Past, present, and future strategies for detecting and quantifying circular RNA variants.

Circular RNAs (circRNAs) are a family of covalently closed RNA transcripts ubiquitous across the eukaryotic kingdom. CircRNAs are generated by a class of alternative splicing called backsplicing, with the resultant circularization of a part of parental RNA producing the characteristic backsplice junction (BSJ). Because of the noncontiguous sequence of the BSJ with respect to the DNA genome, circRNAs remained hidden in plain sight through over a decade of RNA next-generation sequencing, yet over 3 million unique circRNA transcripts have been illuminated in the past decade alone.

Use of synthetic circular RNA spike-ins (SynCRS) for normalization of circular RNA sequencing data.

High-throughput RNA sequencing enables the quantification of transcript abundance and the identification of novel transcripts in biological samples. These include circular RNAs (circRNAs), a family of alternatively spliced RNA molecules that form a continuous loop. However, quantification and comparison of circRNAs between RNA sequencing libraries remain challenging due to confounding errors introduced during exonuclease digestion, library preparation and RNA sequencing itself.

Circular RNA in cancer.

Over the past decade, circular RNA (circRNA) research has evolved into a bona fide research field shedding light on the functional consequence of this unique family of RNA molecules in cancer. Although the method of formation and the abundance of circRNAs can differ from their cognate linear mRNA, the spectrum of interacting partners and their resultant cellular functions in oncogenesis are analogous. However, with 10 times more diversity in circRNA variants compared with linear RNA variants, combined with their hyperstability in the cell, circRNAs are equipped to influence every stage of oncogenesis.

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).

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.

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.

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.

Functional Characterisation of the Circular RNA, , 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 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 pre-mRNA.

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.

Don't go in circles: confounding factors in gene expression profiling.

Quantification of gene expression is a crucial research tool in the life sciences, which makes it important to identify any factors that could compromise its accuracy. One of these factors are non‐polyadenylated (poly(A)) transcripts, including circular RNAs (circRNAs) that can skew quantification of gene expression as they resemble messenger RNAs (mRNAs). Here, we highlight the impact circRNAs and other poly(A) transcripts have on gene expression profiling and the biological conclusions drawn from such experiments.

A circRNA from SEPALLATA3 regulates splicing of its cognate mRNA through R-loop formation.

Circular RNAs (circRNAs) are a diverse and abundant class of hyper-stable, non-canonical RNAs that arise through a form of alternative splicing (AS) called back-splicing. These single-stranded, covalently-closed circRNA molecules have been identified in all eukaryotic kingdoms of life, yet their functions have remained elusive. Here, we report that circRNAs can be used as bona fide biomarkers of functional, exon-skipped AS variants in Arabidopsis, including in the homeotic MADS-box transcription factor family.

The RNA binding protein quaking regulates formation of circRNAs.

Circular RNAs (circRNAs), formed by non-sequential back-splicing of pre-mRNA transcripts, are a widespread form of non-coding RNA in animal cells. However, it is unclear whether the majority of circRNAs represent splicing by-products without function or are produced in a regulated manner to carry out specific cellular functions. We show that hundreds of circRNAs are regulated during human epithelial-mesenchymal transition (EMT) and find that the production of over one-third of abundant circRNAs is dynamically regulated by the alternative splicing factor, Quaking (QKI), which itself is regulated during EMT.

Protocol: a fast and simple in situ PCR method for localising gene expression in plant tissue.

Background: An important step in characterising the function of a gene is identifying the cells in which it is expressed. Traditional methods to determine this include in situ hybridisation, gene promoter-reporter fusions or cell isolation/purification techniques followed by quantitative PCR. These methods, although frequently used, can have limitations including their time-consuming nature, limited specificity, reliance upon well-annotated promoters, high cost, and the need for specialized equipment.

Effect of microbial inoculants on the indigenous actinobacterial endophyte population in the roots of wheat as determined by terminal restriction fragment length polymorphism.

The effect of single actinobacterial endophyte seed inoculants and a mixed microbial soil inoculant on the indigenous endophytic actinobacterial population in wheat roots was investigated by using the molecular technique terminal restriction fragment length polymorphism (T-RFLP). Wheat was cultivated either from seeds coated with the spores of single pure actinobacterial endophytes of Microbispora sp. strain EN2, Streptomyces sp.

Analysis of the endophytic actinobacterial population in the roots of wheat (Triticum aestivum L.) by terminal restriction fragment length polymorphism and sequencing of 16S rRNA clones.

The endophytic actinobacterial population in the roots of wheat grown in three different soils obtained from the southeast part of South Australia was investigated by terminal restriction fragment length polymorphism (T-RFLP) analysis of the amplified 16S rRNA genes. A new, validated approach was applied to the T-RFLP analysis in order to estimate, to the genus level, the actinobacterial population that was identified. Actinobacterium-biased primers were used together with three restriction enzymes to obtain terminal restriction fragments (TRFs).