13-tracing reveals transcriptional memory and limited mRNA export in developing zebrafish embryos.

BACKGROUND: Understanding gene transcription and mRNA-protein (mRNP) dynamics in single cells in a multicellular organism has been challenging. The catalytically dead CRISPR-Cas13 (dCas13) system has been used to visualize RNAs in live cells without genetic manipulation. We optimize this system to track developmentally expressed mRNAs in zebrafish embryos and to understand features of endogenous transcription kinetics and mRNP export. RESULTS: We report that zygotic microinjection of purified CRISPR-dCas13-fluorescent proteins and modified guide RNAs allows single- and dual-color tracking of developmentally expressed mRNAs in zebrafish embryos from zygotic genome activation (ZGA) until early segmentation period without genetic manipulation. Using this approach, we uncover non-synchronized de novo transcription between inter-alleles, synchronized post-mitotic re-activation in pairs of alleles, and transcriptional memory as an extrinsic noise that potentially contributes to synchronized post-mitotic re-activation. We also reveal rapid dCas13-engaged mRNP movement in the nucleus with a corralled and diffusive motion, but a wide varying range of rate-limiting mRNP export, which can be shortened by Alyref and Nxf1 overexpression. CONCLUSIONS: This optimized dCas13-based toolkit enables robust spatial-temporal tracking of endogenous mRNAs and uncovers features of transcription and mRNP motion, providing a powerful toolkit for endogenous RNA visualization in a multicellular developmental organism.

Knockout of circRNAs by base editing back-splice sites of circularized exons.

Many circular RNAs (circRNAs) are produced from back-splicing of exons of precursor mRNAs and are generally co-expressed with cognate linear RNAs. Methods for circRNA-specific knockout are lacking, largely due to sequence overlaps between forms. Here, we use base editors (BEs) for circRNA depletion. By targeting splice sites involved in both back-splicing and canonical splicing, BEs can repress circular and linear RNAs. Targeting sites predominantly for circRNA biogenesis, BEs could efficiently repress the production of circular but not linear RNAs. As hundreds of exons are predominantly back-spliced to produce circRNAs, this provides an efficient method to deplete circRNAs for functional study.

Fast and furious: insights of back splicing regulation during nascent RNA synthesis.

Alternative splicing of eukaryotic precursor (messenger) RNAs in the nucleus not only increases transcriptomic complexity, but also expands proteomic and functional diversity. In addition to basic types of alternative splicing, recent transcriptome-wide analyses have also suggested other new types of non-canonical splicing, such as back splicing and recursive splicing, and their widespread expression across species Increasing lines of evidence have suggested mechanisms for back splicing, including insights from analyses of nascent RNA sequencing. In this review, we discuss our current understanding of back splicing regulation, and highlight its distinct characteristics in processing during nascent RNA synthesis by taking advantage of metabolic tagging nascent RNA sequencing. Features of recursive splicing are also discussed in the perspective of nascent RNA sequencing.

Genome-Wide Annotation of circRNAs and Their Alternative Back-Splicing/Splicing with CIRCexplorer Pipeline.

Circular RNAs (circRNAs) derived from back-spliced exons were sporadically identified about 25 years ago, and have been recently re-discovered genome-wide across different species. Interestingly, one gene locus can generate multiple circRNAs through alternative back-splicing and/or alternative splicing, thus expanding our understanding on the diversity and complexity of transcriptomes. Precise annotation of circRNAs with their alternative back-splicing and alternative splicing events is the basis for the functional characterization of different categories of circRNAs. Here we describe a step-by-step computational scheme to annotate circRNAs from publicly available RNA sequencing datasets with the CIRCexplorer2 pipeline.

CIRCexplorer3: A CLEAR Pipeline for Direct Comparison of Circular and Linear RNA Expression.

Sequences of circular RNAs (circRNAs) produced from back-splicing of exon(s) completely overlap with those from cognate linear RNAs transcribed from the same gene loci with the exception of their back-splicing junction (BSJ) sites. Therefore, examination of global circRNA expression from RNA-seq datasets generally relies on the detection of RNA-seq fragments spanning BSJ sites, which is different from the quantification of linear RNA expression by normalized RNA-seq fragments mapped to whole gene bodies. Thus, direct comparison of circular and linear RNA expression from the same gene loci in a genome-wide manner has remained challenging. Here, we update the previously-reported CIRCexplorer pipeline to version 3 for circular and linear RNA expression analysis from ribosomal-RNA depleted RNA-seq (CIRCexplorer3-CLEAR). A new quantitation parameter, fragments per billion mapped bases (FPB), is applied to evaluate circular and linear RNA expression individually by fragments mapped to circRNA-specific BSJ sites or to linear RNA-specific splicing junction (SJ) sites. Comparison of circular and linear RNA expression levels is directly achieved by dividing FPBcirc by FPBlinear to generate a CIRCscore, which indicates the relative circRNA expression level using linear RNA expression level as the background. Highly-expressed circRNAs with low cognate linear RNA expression background can be readily identified by CIRCexplorer3-CLEAR for further investigation. CIRCexplorer3-CLEAR is publically available at https://github.com/YangLab/CLEAR.

Circular RNAs negatively regulate cancer stem cells by physically binding FMRP against CCAR1 complex in hepatocellular carcinoma.

Circular RNA (circRNA) possesses great pre-clinical diagnostic and therapeutic potentials in multiple cancers. It has been reported playing roles in multiple malignant behaviors including proliferation, migration, metastasis and chemoresistance. However, the underlying correlation between circRNAs and cancer stem cells (CSCs) has not been reported yet. Methods: circZKSCAN1 level was detected in HCC tissue microarrays to clarify its prognostic values. Gain and loss function experiments were applied to investigate the role of circZKSCAN1 in HCC stemness. Bioinformatic analysis was used to predict the possible downstream RNA binding protein and further RNA immunoprecipitation sequencing was carried out to identify the RBP-regulated genes. Results: The absence of circZKSCAN1 endowed several malignant properties including cancer stemness and tightly correlated with worse overall and recurrence-free survival rate in HCC. Bioinformatics analysis and RNA immunoprecipitation-sequencing (RIP-seq) results revealed that circZKSCAN1 exerted its inhibitive role by competitively binding FMRP, therefore, block the binding between FMRP and ß-catenin-binding protein-cell cycle and apoptosis regulator 1 (CCAR1) mRNA, and subsequently restrain the transcriptional activity of Wnt signaling. In addition, RNA-splicing protein Quaking 5 was found downregulated in HCC tissues and responsible for the reduction of circZKSCAN1. Conclusion: Collectively, this study revealed the mechanisms underlying the regulatory role of circZKSCAN1 in HCC CSCs and identified the newly discovered Qki5-circZKSCAN1-FMRP-CCAR1-Wnt signaling axis as a potentially important therapeutic target for HCC treatment.

CIRCpedia v2: An Updated Database for Comprehensive Circular RNA Annotation and Expression Comparison.

Circular RNAs (circRNAs) from back-splicing of exon(s) have been recently identified to be broadly expressed in eukaryotes, in tissue- and species-specific manners. Although functions of most circRNAs remain elusive, some circRNAs are shown to be functional in gene expression regulation and potentially relate to diseases. Due to their stability, circRNAs can also be used as biomarkers for diagnosis. Profiling circRNAs by integrating their expression among different samples thus provides molecular basis for further functional study of circRNAs and their potential application in clinic. Here, we report CIRCpedia v2, an updated database for comprehensive circRNA annotation from over 180 RNA-seq datasets across six different species. This atlas allows users to search, browse, and download circRNAs with expression features in various cell types/tissues, including disease samples. In addition, the updated database incorporates conservation analysis of circRNAs between humans and mice. Finally, the web interface also contains computational tools to compare circRNA expression among samples. CIRCpedia v2 is accessible at http://www.picb.ac.cn/rnomics/circpedia.