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circRNAs are circular noncoding RNAs formed by reverse splicing of pre-mRNAs. circRNAs were
firstly found in viruses in the 1970s. However, due to the extensive use of
the method for enrichment of poly (A) (no 5 'and 3' ends of circRNA) in the early RNA
library preparation, and the calculation algorithm that RNA-seq reading requires linear
alignment with genome, a large number of circRNA information was omitted, which led to the
belief that circRNA is just a byproduct of miss splicing.
With the development of high-throughput sequencing technology and bioinformatics, thousands
of circRNA have been found, and more and more basic researches related to circRNA have been
done. A large number of studies have shown that circRNA is endogenetic, abundant,
conservative and stable in mammalian cells, and often shows tissue or space-time
specificity. It can participate in the regulation of cell growth and development, as well as
the occurrence and development of diseases through a variety of mechanisms. Therefore, in
recent years, circRNA has become popular in the field of non-coding RNA research.
circRNA related services in Ubigene:
what is circRNA
Mechanisms of circRNA formation
According to the origin of circRNA, it can be sorted into
three types:
· Exonic circRNAs: All sequences are
derived from exons.
· EIciRNAs: Sequences are derived from
exons and introns.
· ciRNAs: All sequences are derived from
introns.
Mechanisms of circRNA formation
circRNA is formed by the pre-mRNA by back splicing. At
present, there are three kinds of mechanisms reported as follows:
· Intron reverse
complementary sequence
The flanking introns at both sides of the exon
contain many pairs of reverse complementary sequences. The reverse
complementary sequence promotes the intron sequence pairing, making the
Splice-Donor in the downstream close to the Splice-Acceptor in the
upstream, so as to form a circRNA. (Fig 1. Left)
· RNA binding
protein
The flanking introns at both sides of the exon
contain the motifs recognized by RNA binding proteins (RBPs). RBP, when
combined with the specific motifs of the two flanking introns, will form
dimers, promote the two flanking introns close to each other, and then
connect to form a ring. (Fig 1. Right)
· Lariat-driven
circularization
When the pre-mRNA is spliced, exon skipping occurs,
which results in the formation of a lariat intermediate containing exon
and intron. Then the intermediate is back spliced to form a circRNA.
(Fig 2.)
Functions of circRNA
The most common function of circRNA is to bind
to miRNA as miRNA sponge, thus affecting the regulation of miRNA on
genes.（Fig 3.）
Many circRNAs contain protein binding sites, which can be used as
protein sponges.
In addition to being miRNA and protein sponge, circRNAs can also be used
as a scaffold protein to promote the co-location of enzyme, to inhibit
the target gene expression by binding transcription factors, to
participate in the regulation of parent gene expression, and to
translate polypeptides under specific circumstances. According to the
different functions, the locations of circRNAs are different. For
example, as a miRNA or protein sponge, circRNA needs to be transported
from the nucleus to the cell matrix to play a role. When participating
in the regulation of parent gene expression or binding transcription
factor to inhibit the target gene, circRNA often plays a role in the
cell nucleus.
Reference：
Kristensen, L. S., Andersen, M. S., Stagsted, L. V., Ebbesen, K. K.,
Hansen, T. B., & Kjems, J. (2019). The biogenesis, biology and
characterization of circular RNAs. Nature Reviews Genetics, 20(11),
675-691.
CircRNA knockout
At present, the most studied is the relationship between circRNAs and tumors.
Some circRNAs promote tumor formation, such as circPvt1 in squamous cell
carcinomas of the head and neck, cirs-7 (CDr1as) in colorectal cancer,
esophageal squamous cell carcinoma and hepatocellular carcinoma. Some circRNAs
suppress tumors, such as circsMARCA5 and circ-SHPRH in glioblastoma. Some
circRNAs may play different roles in different tissues or cells, such as
circHiPK3, which is a proto-oncogene in rectal cancer, but suppresses cancer
cells in bladder cancer.
In addition to cancers, circRNA has been found to be closely related to
diabetes, cardiovascular disease, chronic inflammation and nervous system
diseases. It is believed that with the development of biotechnology and more
in-depth researches on circRNA, the formations and mechanisms of circRNAs can be
identified. circRNAs can play important roles in disease prevention, diagnosis
and treatment discovery.
circRNA knockout refers to editing at the level of DNA to achieve the purpose of
a complete knockout. gRNA and Cas9 would be transferred into cells by virus
transduction or nucleofection. After drug screening, single clones would be
generated. Positive clones would be validated by sequencing.
The most common strategies for circRNA knockout：
Strategy 1
The most commonly used method is to design two gRNAs at both ends of the circRNA exon to knockout the whole cyclized exon sequence. Although this strategy can knockout the circRNA, it will also affect the parent gene encoding the protein, and the study on its function is not ideal.
Strategy 2
The ideal method is to knockout the loop forming elements (Alu) in the flanking intron of the exon, so as to destroy the circRNA loop forming without affecting the expression of the coding gene.（Fig 4.
Ubigene is experienced in designing strategy of knockout the
loop forming elements in the flanking intron of circRNA exon, to achieve the
purpose of knockout circRNA without affecting the expression of coding gene.
Combined with CRISPR-U™ technology, the positive clones of circRNA knockout can
be generated 10x faster than other common methods.
Case study：
circ-HIPK3 is a kind of circRNA rich in human cells, which can combine with a
variety of miRNAs as a regulator of cell growth and affect the formation of
tumors. In order to verify how circ-HIPK3 forms into circle, it is necessary to find the
loop forming elements in flanking intron. A pair of sgRNA is designed for the two
Alu elements predicted at upstream and downstream respectively. The predicted loop
forming elements are knockout by CRISPR/Cas9 system to detect whether the expression
of circRNA changes. After PCR and RT-qPCR verification, it was found that the
expression of circ-HIPK3 was significantly down-regulated after knockout of
downstream loop forming elements, while the expression of circ-HIPK3 was not
decreased but increased after knockout of upstream loop forming elements. It was
speculated that there were too many loop forming elements in the upstream and the
prediction was not accurate. In order to further verify the RNA circulation driven
by other elements, the large fragment of intron in the upstream of the element was
knockout by co-injection of gRNA3 or gRNA4 with gRNA5 or gRNA6. RT-qPCR results
showed that the expression of circ-HIPK3 decreased, indicating that other loop
forming elements of circ-HIPK3 exist.
Reference：
Zheng, Q., Bao, C., Guo, W., Li, S., Chen, J., Chen, B., ... & Liang, L. (2016).
Circular RNA profiling reveals an abundant circHIPK3 that regulates cell growth by
sponging multiple miRNAs. Nature communications, 7(1), 1-13.
CircRNA knockdown (RNAi)
Among the methods to study the function of circRNA, the most classical way to
inhibit circRNA is to knockdown it by RNAi (shRNA or siRNA). In order to avoid
affecting mRNAs of coding genes, the shRNA should be designed at the back
splicing
site (BSS).
Ubigene can design high-score shRNA and use lentivirus to transfer the RNA
interference vector into the cells. Cells were screened according to the drug
screening, and the stable cell lines with circRNA knockdown were obtained.
Case study：
Cell proliferation and apoptosis were detected by CCK-8 and
EdU
assays. The results showed that the knockdown circ-HIPK3 significantly inhibited
cell proliferation.
Reference：
Zheng, Q., Bao, C., Guo, W., Li, S., Chen, J., Chen, B., ... & Liang, L. (2016).
Circular RNA profiling reveals an abundant circHIPK3 that regulates cell growth
by
sponging multiple miRNAs. Nature communications, 7(1), 1-13.
CircRNA overexpression
Overexpression of circRNA is not easy because of its low efficiency of loop
forming,
and it is easy to mismatch. By optimizing the binding sites of RBP, such as Alu
elements and QKI, the circRNA can be formed accurately and efficiently. After
overexpression, it is necessary to detect successful loop-forming and linear
mRNA
expression. In order to study the loop-forming efficiency of a new circRNA
expression system, the mouse circrtn4 gene was selected to express in a variety
of
cell lines (including HeLa, N2a, HEK293). According to the RT-qPCR results in
different cell lines, the efficiency of the new vector system pCircRNA-DMo-Rtn4
is
much higher than that of the common vector system (pCircRNA-BE-Rtn4) in these
cell
lines.
CircRNA testing and identification
Northern blotting is the gold standard for the detection of circRNA, and probes
are
usually designed across back splicing sites. However, due to the large amount of
circRNA required by Northern blotting, which takes time and energy, and the
probes
are generally radioactive markers, it is difficult to handle. RT-PCR or RT-qPCR
are
the common detection methods as well, and the primers are designed at both ends
of
the back splicing site. (Fig 9)
