User:Ysobhi/Circular RNA

Classes of CircRNA

Hepatitis delta virus containing a circular single-stranded RNA genome.

Circular RNAs can be separated into five classes:

Classes of Circular RNAs	Description
Viroids and the hepatitis delta virus (HDV)	In viroids and HDV, single-stranded circRNAs are vital in RNA replication. Circularity allows for one initiation event to lead to multiple genomic copies in a process otherwise known as rolling circle RNA replication.
CircRNAs from introns	Circular molecules are produced by introns produced from spliceosomal splicing, tRNA splicing, and group I and group II (self-splicing ribozymes) introns. Group I introns form circRNAs through autocatalytic ribozymal action, and while they can be detected in vivo, their function is yet to be determined. Group II introns also generate circRNAs in vivo. The final class of circular introns is produced from eukaryotic spliceosomal splicing and are circularized intron lariats known as circular intronic RNAs (ciRNAs). Due to circularization, ciRNAs can avoid degradation and are believed to be highly overrepresented. CiRNA function is currently unknown; however, it is speculated they may play a role in enhancing the transcription of genes they are produced from, as they interact with RNA polymerase II.
CircRNAs from intermediates in RNA processing reactions	These are first spliced from precursors as linear molecules and then circularized with a ligase. They are essential in allowing for the rearrangement in RNA sequence order and vital in the biogenesis of permuted tRNA genes in certain algae and archaea.
Noncoding circRNAs in archaea	Certain archaeal species have circRNAs that are produced from excised circularized tRNA introns. Circularization of functional noncoding RNAs is thought to work as a protective mechanism against exonucleases and to promote proper folding.
CircRNAs in eukaryotes produced by back-splicing	Circular RNAs produced by back-splicing (a form of exon scrambling) occur when a 5′ splice site is joined to an upstream 3′ splice site. Currently, more than 25,000 different circRNAs have been identified in humans.

Early discoveries of circRNAs

Early discoveries of circular RNAs led to the belief that they lacked significance due to their rarity. These early discoveries included the analysis of genes like the DCC and Sry genes, and the recent discovery of the human non-coding RNA ANRIL, all of which expressed circular isoforms. CircRNA producing genes like the human ETS-1 gene, the human and rat cytochrome P450 genes, the rat androgen binding protein gene (Shbg), and the human dystrophin gene were also discovered.

Genome-wide identification of circRNAs

Scrambled isoforms and circRNAs

In 2012, in an effort to initially identify cancer-specific exon scrambling events, scrambled exons were discovered in large numbers in both normal and cancer cells. It was found that scrambled exon isoforms comprised about 10% of the total transcript isoforms in leukocytes, with 2,748 scrambled isoforms in HeLa and H9 embryonic stem cells being identified. Additionally, about 1 in 50 expressed genes produced scrambled transcript isoforms at least 10% of the time. Tests used to recognize circularity included treating samples with RNase R, an enzyme that degrades linear but not circular RNAs, and testing for the presence of poly-A tails, which are not present in circular molecules. Overall, 98% of scrambled isoforms were found to represent circRNAs, circRNAs were found to be located in the cytoplasm, and circRNAs were found to be abundant.

Discovery of a higher abundance of circRNAs

In 2013, a higher abundance of circRNAs was discovered. Human fibroblast RNA was treated with RNase R to enrich for circular RNAs, followed by the categorization of circular transcripts based on their abundance (low, medium, high). Approximately 1 in 8 expressed genes were found to produce detectable levels of circRNAs, including those of low abundance, which was significantly higher than previously suspected, and was attributed to greater sequencing depth.

CircRNAs tissue specificity and antagonist activity

At the same time, a computational method to detect circRNAs was developed, leading to de novo detection of circRNAs in humans, mice, and C. elegans, and extensively validating them. The expression of circRNAs was often found to be tissue/developmental stage specific. Additionally, circRNAs were found to have the ability to act as antagonists of miRNAs, microRNAs which interfere with translation of mRNAs, as exemplified by the circRNA CDR1as, which has miRNA binding sites (as seen below).

CircRNAs and ENCODE Ribozero RNA-seq data

In 2014, human circRNAs were identified and quantified from ENCODE Ribozero RNA-seq data. Most circRNAs were found to be minor splice isoforms and to be expressed in only a few cell types, with 7,112 human circRNAs having circular fractions (the fraction of similarity an isoform has to transcripts the same locus) of at least 10%. CircRNAs were also found to be no more conserved than their linear controls and, according to ribosome profiling, are not translated. As previously noted, circRNAs have the ability to act as antagonists of miRNA, which is also known as the potential to act as microRNA sponges. Aside from CDR1as, very few circRNAs have the potential to act as microRNA sponges. As a whole, the majority of circular RNAs were found to be inconsequential side-products of imperfect splicing.

CircRNAs and CIRCexplorer

In the same year, CIRCexplorer, a tool used to identify thousands of circRNAs in humans without RNase R RNA-seq data, was developed. The vast majority of identified highly expressed exonic circular RNAs were found to be processed from exons located in the middle of RefSeq genes, suggesting that the circular RNA formation is generally coupled to RNA splicing. It was determined that most circular RNAs contain multiple, most commonly, two to three, exons. Exons from circRNAs with only one circularized exon were found to be much longer than those from circRNAs with multiple circularized exons, indicating that processing may prefer a certain length to maximize exon(s) circularization. The introns of circularized exons generally contain high Alu densities that can form inverted repeated Alu pairs (IRAlus). IRAlus, either convergent or divergent, are juxtaposed across flanking introns of circRNAs in a parallel way with similar distances to adjacent exons. IRAlus, and other non-repetitive, but complementary, sequences were also found to promote circular RNA formation. On the other hand, exon circularization efficiency was determined to be affected by the competition of RNA pairing, such that alternative RNA pairing, and its competition, leads to alternative circularization. Finally, both exon circularization and its regulation were found to be evolutionarily dynamic.

Genome-wide calling of circRNA in Alzheimer disease cases

Alzheimer disease (AD) cases demonstrating the role of circRNAs in health and disease, and optimizing and validating a pipeline for calling circRNA from human ribo-depleted RNA-seq. An association between circRNAs and neurodegenerative diseases like AD and clinical dementia was elucidated, with a total of 148 circRNAs being significantly correlated with clinical dementia ratings at expiration/death (CDR) after false discovery rate (FDR) correction. The expression of circRNAs was independent of the lineal form and that circRNA expression was also corrected by cell proportion. CircRNAs were also found to be co-expressed with known causal Alzheimer genes, such as APP and PSEN1, indicating that some circRNAs are also part of the causal pathway. Altogether, circRNA brain expression was found to explain more about Alzheimer’s clinical manifestations than the number of APOε4 alleles, suggesting that circRNAs could be used as a potential biomarker for Alzheimer’s.