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.

ESRP1 controls biogenesis and function of a large abundant multiexon circRNA.

While the majority of circRNAs are formed from infrequent back-splicing of exons from protein coding genes, some can be produced at quite high level and in a regulated manner. We describe the regulation, biogenesis and function of circDOCK1(2-27), a large, abundant circular RNA that is highly regulated during epithelial-mesenchymal transition (EMT) and whose formation depends on the epithelial splicing regulator ESRP1. CircDOCK1(2-27) synthesis in epithelial cells represses cell motility both by diverting transcripts from DOCK1 mRNA production to circRNA formation and by direct inhibition of migration by the circRNA. HITS-CLIP analysis and CRISPR-mediated deletions indicate ESRP1 controls circDOCK1(2-27) biosynthesis by binding a GGU-containing repeat region in intron 1 and detaining its splicing until Pol II completes its 157 kb journey to exon 27. Proximity-dependent biotinylation (BioID) assay suggests ESRP1 may modify the RNP landscape of intron 1 in a way that disfavours communication of exon 1 with exon 2, rather than physically bridging exon 2 to exon 27. The X-ray crystal structure of RNA-bound ESRP1 qRRM2 domain reveals it binds to GGU motifs, with the guanines embedded in clamp-like aromatic pockets in the protein.

Versatile toolkit for highly-efficient and scarless overexpression of circular RNAs.

Circular RNAs (circRNAs) are a class of single-stranded, covalently closed RNA that contain a unique back-splice junction (bsj) sequence created by the ligation of their 5' and 3' ends via spliceosome-catalyzed back-splicing. A key step in illuminating the cellular roles of specific circRNAs is via increasing their expression. This is frequently done by transfecting cells with plasmid DNA containing cloned exons from which the circRNA is transcribed, flanked by sequences that promote back-splicing. We observed that commonly used plasmids lead to the production of circRNAs with molecular scars at the circRNA bsj. Stepwise redesign of the cloning vector corrected this problem, ensuring bona fide circRNAs are produced with their natural bsj at high efficiency. The fidelity of circRNAs produced from this new construct was validated by RNA sequencing and also functionally validated. To increase the utility of this modified resource for expressing circRNA, we developed an expanded set of vectors incorporating this design that (i) enables selection with a variety of antibiotics and fluorescent proteins, (ii) employs a range of promoters varying in promoter strength and (iii) generated a complementary set of lentiviral plasmids for difficult-to-transfect cells. These resources provide a novel and versatile toolkit for high-efficiency and scarless overexpression of circular RNAs that fulfill a critical need for the investigation of circRNA function.

Human cerebrospinal fluid affects chemoradiotherapy sensitivities in tumor cells from patients with glioblastoma.

Cancers in the central nervous system resist therapies effective in other cancers, possibly due to the unique biochemistry of the human brain microenvironment composed of cerebrospinal fluid (CSF). However, the impact of CSF on cancer cells and therapeutic efficacy is unknown. Here, we examined the effect of human CSF on glioblastoma (GBM) tumors from 25 patients. We found that CSF induces tumor cell plasticity and resistance to standard GBM treatments (temozolomide and irradiation). We identified nuclear protein 1 (NUPR1), a transcription factor hampering ferroptosis, as a mediator of therapeutic resistance in CSF. NUPR1 inhibition with a repurposed antipsychotic, trifluoperazine, enhanced the killing of GBM cells resistant to chemoradiation in CSF. The same chemo-effective doses of trifluoperazine were safe for human neurons and astrocytes derived from pluripotent stem cells. These findings reveal that chemoradiation efficacy decreases in human CSF and suggest that combining trifluoperazine with standard care may improve the survival of patients with GBM.

Intra- and interchromosomal contact mapping reveals the Igh locus has extensive conformational heterogeneity and interacts with B-lineage genes.

To produce a diverse antibody repertoire, immunoglobulin heavy-chain (Igh) loci undergo large-scale alterations in structure to facilitate juxtaposition and recombination of spatially separated variable (VH), diversity (DH), and joining (JH) genes. These chromosomal alterations are poorly understood. Uncovering their patterns shows how chromosome dynamics underpins antibody diversity. Using tiled Capture Hi-C, we produce a comprehensive map of chromatin interactions throughout the 2.8-Mb Igh locus in progenitor B cells. We find that the Igh locus folds into semi-rigid subdomains and undergoes flexible looping of the VH genes to its 3' end, reconciling two views of locus organization. Deconvolution of single Igh locus conformations using polymer simulations identifies thousands of different structures. This heterogeneity may underpin the diversity of V(D)J recombination events. All three immunoglobulin loci also participate in a highly specific, developmentally regulated network of interchromosomal interactions with genes encoding B cell-lineage factors. This suggests a model of interchromosomal coordination of B cell development.

Circular RNAs drive oncogenic chromosomal translocations within the MLL recombinome in leukemia.

The first step of oncogenesis is the acquisition of a repertoire of genetic mutations to initiate and sustain the malignancy. An important example of this initiation phase in acute leukemias is the formation of a potent oncogene by chromosomal translocations between the mixed lineage leukemia (MLL) gene and one of 100 translocation partners, known as the MLL recombinome. Here, we show that circular RNAs (circRNAs)-a family of covalently closed, alternatively spliced RNA molecules-are enriched within the MLL recombinome and can bind DNA, forming circRNA:DNA hybrids (circR loops) at their cognate loci. These circR loops promote transcriptional pausing, proteasome inhibition, chromatin re-organization, and DNA breakage. Importantly, overexpressing circRNAs in mouse leukemia xenograft models results in co-localization of genomic loci, de novo generation of clinically relevant chromosomal translocations mimicking the MLL recombinome, and hastening of disease onset. Our findings provide fundamental insight into the acquisition of chromosomal translocations by endogenous RNA carcinogens in leukemia.

Functional Characterisation of the Circular RNA, circHTT(2-6), in Huntington's Disease.

Trinucleotide repeat disorders comprise ~20 severe, inherited, human neuromuscular and neurodegenerative disorders, which result from an abnormal expansion of repetitive sequences in the DNA. The most common of these, Huntington's disease (HD), results from expansion of the CAG repeat region in exon 1 of the HTT gene via an unknown mechanism. Since non-coding RNAs have been implicated in the initiation and progression of many diseases, herein we focused on a circular RNA (circRNA) molecule arising from non-canonical splicing (backsplicing) of HTT pre-mRNA. The most abundant circRNA from HTT, circHTT(2-6), was found to be more highly expressed in the frontal cortex of HD patients, compared with healthy controls, and positively correlated with CAG repeat tract length. Furthermore, the mouse orthologue (mmu_circHTT(2-6)) was found to be enriched within the brain and specifically the striatum, a region enriched for medium spiny neurons that are preferentially lost in HD. Transgenic overexpression of circHTT(2-6) in two human cell lines-SH-SY5Y and HEK293-reduced cell proliferation and nuclear size without affecting cell cycle progression or cellular size, or altering the CAG repeat region length within HTT. CircHTT(2-6) overexpression did not alter total HTT protein levels, but reduced its nuclear localisation. As these phenotypic and genotypic changes resemble those observed in HD patients, our results suggest that circHTT(2-6) may play a functional role in the pathophysiology of this disease.

The RNA interactome in the Hallmarks of Cancer.

Ribonucleic acid (RNA) molecules are indispensable for cellular homeostasis in healthy and malignant cells. However, the functions of RNA extend well beyond that of a protein-coding template. Rather, both coding and non-coding RNA molecules function through critical interactions with a plethora of cellular molecules, including other RNAs, DNA, and proteins. Deconvoluting this RNA interactome, including the interacting partners, the nature of the interaction, and dynamic changes of these interactions in malignancies has yielded fundamental advances in knowledge and are emerging as a novel therapeutic strategy in cancer. Here, we present an RNA-centric review of recent advances in the field of RNA-RNA, RNA-protein, and RNA-DNA interactomic network analysis and their impact across the Hallmarks of Cancer. This article is categorized under: RNA in Disease and Development > RNA in Disease RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes.

The Non-Coding RNA Journal Club: Highlights on Recent Papers-12.

We are delighted to share with you our twelfth Journal Club and highlight some of the most interesting papers published recently [...].

Circular RNAs: Non-Canonical Observations on Non-Canonical RNAs.

The existence of circular RNA (circRNA) research in mainstream science can be attributed to the contemporary synergism of big data and keen attention to detail by several research groups worldwide. Since the re-emergence of these non-canonical RNA transcripts, seminal advances have been made in understanding their biogenesis, interactome, and functions in diverse fields and a myriad of human diseases. However, most research outputs to date have focused on the ability of highly stable circRNAs to interact with, and impact signalling through, microRNAs. This is likely to be the result of seminal papers in the field ascribing a few remarkable circRNAs as "miRNA sponges". However, the stoichiometric ratio between the (often-lowly-expressed) circRNA and their (commonly-more-abundant) target is rarely in favour of a biologically relevant and functional consequence of these interactions. It is time for yet another revolution in circRNA research to uncover functions beyond their documented ability to bind miRNAs. This Special Issue aims to highlight non-canonical functions for this non-canonical family of RNA molecules.

The Suitability of Glioblastoma Cell Lines as Models for Primary Glioblastoma Cell Metabolism.

In contrast to most non-malignant tissue, cells comprising the brain tumour glioblastoma (GBM) preferentially utilise glycolysis for metabolism via "the Warburg effect". Research into therapeutics targeting the disease's highly glycolytic state offer a promising avenue to improve patient survival. These studies often employ GBM cell lines for in vitro studies which translate poorly to the in vivo patient context. The metabolic traits of five of the most used GBM cell lines were assessed and compared to primary GBM and matched, healthy brain tissue. In patient-derived GBM cell lines, the basal mitochondrial rate (p = 0.043) and ATP-linked respiration (p < 0.001) were lower than primary adjacent normal cells from the same patient, while reserve capacity (p = 0.037) and Krebs cycle capacity (p = 0.002) were higher. Three cell lines, U251MG, U373MG and D54, replicate the mitochondrial metabolism of primary GBM cells. Surprisingly, glycolytic capacity is not different between healthy and GBM tissue. The T98G cell line recapitulated glycolysis-related metabolic parameters of the primary GBM cells and is recommended for research relating to glycolysis. These findings can guide preclinical research into the development of novel therapeutics targeting metabolic pathways in GBM.

SRRM4 Expands the Repertoire of Circular RNAs by Regulating Microexon Inclusion.

High-throughput RNA sequencing (RNA-seq) and dedicated bioinformatics pipelines have synergized to identify an expansive repertoire of unique circular RNAs (circRNAs), exceeding 100,000 variants. While the vast majority of these circRNAs comprise canonical exonic and intronic sequences, microexons (MEs)-which occur in 30% of functional mRNA transcripts-have been entirely overlooked. CircRNAs which contain these known MEs (ME-circRNAs) could be identified with commonly utilized circRNA prediction pipelines, CIRCexplorer2 and CIRI2, but were not previously recognized as ME-circRNAs. In addition, when employing a bespoke bioinformatics pipeline for identifying RNA chimeras, called Hyb, we could also identify over 2000 ME-circRNAs which contain novel MEs at their backsplice junctions, that are uncalled by either CIRCexplorer2 or CIRI2. Analysis of circRNA-seq datasets from gliomas of varying clinical grades compared with matched control tissue has shown circRNAs have potential as prognostic markers for stratifying tumor from healthy tissue. Furthermore, the abundance of microexon-containing circRNAs (ME-circRNAs) between tumor and normal tissues is correlated with the expression of a splicing associated factor, Serine/arginine repetitive matrix 4 (SRRM4). Overexpressing SRRM4, known for regulating ME inclusion in mRNAs critical for neural differentiation, in human HEK293 cells resulted in the biogenesis of over 2000 novel ME-circRNAs, including ME-circEIF4G3, and changes in the abundance of many canonical circRNAs, including circSETDB2 and circLBRA. This shows SRRM4, in which its expression is correlated with poor prognosis in gliomas, acts as a bona fide circRNA biogenesis factor. Given the known roles of MEs and circRNAs in oncogenesis, the identification of these previously unrecognized ME-circRNAs further increases the complexity and functional purview of this non-coding RNA family.

SplintQuant: a method for accurately quantifying circular RNA transcript abundance without reverse transcription bias.

Reverse transcription of RNA is fallible, introducing biases and confounding the quantification of transcript abundance. We demonstrate that circular RNAs (circRNAs) are more subjective to overestimation of transcript abundance than cognate linear RNAs due to their covalently closed, circular form, producing multiple concatameric products from a single priming of reverse transcriptase. We developed SplintQuant, where custom DNA oligonucleotides are ligated by PBCV-1 DNA ligase only when bound to their target RNA. These circRNA-specific DNA oligonucleotides are terminally tagged with universal primers, allowing SplintQuant to accurately quantify even lowly abundant circRNAs through highly specific quantitative PCR (qPCR) in the absence of reverse transcription. SplintQuant is sensitive, specific, highly reproducible, and applicable to the quantification of canonical and noncanonical RNA transcripts including alternative splice variants, gene fusions, and offers a gold-standard approach for accurately quantifying circRNAs.

The Non-Coding RNA Journal Club: Highlights on Recent Papers-7.

We are delighted to share with you our seventh Journal Club and highlight some of the most interesting papers published recently [...].

The Non-Coding RNA Journal Club: Highlights on Recent Papers-6.

We are delighted to share with you our sixth Journal Club and highlight some of the most interesting papers published recently [...].

CircRNAs in Plants.

Circular RNAs (circRNAs) are covalently closed, single-stranded transcripts that are ubiquitously expressed in all eukaryotes and even prokaryotic archaea. Although once regarded as splicing artifacts, circRNAs are a novel class of regulatory molecules with diverse biological functions, including regulation of transcription, modulation of alternative splicing, and binding of miRNAs and proteins. The majority of studies of circRNAs have been performed in animals with a focus on the biogenesis, function, and mechanistic characterization of these molecules. In contrast, the study of circRNAs in plants is just emerging. Interestingly, recent circRNA profiling studies in model plant systems show distinct features of plant circRNAs compared with those from animals, including putative roles in stress response, differences in expression patterns, and novel biogenesis mechanisms. This provides a great opportunity to broaden our knowledge of circRNAs using plant model systems, such as Arabidopsis and rice, which are ideal for phenotypic characterization and genetic studies. In this review, we summarize current knowledge of plant circRNAs, discuss their identification and biogenesis, describe potential functions, and propose future perspectives for plant circRNA study.

miR-200/375 control epithelial plasticity-associated alternative splicing by repressing the RNA-binding protein Quaking.

Members of the miR-200 family are critical gatekeepers of the epithelial state, restraining expression of pro-mesenchymal genes that drive epithelial-mesenchymal transition (EMT) and contribute to metastatic cancer progression. Here, we show that miR-200c and another epithelial-enriched miRNA, miR-375, exert widespread control of alternative splicing in cancer cells by suppressing the RNA-binding protein Quaking (QKI). During EMT, QKI-5 directly binds to and regulates hundreds of alternative splicing targets and exerts pleiotropic effects, such as increasing cell migration and invasion and restraining tumour growth, without appreciably affecting mRNA levels. QKI-5 is both necessary and sufficient to direct EMT-associated alternative splicing changes, and this splicing signature is broadly conserved across many epithelial-derived cancer types. Importantly, several actin cytoskeleton-associated genes are directly targeted by both QKI and miR-200c, revealing coordinated control of alternative splicing and mRNA abundance during EMT These findings demonstrate the existence of a miR-200/miR-375/QKI axis that impacts cancer-associated epithelial cell plasticity through widespread control of alternative splicing.

Tetramerization of MADS family transcription factors SEPALLATA3 and AGAMOUS is required for floral meristem determinacy in Arabidopsis.

The MADS transcription factors (TF) constitute an ancient family of TF found in all eukaryotes that bind DNA as obligate dimers. Plants have dramatically expanded the functional diversity of the MADS family during evolution by adding protein-protein interaction domains to the core DNA-binding domain, allowing the formation of heterotetrameric complexes. Tetramerization of plant MADS TFs is believed to play a central role in the evolution of higher plants by acting as one of the main determinants of flower formation and floral organ specification. The MADS TF, SEPALLATA3 (SEP3), functions as a central protein-protein interaction hub, driving tetramerization with other MADS TFs. Here, we use a SEP3 splice variant, SEP3Δtet, which has dramatically abrogated tetramerization capacity to decouple SEP3 tetramerization and DNA-binding activities. We unexpectedly demonstrate that SEP3 heterotetramer formation is required for correct termination of the floral meristem, but plays a lesser role in floral organogenesis. The heterotetramer formed by SEP3 and the MADS protein, AGAMOUS, is necessary to activate two target genes, KNUCKLES and CRABSCLAW, which are required for meristem determinacy. These studies reveal unique and highly specific roles of tetramerization in flower development and suggest tetramerization may be required to activate only a subset of target genes in closed chromatin regions.