Native Circular RNA Pulldown Method to Simultaneously Profile RNA and Protein Interactions.

Circular RNAs (circRNAs) are a widespread, cell-, tissue-, and disease-specific class of largely non-coding RNA transcripts. These single-stranded, covalently-closed transcripts arise through non-canonical splicing of pre-mRNA, a process called back-splicing. Back-splicing results in circRNAs which are distinguishable from their cognate mRNA as they possess a unique sequence of nucleic acids called the backsplice junction (BSJ). CircRNAs have been shown to play key functional roles in various cellular contexts and achieve this through their interaction with other macromolecules, particularly other RNA molecules and proteins. To elucidate the molecular mechanisms underlying circRNA function, it is necessary to identify these interacting partners. Herein, we present an optimized strategy for the simultaneous purification of the circRNA interactome within eukaryotic cells, allowing the identification of both circRNA-RNA and circRNA-protein interactions.

Nucleotide-level prediction of CircRNA-protein binding based on fully convolutional neural network.

Introduction: CircRNA-protein binding plays a critical role in complex biological activity and disease. Various deep learning-based algorithms have been proposed to identify CircRNA-protein binding sites. These methods predict whether the CircRNA sequence includes protein binding sites from the sequence level, and primarily concentrate on analysing the sequence specificity of CircRNA-protein binding. For model performance, these methods are unsatisfactory in accurately predicting motif sites that have special functions in gene expression. Methods: In this study, based on the deep learning models that implement pixel-level binary classification prediction in computer vision, we viewed the CircRNA-protein binding sites prediction as a nucleotide-level binary classification task, and use a fully convolutional neural networks to identify CircRNA-protein binding motif sites (CPBFCN). Results: CPBFCN provides a new path to predict CircRNA motifs. Based on the MEME tool, the existing CircRNA-related and protein-related database, we analysed the motif functions discovered by CPBFCN. We also investigated the correlation between CircRNA sponge and motif distribution. Furthermore, by comparing the motif distribution with different input sequence lengths, we found that some motifs in the flanking sequences of CircRNA-protein binding region may contribute to CircRNA-protein binding. Conclusion: This study contributes to identify circRNA-protein binding and provides help in understanding the role of circRNA-protein binding in gene expression regulation.

Circular RNAs: Emerging regulators of glucose metabolism in cancer.

Aberrant glucose metabolism is one of the most striking characteristics of metabolic reprogramming in cancer. Thus, clarifying the regulatory mechanism of glucose metabolism is crucial to understanding tumor progression and developing novel therapeutic strategies for cancer patients. Recent developments in circular RNAs have explained the regulatory mechanism of glucose metabolism from a new dimension. In this review, we briefly summarize the recent advances in circRNA research on cancer glucose metabolism and emphasize the different regulatory mechanisms, including acting as miRNA sponges, interacting with proteins and being translated into proteins. Additionally, we discuss the future research directions of circular RNAs in the field of glucose metabolism.

Hsa_circ_0005050 interacts with ILF3 and affects cell apoptosis and proliferation by disrupting the balance between p53 and p65.

The regulatory network between arsenic, genes and signaling pathways has been reported in arsenic carcinogenesis. Studies on circRNA represent a growing field, but the extent to circRNA potential mechanisms remains poorly understood. So this study we explore the systematic function of hsa_circ_0005050 in mediating the cell apoptosis and proliferation. We demonstrated that hsa_circ_0005050 was highly expressed in subjects who are long-term exposed to arsenic, and could be induced by NaAs2O3 in A549 and 16HBE. Knockdown of hsa_circ_0005050 promotes A549 cell viability, whereas exerts the opposite effects in 16HBE. Mechanistically, hsa_circ_0005050 regulates the p53 and NF-κB signaling pathway involved in the apoptosis and proliferation. And we found that hsa_circ_0005050 could directly bind to the RNA binding protein ILF3 and mutually influence each other's formation. Upon si-hsa_circ_0005050, ILF3 export to the cytoplasm resulting the formation of a ternary complex ILF3-p65-IκBA, breaks the balance of p53 and NF-κB pathway and induces A549 apoptosis and leads to 16HBE proliferation. As a result of these investigations, suggestions were identified for future research.

Identification of circRNA-Interacting Proteins by Affinity Pulldown.

Circular RNAs (circRNAs) comprise a vast class of covalently closed transcripts, generated primarily via backsplicing. Most circRNAs arise from full or partial exons, but they can also arise from introns, and from combinations of introns and exons. While high-throughput RNA-sequencing analysis has identified tens of thousands of circRNAs expressed in different tissues and growth conditions, the function of circRNAs has only been described for a handful of them. As most circRNAs appear not to encode peptides, their function is presumed to be linked to their interaction with a range of molecules, particularly other nucleic acids (notably microRNAs) and proteins. A major impediment to identifying circRNA-associated molecules is a lack of suitable methodologies capable of analyzing specifically circRNAs and not their linear RNA counterparts with which they share most of their sequence. Here, we describe a flexible and robust method for identifying the proteins that associate with a given circRNA. The affinity pulldown assay is based on the use of a biotinylated antisense oligomer that recognizes the circRNA-specific junction sequence. Following pulldown using streptavidin beads, the proteins are eluted from the circRNP (circribonucleoprotein) complex and identified by mass spectroscopy; validation by Western blot analysis and other methods would then confirm the identity of the circRNA-associated proteins. We present a detailed step-by-step protocol, tips to optimize the analysis, troubleshooting suggestions, and assistance in interpreting the results. In sum, this protocol enables the discovery of proteins present in circRNPs, a critical effort toward elucidating circRNA function.

Silencing circRNA protein kinase C iota (circ-PRKCI) suppresses cell progression and glycolysis of human papillary thyroid cancer through circ-PRKCI/miR-335/E2F3 ceRNA axis.

The circular RNA PRKCI (circ-PRKCI; ID: hsa_circ_0122683) is highly expressed in human papillary thyroid cancer (PTC) tumors according to GSE93522 dataset. However, its role in PTC tumorigenesis remains to be documented. Here, quantitative real-time PCR showed that expression of circ-PRKCI was abnormally upregulated in human PTC patients' tumors and cells, and higher circ-PRKCI might predict lymph node metastasis and recurrence. Functionally, cell behaviors were measured by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide assay, colony formation assay, fluorescence-activated cell sorting method, scratch wound assay, transwell assay, western blotting, and assay kits for glucose and lactate. As a result, circ-PRKCI knockdown could suppress cell cycle progression of PTC cells and restrain the abilities of cell proliferation, colony formation, wound closure, invasion, glucose consumption and lactate production, accompanied with decreased levels of matrix metalloproteinase-2 (MMP2), MMP9 and Snail. Moreover, above-mentioned inhibition could be imitated by overexpressing microRNA-335-5p (miR-335). Molecularly, circ-PRKCI functioned as a sponge for miR-335 and miR-335 could further targeted E2F transcription factor-3 (E2F3), according to dual-luciferase reporter assay and RNA immunoprecipitation. However, downregulating miR-335 diminished the effects of circ-PRKCI role on cell growth, metastasis and glycolysis in PTC cells; besides, there was a counteractive effect between miR-335 upregulation and E2F3 upregulation in PTC cells as well. Furthermore, xenograft experiment revealed that silencing circ-PRKCI could retard tumor growth of PTC cells in vivo. Collectively, circ-PRKCI exerted oncogenic role in PTC by antagonizing cell progression and glycolysis via regulating miR-335/E2F3 axis, suggesting circ-PRKCI was a potential biomarker and target for PTC.

Circular RNA: metabolism, functions and interactions with proteins.

Circular RNAs (CircRNAs) are single-stranded, covalently closed RNA molecules that are ubiquitous across species ranging from viruses to mammals. Important advances have been made in the biogenesis, regulation, localization, degradation and modification of circRNAs. CircRNAs exert biological functions by acting as transcriptional regulators, microRNA (miR) sponges and protein templates. Moreover, emerging evidence has revealed that a group of circRNAs can serve as protein decoys, scaffolds and recruiters. However, the existing research on circRNA-protein interactions is quite limited. Hence, in this review, we briefly summarize recent progress in the metabolism and functions of circRNAs and elaborately discuss the patterns of circRNA-protein interactions, including altering interactions between proteins, tethering or sequestering proteins, recruiting proteins to chromatin, forming circRNA-protein-mRNA ternary complexes and translocating or redistributing proteins. Many discoveries have revealed that circRNAs have unique expression signatures and play crucial roles in a variety of diseases, enabling them to potentially act as diagnostic biomarkers and therapeutic targets. This review systematically evaluates the roles and mechanisms of circRNAs, with the hope of advancing translational medicine involving circRNAs.

Guidance of circular RNAs to proteins' behavior as binding partners.

Circular RNAs (circRNAs) are single-stranded and covalently closed back-splicing products of pre-mRNAs. They can be derived from exons, introns, or exons with intron retained between exons of transcripts, as well as antisense transcripts. CircRNAs have been reported to function as microRNA sponges, regulate gene transcription mediated by RNA polymerase II, and modulate the splicing or stability of mRNA. However, emerging studies demonstrate that they affect the behavior of proteins via direct interactions with them. Here, we summarize that by binding directly with proteins; circRNAs can facilitate their nuclear or cytoplasmic localizations, regulate their functions or stability, promote or inhibit the interactions between them, or influence the interactions between them and DNA. Furthermore, these circRNA-binding proteins contain transcription factors, RNA processing proteins, proteases, and some other RNA-binding proteins. As a consequence, circRNAs are involved in the regulation of multiple physiological or pathological processes, including tumorigenesis, atherosclerosis, wound repair, cardiac senescence, myocardial ischemia/reperfusion injury, and so forth. Nonetheless, it is worthwhile to further explore more types of proteins that interact with circRNAs, which would be helpful in revealing other unknown biological functions of circRNAs that guide the variation in behavior of cellular proteins.