Biochimica et Biophysica Acta 1859 (2016) 12451251

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Biochimica et Biophysica Acta

journal homepage: www.elsevier.com/locate/bbagrm

Review

The complexity of the translation ability of circRNAs

Javier T. Granados-Riveron, Guillermo Aquino-Jarquin
Laboratorio de Investigacin en Genmica, Gentica y Bioinformtica, Torre de Hemato-Oncologa, 4to Piso, Seccin 2, Hospital Infantil de Mxico, Federico Gmez, Mexico

a r t i c l e

i n f o

Article history:
Received 24 March 2016
Received in revised form 21 June 2016
Accepted 15 July 2016
Available online 19 July 2016
Keywords:
Circular RNAs
MicroRNA sponges
IRES sequences
CircRNA-derived protein
Translation

a b s t r a c t
Circular RNAs (circRNAs) are a new class of long non-coding RNAs that play a potential role in gene expression
regulation, acting as efcient microRNAs sponges. The latest surprise concerning circRNAs is that we now
know that they can serve as transcriptional activators in human cells, indicating that circRNAs are involved in important regulatory tasks. Recently, new insight has been gained about the coding potential of circular viroid RNAs,
as well as the presence of Internal Ribosomal Entry Sites (IRES) allowing the formation of peptides or proteins
from circular RNA. Here, we discuss the current state of our knowledge regarding evidence supporting the hypothesis that circRNAs serve as protein-coding sequences in vitro and in vivo. Also, we remark on the difculties
of their identication and highlight some tools currently available for exploring the coding potential of circRNA.
2016 Elsevier B.V. All rights reserved.

1. Introduction
Alternative RNA splicing represents an additional pathway to increase the complexity of the transcriptome by modulating the sequential arrangement of exons, in which particular exons of a gene may be
included or excluded from the nal processed messenger RNA
(mRNA) [1]. The versatility of RNA appears unlimited, and among the
variety of tasks performed by RNA, it can form circular molecules. In
the late-seventies, plant viroids were the rst described naturally
occurring circular RNA molecules [2,3]. Since then, several mammalian
species of circular RNA molecules have been discovered [46]. The
circular RNA molecules derived from lariat structures (ciRNAs), a
by-product of intron removal during splicing, are formed through
end-joining by a 2-5 phosphodiester bond, which can be cleaved by
the debranching enzyme DBR1, making the molecule available to
degradation by exonucleases [7]. CiRNAs that fail to be debranched can
accumulate in the nucleus, and some have displayed a cis-regulatory
role on their parent coding genes [8].
In contrast, another class of circular RNA molecules, the circRNAs,
comprise an abundant class of highly stable RNAs that consist of 24
exons of protein-coding genes, although these can also derive from
non-coding exons, intergenic regions or transcripts antisense to 5 and
3 UnTranslated Regions (UTRs) [911]. Additionally, circRNAs are
widely expressed in a complex tissue-, cell-type- or developmentalstage-specic manner, and their expression levels can be approximately
10-fold compared with their linear isoform, suggesting that their
Corresponding author at: Dr. Mrquez No. 162, Col. Doctores, Delegacin:
Cuauhtmoc, Mxico D.F. C.P 06720, Mexico.
E-mail address: guillaqui@himfg.edu.mx (G. Aquino-Jarquin).

http://dx.doi.org/10.1016/j.bbagrm.2016.07.009
1874-9399/ 2016 Elsevier B.V. All rights reserved.

formation may be nely regulated [12,13]. Canonical eukaryotic premRNA splicing is catalyzed by the spliceosomal machinery to remove
introns and join exons, leading to the formation of a linear RNA
transcript with 5 to 3 polarity [14]. The biogenesis of circRNAs usually
involves the transcription and splicing of the parent gene, and it has
been suggested that their formation may compete with linear splicing
[15]. Sequence annotation suggests that a subset of circRNAs results
from the direct ligation of the 5 and 3 ends of linear RNA molecules
[16]. They can also originate from intermediates in RNA-processing
reactions, or be the result of the back-splicing of protein-coding
genes, whereby a 5 splice site (the splice donor) is joined to a 3 splice
site (the splice acceptor) located upstream in the same pre-mRNA
molecule [16] (Fig. 1).
Protein-coding regions have been dened according to simple rules
concerning the nature of translation, for example, the presence of Open
Reading Frames (ORFs) with minimal length, practical use of codonusage biases, and the use of AUG as the initiation codon [17]. Recently,
new insight has been gained about the coding potential of circular viroid
RNAs can exhibit coding potential [18], as well as the presence of
Internal Ribosomal Entry Sites (IRES) sequences, which might allow
the formation of peptides or proteins from circular RNA [19,20].
However, because no current substantial evidence is available about
endogenous circRNA-derived protein in eukarya, it has been generally
accepted that circRNAs have a tendency to function as a new class of
long non-coding RNAs (lncRNAs). For now, there is limited knowledge
related to the biological roles of circRNAs in translation; nonetheless,
at present, the possibility that some circRNAs can function as proteincoding RNA transcripts cannot be discarded. Here, our aim was to
highlight some relevant aspects in circRNAs biology and, particularly,
we discuss the possible scenarios by which circRNAs can serve as

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Fig. 1. Backsplicing for circRNA formation. Different classes of circular RNAs (circRNAs) can be originated from a particular gene. These circRNAs are generated through a non-canonical
splicing process known as backsplicing in which a downstream splice donor is joined to an upstream splice acceptor. Such circRNAs can be formed by one or more exons and can even
contain unspliced intronic sequences (exon-intron(s)-exon intermediate). ss, splice site [67].

translational templates in vitro and in vivo, based on lines of evidence

supporting this hypothesis. Also, we denote the difculties of their identication and some strategies that are currently available for exploring
the circRNA-coding potential.
2. CircRNAs function as microRNA sponges
Circular RNAs (circRNAs) possessing circularized exons (exonic
circRNAs) are mainly found in the cytoplasm, while those containing
circularized introns (intronic circRNAs), are predominantly detected in
the nucleus [21]. With regard to function, it has been reported that
exons can act as sponges for microRNAs (miRNAs), whereas those
containing an intronic fragment, such as intronic circRNAs, can take
part in absorbing RNA-Binding Proteins (RBPs) [8].
The rst functional circular RNA types described in mammals are
derived from the mouse linear Sex determining region Y (Sry) RNA,
which is required for male sex determination during development
[22]. Recently, in two back-to-back papers published in Nature, Hansen
et al. and Memczak et al. described a new class of post-transcriptional
regulatory RNAs that behave as circular, endogenous RNA sponges [9,
23]. In both reports, the authors demonstrated that human CDR1as or
ciRS-7, a ~1.5-kb single-stranded, antisense circRNA molecule containing multiple miR-7 densely arranged binding sites, acts as a natural
miRNA sponge by capturing complexes formed by miR-7/Ago2. Hansen
et al. also showed that the circular RNA molecule, transcribed from the
mouse Sry gene, contains 16 binding sites for miR-138 and demonstrated in vitro that Sry circRNA (circSry) act as a sponge for this specic
miRNA [23].
Although it has been reported by computational data-sequencing
analysis that thousands of circRNAs in mammals contain spatially distinct miRNA sites for the control of gene expression, only CDR1as and
circSry have been veried as acting as true decoys for miRNAs as their
main function. To our knowledge, these circRNAs are two of the most
extreme examples of miRNA sponges, and it is likely that circRNAs
with only a few binding sites for miRNAs are not sufcient to possess
a biologically relevant sponge effect. In this regard, to demonstrate the
potential of other circRNAs to act as miRNA sponges, Bartel et al.
searched miRNA-loaded Argonaute (AGO) proteins, based on the
density of clusters of AGO2-crosslinking sites, which indicate AGO2
binding. Using data from high-throughput in vivo crosslinking
experiments in HEK-293 cells, the authors concatenated the annotated
exons within each circRNA, and counted the number of canonical
7- and 8-nucleotide target sites for each one of the miRNA families
conserved across vertebrates [24]. By this analysis, they found that a

circRNA derivative from the C2H2 zinc nger 91 locus (circRNAZNF91) contains 24 conserved miR-23 sites, surpassing circSry, which
has 16 sites for miR-138. However, even with 24 binding sites,
circRNA-ZNF-91 is far from resembling CDR1as, which contains 71
miR-7 sites, based on the analysis of miRNA site number performed
[24]. This allowed the authors to conclude that no other human circRNA
shows the strong potential capability of CDR1as as a sponge for any
conserved miRNA seed family [24], which denotes that we are only
beginning to understand the magnitude of the biological signicance
of circRNAs.
3. Role of circular RNAs in transcription regulation: more than
sponges
Important advances have been made in identifying and understanding the interconnections within and among the different processes that
regulate gene expression [25]. For many years, it has been known that
small nuclear RNA (snRNAs) and nucleolar RNAs exert their functions
through direct RNARNA interactions [16]. In this regard, one of the
rst evidences on this interaction was from Kwek et al., who reported
that TFIIH specically associates with U1 snRNA in the coordinated
and efcient control of transcriptional initiation and in early mRNA processing [26]. The discovery of the competing endogenous RNA interplay
of circRNAs has increased the likelihood that these circular RNAs may
have an integral role in regulatory RNA networks [27]. Recently, Li
et al. established that circRNAs containing retained intronic sequences
can function as positive regulators of the RNA Pol II transcription of
their parental genes in a U1 snRNA-dependent manner in HEK-293
human cells [28] (Fig. 2, left panel). The authors identied 111 Pol IIinteracting circRNAs, and only 15 were conrmed to contain introns
among the circularized exons, which were referred as ExonIntron
circular RNAs (EIciRNAs). Li et al. demonstrated that EIciEIF3J and
EIciPAIP2, the two most abundant nuclear species of EIciRNAs in
HEK-293, are enriched at transcription sites and may promote the
transcription of their parent mRNAs. These results provide evidence
for cross-talk between initiation of transcription and mRNA splicing
and a novel circRNAs-based synergistic approach for transcriptional
control in a highly orchestrated manner [28].
Although it is not well known what determines which circRNAs can
shuttle between nucleus and cytoplasm, this dynamic exchange might
be facilitated by nuclear export sequences or nuclear localization
sequences, positioned in exons or retained introns, similar to the
nuclear localization of some miRNAs, e.g., miR-29b [29]. Alternatively,
they might be exported by mRNA export factors [30]. Thus, in addition,

J.T. Granados-Riveron, G. Aquino-Jarquin / Biochimica et Biophysica Acta 1859 (2016) 12451251

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Fig. 2. Diagrammatic depiction of circRNAs in transcriptional regulation and their protein-coding potential. A) Positive feedback loop caused by crosstalk between U1 snRNA-EIciRNAs
interactions. In the nucleus of eukaryotic cells, EIciRNAs (e.g., EIciEIF3J and EIciPAIP2) interact with U1 snRNP, RNA Pol II transcription complex and promoter regions to modulate the
expression of the parental genes in cis, thus augmenting the levels of both circRNA and mRNA. B) Translation directed by circular RNA showing the sequence features within a
transcript's ORF that might be involved in protein synthesis, such as IRES elements, RBPs, AUG initiator codon, and stop codon (in some cases). TSS, Transcription Start Site; TFIID, IID;
TFIIA, IIA; TFIIB, IIB; TFIIE, IIE, TFIIF, IIF; U1 RNP, U1 small nuclear ribonucleoprotein; circRNA, circular RNA; mRNA, messenger RNA; ORF, open reading frame; RBPs, RNA-binding
proteins; IRES, internal ribosomal entry site.

it could be possible to classify circRNAs into subclasses or subtypes,

based on nucleocytoplasmic shuttling and also depending on whether
there are introns retained among circularized exons, or whether these
function as transcription activators.
Interestingly, from the 15 EIciRNAs identied by the authors, we
once again face only two prominent examples of the role of this
circRNAs subclass in transcriptional activation, and some challenging
questions remain: How is the retention of introns in circRNAs regulated? Are EIciRNAs expressed in a tissue-specic or a cell-specic
manner? Could circRNAs also function as transcriptional repressors? If
so, what could the mechanism be?
Whereas the global transcriptional regulation processes and the
mechanisms of transcriptional activation are understood in considerable detail, much less is known about the novel roles of circRNAs in
the modulation of transcription. In this regard, it will be important to
identify, step by step, interactions among other components of the
transcriptional core and the splicing machinery for regulation of gene
expression. On the other hand, one exciting prospect is that EIciRNAs
may elicit transcriptional repression, for example, through the recruitment of components of the epigenomic machinery at the promoter
level, or by complementarity to promoter-generated antisense small
ncRNA to induce transcriptional gene silencing in a sequence-specic
manner, as it happens with miRNAs (e.g., Progesterone Receptor (PR)
gene silencing by miR-423-5p), inducing Transcriptional Gene Silencing
(TGS) [31]. While U1 snRNA-EIciRNAs interaction modulates transcription, it is tempting to speculate that cross-talk may also occur between
circRNAs and other non-coding RNAs (e.g., ribosomal RNA or transfer
RNAs), involving other important biological functions in cells.
4. Translational role of circular RNAs: controversial issue
The main function described for circRNAs to date is to act like
sponges to absorb miRNAs, to regulate alternative splicing, and to
modulate the expression of parental genes [23,28,32].

Currently, it is accepted that circRNAs have a tendency to function as

a new class of lncRNAs because at present, no sufcient evidence has
been produced on endogenous circRNA-derived protein in eukarya.
One of the rst observations of a circRNA behaving as a translational
templates is the case of the single-stranded circular RNA genome of
the hepatitis virus which is a satellite virus of the hepatitis B virus
where encapsulation of HDV with HBV virions results in the production of a single viral protein of 122 amino acids, in a non-canonical
manner [33] (Table 1). This study supports the notion that naturally
occurring circRNA can be translated into a protein in a mammalian cell.
Another recent study indicated that a replicating covalently closed
circRNA of 220 nucleotides (nt) is fully translated into a 16-kDa highly
basic protein in rice plants infected with this circular virusoid [18]
(Table 1). In this conguration, initiation and termination codons are
combined in a UGAUGA sequence, and read-through to generate larger
proteins by eukaryotic ribosomes via the two or three overlapping
reading-frames [18]. However, in eukaryotic sequences, the translated
regions may be very short and the absence of stop codons possesses
no meaning or signicance [36].
Translation initiation in eukaryotes employs the 7-methylguanosine
cap moiety bound to the 5-end of mRNA to recruit transcripts to an
assembling ribosome and initiate protein synthesis [37]. However,
cap-independent translation is an alternative resource of translation
initiation in eukaryotes that depends on the presence of particular
elements that recruit the small ribosomal subunit to an internal initiator
codon in the mRNA, such as an IRES sequence [20]. Interestingly, the
presence of IRES might allow the formation of peptides or proteins
from RNA in circular conformation. Some reports have suggested that
engineered circRNAs in articial constructs, with an IRES sequence
located upstream of the classical AUG start codon of an ORF, could be
translated in vitro [19,34,38] (Fig. 2, right panel). In this regard,
Perriman and Ares reported that an engineered circular messenger
RNA (mRNA) containing a innite Green Fluorescent Protein (GFP)
ORF produces very long protein chains in Escherichia coli, suggesting

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Table 1
CircRNAs with experimental data supporting the potential coding in vitro and in vivo.
Model
Virus
Hepatitis
delta ()
virus
(HDV)
Rice yellow
mottle
virus
Bacteria
Escherichia
coli

CircRNA feature

Sequence feature

Peptide/protein

Source

Function

Ref.

Circular
single-stranded
RNA

OFR with a TGA stop codon

Protein of 122
amino acids

Exogenous/infectious

Unknown

[33]

Covalently
closed circular
RNA (220 nt)

Innite ORF, IRES-dependent sequence

16-kDa highly basic

protein

Ectopic, plasmid-encoded

Unknown

[18]

795-nt circular
mRNA

Innite GFP ORF, IRES-independent

sequence

GFP

Ectopic plasmid-encoded

Only the monomeric forms of GFP

produced from circular mRNA
were uorescent.

[34]

IRES-dependent sequence

GFP

Ectopic plasmid-encoded

Reporter for protein localization

[19]

Poly-A tail-independent translation

NH2-terminal
portion of NCX1
protein (70-kDa)
EGF, IGF-1, IGF-2

Endogenous

Na/Ca exchange activity

[35]

In-vitro
transcription/translation
process, transfection of
synthetic circRNA

Human growth factors

[20]

Mammals
HEK-293 cells Single exon
minigene
HEK-293 cells Exonic

Rabbit
Exonic
reticulocyte
lysate
HeLa cells
Exonic

Cap-independent translation,
IRES-independent sequence, poly-A
tail-independent translation

CircRNA, Circular RNA; nt, nucleotide; ORF, open reading frame; GFP, green uorescent protein; IRES, internal ribosomal entry sites; EGF, epidermal growth factor; NCX1, sodium-calcium
exchanger; IGF-1, insulin-like growth factor 1; insulin-like growth factor 2.

that bacterial ribosomes can repeatedly direct the GFP expression from
a circular mRNA [34] (Table 1). In this case, a circular mRNA of 795nucleotides in length, with an innite 30-kDa-encoding ORF, produced
proteins N 300 kDa in size, indicating that the bacterial ribosomes
scanned the circle N10 times [34]. Similarly, Wang et al. evidenced
that a single exon minigene containing split GFP can be efciently
backspliced to generate circRNA and that it can direct the translation
of an entire functional GFP protein inside HEK-293 human cells [19]
(Table 1). Although synthetic circRNAs containing IRES elements can
be translated, currently no concrete evidence is available to suggest
that endogenous circRNAs in eukaryotic cells, containing or not IRES
elements, might encode proteins with functions distinct from those of
their canonical linear counterparts [39].
Thus, the fact that synthetic circRNAs can be efciently translated
in vitro supports the notion that some circRNAs can be translatable molecules in vivo (Fig. 2). Previously, Li et al. described a truncated protein
originating from a circRNA of the Na+/Ca2+ exchanger gene 1 (NCX1)
into HEK-293 transfected cells. When a circular NCX1 exon 2 transcript
was overexpressed ectopically, the size of the truncated protein was
consistent with the predicted molecular weight of approximately
70 kDa, and surprisingly, this protein exhibited Na+/Ca2 + exchange
activity, denoting its function [35]. However, Li et al. could not identify
the same-sized proteins in native tissue, observing a prominent and
slightly smaller band, possibly as a result of the hydrolysis residual of
the circRNA-derived protein, coupled with discrepancies in terms of
antibody detection (length and antigenicity) [35]. Perhaps this is the
only putative example of an endogenous peptide derived from circRNA
identied so far in mammals; however, these inconsistencies do not
support entirely the existence of endogenous circRNA-directed
translation in vivo.
Although it can be assumed that linear lncRNAs are not generally
translated into proteins, growing evidence shows that a subset of
these can be a source of functional small or micropeptides due to the
presence of short ORFs [40]. For example, Anderson et al. discovered a
highly conserved 46 amino-acid micropeptide, which the authors
denominated myoregulin, encoded by a skeletal muscle-specic RNA
annotated as a putative long non-coding RNA in human and mice.
Interestingly, this micropeptide was identied as an important
regulator of the Sarco-Endoplasmic Reticulum Ca2 +-ATPase (SERCA)
calcium pump in adult skeletal muscle [41].

Furthermore, strong evidence concerning circRNAs serving as templates for translation in vivo derived from Abe et al., who demonstrated
that an exonic circular RNA with an innite ORF is efciently translated
into living human cells to produce an abundant protein product by a
rolling-circle amplication mechanism [20] (Table 1). It is noteworthy
that translation of this circular RNA take place in the absence of any
particular element for internal ribosome entry, a poly-A tail, or a cap
structure in a eukaryotic system [20] (Fig. 2, right panel). This suggests
that there must be some elements within circularized eukaryotic
transcripts that can promote cap-independent translation initiation,
other than IRESs [42]. Furthermore, protein synthesis could occur
through a mechanism different from the canonical translation process,
such as initiation at non-AUG codons (e.g., initiation at CUG and GUG codons), leaky scanning, translational reinitiation, and/or translational
frameshifts [17,43], which also should be considered.

4.1. Difculties encountered in the identication of potential circRNA

translation
Some reports show that the translational ability of circRNA is controversial. Theoretically, some circRNAs may encode and be translated into
protein by eukaryotic ribosomes in living cells [20,38]. However,
different approaches are needed for studying the structural and
functional features of circular RNA and for clearly demonstrating its
coding potential in vivo [44].
It stands to reason that the absence of a 5 cap and 3 poly-(A) tail in
a circular conguration limits accessibility to the ribosome [13]. However, it has been suggested that a circular structure can be favorable for
ribosome recycling once translation has been initiated, which would
facilitate the production of more proteins molecules than the linear
version [34].
Even though many circRNAs overlap with coding exons, several
arguments favor the notion that a circular RNA may be inefciently
translatable. For example, in a circular conformation, the beginning
and the end of the same ORF are topologically close to each another,
which may create a unique scenario in which initiation and termination
of translational process are occurring simultaneously in the same space.
In addition, efcient translation elongation, and termination steps
require auxiliary factors that bind to the 5 end and poly(A) tail of

J.T. Granados-Riveron, G. Aquino-Jarquin / Biochimica et Biophysica Acta 1859 (2016) 12451251

mRNAs, and correct folding of nascent chains impacts on its termination

and release [19,34].
On the other hand, determining whether lncRNAs possess the ability
for coding proteins is challenging because it is unlikely that they contain
a typical ORF [45]. Thus, the absence of evidence of the coding potential
of circRNAs could, at least partially, due to the lack of evolutionary
conservation of identied ORFs, as well as lack of homology of their
products with known protein domains from other species catalogued
even in large databases [4648]. For example, the Drosophila tarsalless/polished rice gene was described as a lncRNA [49]; however, it encodes a series of short peptides (11 amino acids residues) that modulate
the activity of the shavenbaby transcription factor [50,51]. However,
conservation-based methods may not be appropriate for identifying
newly evolved proteins, despite their sensitivity, because the absence
of conserved ORF sequences [47,48].
A useful method for distinguishing between coding and non-coding
RNAs known as ribosome proling, has been developed recently. It can
provide, through deep sequencing, a single-nucleotide resolution
measurement of mRNA fragments protected by the 80S ribosome
(ribosome-protected fragments, RFPs) after nuclease digestion [52]. By
this approach Ingolia et al. dened a class of short, polycistronic,
ribosome-associated coding RNAs (sprcRNAs) that encode small
proteins in mouse embryonic stem cells [17]. However, ribosomeproling biochemical techniques solely indicate the ribosome positions,
but do not demonstrate the process of translational elongation or distinguish stalled ribosomes from those engaged in active elongation [17,53].
To systematically analyze the pattern of ribosome occupancy across
different classes of RNAs, Guttman et al. developed a metric that they
named the ribosome release score (RRS), which identies functional
protein-coding transcripts with greater sensitivity by detecting the
termination of translation at the end of an ORF [54]. An interesting
aspect to consider is that any capped ncRNA present in the cytosol
could engage the translation machinery. Such ribosome engagement
may produce 80S footprints after nuclease digestion but without the
translation of a predominant functional reading frame [54]. In this
respect, the RRS metric is well suited to distinguish real translation
from nonribosomal contamination because it robustly discriminates
the latter, as such protection should show no bias for the presence of a
stop codon [54].
On the other hand, the discrete movement along the message in
three-nucleotide steps (phased ribosome binding), is an important
property that actively translating ribosomes possess [52], such that
phased ribosome binding is a direct consequence of active translation.
Based on the above, Bazzini et al. reasoned that screening for phased
binding would identify putative small ORFs (smORFs, 100 amino
acids) undergoing translation in vivo [40]. By this approach, the authors
identied protein products from six new small ORFs in zebrash in
annotated lncRNAs transcripts [40]. As a major methodological
advancement, Bazzini et al. development the ORFscore method which
takes advantage of the periodicity of high-quality RPFs observed along
the dened coding sequence (CDS), reecting the stepwise translocation of active ribosomes and hence denes small translated ORFs [40].
For example, when the distribution of RPFs is inconsistent with the
frame of the CDS, the resulting ORFscore is assigned a negative value.
Thus, this pattern could be used to dene actively translated regions
and distinguish them from background signal [40].
For example, You et al. found that brain expressed circRNAs are
derived from genes coding for synaptic proteins and are enriched in
synaptic tissues [55]. Because neuronal circRNAs are mostly composed
of protein coding exons, the authors investigated their potential to be
translated into peptides using a mass spectrometry data set obtained
from hippocampal neurons. Thus, by polysome proling (see below)
on mouse brain and ribosome footprinting on rat brain, the authors
were unable to detect a single ribosome-protected fragment mapping
to a circRNA head-to-tail junction. It is important to mention that peptides crossing the circular junction position strongly indicate that

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these could only arise from circRNA translation; thus, the negative
ndings allowed to conclude that these brain expressed circRNAs, are
unlikely to be translated into peptides [55].
Practically all of the transcripts that have been suggested to encode
small peptides by ribosome proling [17] lack the evolutionary
conservation of their proposed coding regions [56,57], in contrast with
known protein-coding genes [58], including the few, well-characterized,
functional small peptides [53,59,60].
Accordingly, identication of any new protein-coding gene requires
additional biological information; therefore, further experimentation is
needed for demonstrating the existence and the role of the circRNAsderived protein in vivo [51,53].
4.2. Some bioinformatic tools for exploring circRNA coding potential
At present, to our knowledge, no computational resource is available
to systematically search for circRNAs with coding potential. The search
for ORFs into the circRNA sequence could provide insights into the function of circRNA-derived proteins. For this, the ORF Finder comprises a
graphical analysis tool that localizes all ORFs of a selectable minimal
size in a sequence provided by the user or in a sequence already located
in the PubMed database (Table 2).
Another approach could include homology search of a putative
product sequence within protein-domain databases (e.g., Pfam 29.0),
in an attempt to nd matches with reference proteomes sequences.
Thus, the identication of domains that occur within known proteins
can provide insights into their function [62] (Table 2). Additionally, bioinformatics tools, such as the PhyloCSF method [48], represent a useful
strategy for examining the coding potential of circRNAs through
evolutionary signatures characteristic of alignments of conserved
coding regions. This algorithm searches for matches with high
frequency of synonymous codon substitutions and conservative
amino-acid substitutions [47,48] (Table 2). Due to the ever-increasing
number of available genome sequences, these methods have been
successfully employed for accurate determination of conserved coding
potential in very small regions (of only ve amino acids) [56].
On the other hand, cellular IRES exist to play a crucial role at some
critical moments of cell life when cap-dependent translation initiation
is compromised [42]. Structures of many IRES are well-known and are
hypothesized to be important due to their function in translation
initiation [37]. Very recently, Dudekula et al. developed a new
computational resource named the CircInteractome that, in addition
to facilitate the search for possible interactions of human circRNAs
with RNA-binding proteins and miRNAs, allows the investigation of
potential circRNA translation through IRES sequences (Table 2). To
perform this, the tool compares all experimentally validated IRES
sequences [37] against mature circRNA sequences in order to identify instances of these cis-acting elements in circRNAs [63]. By means
of this resource, it was possible to nd that hsa_circ_0041407
contains an IRES and partial coding sequences of the MAX network
transcriptional repressor, and it was suggested that this circRNA
could give rise to a small chimeric protein of ~ 31 kDa [63]. With
this Web tool, a researcher is also assisted to design junctionspanning primers for the specic detection of circRNAs of
interest and to design small interfering RNAs for circRNA silencing.
Thus, with the data from current databases taken together, this
represents a great advantage for the analysis of the translational
role of circRNAs in vitro and in vivo (Table 2). However,
nucleotide composition and especially (G + C) content (introns
being more A/T-rich than exons, particularly in plants), codon
composition, hexamer frequency, base occurrence periodicity,
among others, are other measures that have been considered for
attempting the ne characterization of a sequence possessing coding
potential for translation into detectable peptides. Therefore, this
feature must be exploited by diverse genomic algorithms through
different methods [64].

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Table 2
Algorithms and tools for identifying circRNA-translation potential.
Resource
names

Web links

ORF Finder
IRESite

http://www.ncbi.nlm.nih.gov/gorf/gorf.html
http://iresite.org/

Remarks

Refs.

This tool identies all ORFs that a transcript possesses using the standard or alternative genetic codes
This database could be employed to evaluate which of the many IRESs published to datethat are
experimentally validatedcan be aligned with one or more mature circRNA sequences, supporting
their existence
CPAT
http://lilab.research.bcm.edu/cpat/index.php Calculates the likelihood with which a presumed ncRNA could encode for a peptide
Pfam 29.0
http://pfam.xfam.org/
This tool is useful for analyses of the sequence features of ORFs and also screens for the presence of
known protein motifs within an ORF
PhyloCSF
http://compbio.mit.edu/PhyloCSF
PhyloCSF is helpful for evaluating the coding potential of transcript models or individual exons in an
assembled genome that can be aligned with one or more informant genomes at appropriate
phylogenetic distances
CircInteractome http://circinteractome.nia.nih.gov/index.html This computational tool enables the prediction and mapping of binding sites for RBPs and
miRNAs on reported circRNAs

[37]

[61]
[62]
[48]

[63]

CAPT, Coding-Potential Assessment Tool; ORF, open reading frame; IRES, internal ribosomal entry site; ncRNA, non-coding RNA; RBPs, RNA-binding proteins; miRNA, microRNA; circRNA,
circular RNA.

4.3. Some experimental approaches for evaluating the circRNA-templated

translation
Once a specic circRNA has been identied employing different computational lters, it is desirable to test experimentally whether it is indeed
translatable. For this purpose, the coding potential of circRNAs could be
interrogated by ribosome proling and polysomal fractionation, which
are two complementary methods that enable translational global analysis. Whereas ribosome proling allows to obtain a global map of the positions within eukaryotic RNAs occupied by 80S ribosomes that is,
obtaining footprints of actively translating ribosomes [17,52] polysomal
fractionation indicates the fraction of the copies of a transcript that are actively translated. With polysomal fractionation, transcripts are separated
according to the number of ribosomes associated to them using a sucrose
gradient, and then high-throughput methods such as microarrays or
Next-generation RNA sequencing (RNA-seq) are employed to identify
and count the transcripts in each fraction [52]. However, it is important
to have on mind that association with polysomes does not necessarily
imply that the protein products are functional.
On the other hand, although a sequence which can be translated
in vitro does not necessarily undergo the same process in vivo. Thereby,
for condently establishing circRNA-templated translation, one approach
would be to assess whether the circRNA yields peptides by in vitro translation assays. Furthermore, predicted peptides can also be synthesized
chemically and used to produce antibodies, which can then be employed
to detect the putative peptide by different methods such as immunohistochemistry or western blot. For peptides suspected to be encoded by
circRNAs another attractive alternative would be to add a peptide Tag
(FLAG or uorescent protein) at the C terminus of the predicted ORF
that can then be used for detection using Western blotting or
epiuorescence microscopy, respectively on cells transfected with a plasmid encoding the tagged circRNA. The trouble here is that fusion of a peptide Tag can generate unstable protein [65]. In addition, mass
spectrometry (MS) is currently the leading proteomic platform for peptide detection in different tissues. However, experimental biases against
detection of peptides smaller than 10 kDa, paucity of peptides sequence
data coming from unannotated transcripts in databases used for spectra
search, as well as the inherent limited sensitivity of MS which leads to
bias against detection of lowly expressed genes [65]. These aspects
could be some of the constraints to condently establishing circRNAtemplated translation. The reader is referred to excellent reviews describing methods for distinguishing between protein-coding and long noncoding RNAs in detail [65,66], which may also be applied for the study of
circular RNAs.
5. Concluding remarks and future challenges
One of the challenges for identifying translatable sequence elements
located within circRNAs is the absence of relatively strong sequence

patterns that identify true translation-initiation sites or motifs related

with translation, coupled with a limited number of experimentally
validated start translation sites. Detecting a circRNA as a protein-coding
element requires full availability of information on gene annotation. For
example, the analysis of consensus Ribosome Binding Site (RBS) sequences and other consensus sequences necessary for the translation of
validated circRNA-encoded peptides, as well as additional analysis of several datasets, may facilitate the identication by data mining of new coding circRNAs, and, in the best case scenario, a better understanding of the
translation mechanisms of these molecules. In this regard, there is still a
real need for accurate, fast, and reliable bioinformatic tools to analyze
circRNAs sequences; suitable models are required as well for complete
structural and functional understanding of the coding potential of
circRNAs. Furthermore, advances in synthetic biology are improving
mass spectrometry approaches, and progress in sequencing methods
might conrm the existence of proteins deriving from circRNA sequences. For now, it appears that we are only beginning to catch a
glimpse of the whole complexity of the regulatory mechanisms based
on circRNAs. Thus, we must encourage research efforts to investigate
the potential roles of circRNAs in translation that might contribute to
the elucidation of gene function.
Transparency document
The Transparency document associated with this article can be
found, in online version.
Acknowledgments
The authors would like to thank Dr Julia D. Toscano-Garibay for
helpful discussion and critical reading of this manuscript. We also
thank Maggie Brunner, M.A., for improving the style and language
editing of the manuscript. GA-J is supported by grants CB-168661
from the Mexican Council of Sciences and Technology (CONACyT) and
Mexican Federal Funds (HIM/2015/004-SSA 1175).
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