Briefings in Bioinformatics, 2022, 23(2), 1–14

https://doi.org/10.1093/bib/bbab601
Problem Solving Protocol

CRAFT: a bioinformatics software for custom prediction

of circular RNA functions
Anna Dal Molin, Enrico Gaffo , Valeria Difilippo, Alessia Buratin, Caterina Tretti Parenzan, Silvia Bresolin and
Stefania Bortoluzzi
Corresponding authors: Stefania Bortoluzzi, Department of Molecular Medicine, University of Padova, Padova, Italy; Interdepartmental Research Center for

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Innovative Biotechnologies (CRIBI), University of Padova, Padova, Italy. Tel.: +39-049-827-6502; Fax: +39-049-827-6208; E-mail: [email protected];
Anna Dal Molin, Department of Molecular Medicine, University of Padova, Padova, Italy. Tel.: +39-049-827-6502; Fax: +39-049-827-6208;
E-mail: [email protected]

Abstract
Circular RNAs (circRNAs), transcripts generated by backsplicing, are particularly stable and pleiotropic molecules, whose dysregulation
drives human diseases and cancer by modulating gene expression and signaling pathways. CircRNAs can regulate cellular processes
by different mechanisms, including interaction with microRNAs (miRNAs) and RNA-binding proteins (RBP), and encoding specific
peptides. The prediction of circRNA functions is instrumental to interpret their impact in diseases, and to prioritize circRNAs
for functional investigation. Currently, circRNA functional predictions are provided by web databases that do not allow custom
analyses, while self-standing circRNA prediction tools are mostly limited to predict only one type of function, mainly focusing on
the miRNA sponge activity of circRNAs. To solve these issues, we developed CRAFT (CircRNA Function prediction Tool), a freely
available computational pipeline that predicts circRNA sequence and molecular interactions with miRNAs and RBP, along with their
coding potential. Analysis of a set of circRNAs with known functions has been used to appraise CRAFT predictions and to optimize its
setting. CRAFT provides a comprehensive graphical visualization of the results, links to several knowledge databases, and extensive
functional enrichment analysis. Moreover, it originally combines the predictions for different circRNAs. CRAFT is a useful tool to help
the user explore the potential regulatory networks involving the circRNAs of interest and generate hypotheses about the cooperation
of circRNAs into the modulation of biological processes.

Keywords: CircRNA, bioinformatics, computational pipeline, functional prediction, regulatory network

Introduction
in disease, cancer and when genomic translocations
Circular RNAs (circRNAs) are RNA molecules in which, in occur [4–6]. They represent stable diagnostic and
a process called backsplicing, a downstream 5 splice site prognostic markers [7, 8] and, most interesting, the
is covalently linked to an upstream 3 splice site giving identification of new disease mechanisms involving
rise to a circle. The backsplice most frequently, although circRNAs has the potential to indicate novel therapeutic
not always, joins canonical exons, with respect to linear targets.
transcript annotation [1]. CircRNA functions mostly involve sequence-specific
These peculiar RNAs often exhibit cell type- and binding with other nucleic acids or proteins, or specific
tissue-specific expression [2, 3] and can regulate several coding potential. One prominent mechanism whereby
biological processes with different mechanisms. More- circRNAs function is by sponging microRNAs (miRNAs),
over, circRNAs are emerging as key oncogenic or tumor through one or multiple miRNA-response elements
suppressor molecules whose expression is dysregulated (MRE). CircRNAs thus act as competitive endogenous
Anna Dal Molin is post-doc at the Computational Genomics Laboratory at the Department of Molecular Medicine, University of Padova. Her research interests
include bioinformatics and transcriptomics, circular RNAs in leukemias and development of computational methods for circular RNA function prediction.
Enrico Gaffo is post-doc at the Computational Genomics Laboratory at the Department of Molecular Medicine, University of Padova. His research interests include
circular RNA, microRNA, advanced methods for RNA-seq data analysis and bioinformatics applied to cancer research.
Valeria Difilippo graduated in Industrial Biotechnologies at the University of Padova working on circRNA prediction for her master thesis.
Alessia Buratin is PhD student in Biosciences (curriculum Genetics, Genomics and Bioinformatics) of the University of Padova. Her main interests are biostatistics
and bioinformatics, transcriptomics of hematologic malignancies and circular RNA biogenesis.
Caterina Tretti Parenzan is a PhD student in Pediatric Oncoematology of the University of Padua. Her research is mostly focused on the study of the oncogenic
molecular mechanisms involving circRNAs in pediatric acute lymphoblastic leukemia.
Silvia Bresolin is assistant professor of Molecular Biology at the Department of Women and Child Health Department of the University of Padova. Her field of
interest is focused on pediatric leukemia transcriptomics and genomics alterations, as well as functional genomics and development of novel disease models.
Stefania Bortoluzzi is associate professor of Applied Biology at the Department of Molecular Medicine of the University of Padova, where she leads the
Computational Genomics Laboratory. Her research interests include cancer genomics and transcriptomics, bioinformatics, systems biology, noncoding RNAs,
circular RNAs, exosomal RNAs and hematologic malignancies.
Received: November 10, 2021. Revised: December 10, 2021. Accepted: December 26, 2021
© The Author(s) 2022. Published by Oxford University Press.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial
re-use, please contact [email protected]
2 | Dal Molin et al.

RNAs (ceRNA) and regulate the expression of miRNA- Instead, CircRNAprofiler [36] and CircCode [37] are both
target genes (TG) [9]. Several key oncogenic circRNA- dedicated to the coding potential of circRNAs, and the
miRNA-gene axes have been described, impacting all second needs ribosome profiling (Ribo-seq) data as input.
cancer hallmarks [10]. With this mechanism, circRNAs In summary, all of the available methods allow the
also regulate key epigenetic ‘writers’ [11] controlling DNA prediction of a single function of circRNAs and there
methylation and histone modifications. is a high need for software tools to comprehensively
CircRNAs can also modulate the activity of RNA- predict circRNA functions. The circRNA sequence is
binding proteins (RBP), a large class of molecules a prerequisite for functional predictions, but most
involved in most biological processes, since they reg- methods to detect circRNA from RNA-seq data return
ulate gene expression at the transcriptional and post- only the backsplice junction coordinates. FcircSEC R
transcriptional levels [12–14]. The protein-circRNA inter- package [38] retrieves circRNA sequence using the longer
action is sequence-specific and RBP-response elements transcript of the gene as reference.
(RRE) can be predicted using pattern recognition [15]. To fill this gap, we developed CRAFT (CircRNA Function

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Finally, beyond exerting functions typical of long non- prediction Tool), a new computational pipeline to pre-
coding RNAs, circRNAs can be translated into circRNA- dict circRNA putative molecular interactions with miR-
encoded peptides (CEP) [16–18]. CEP generated by trans- NAs and RBP, and circRNA coding potential. Plus, CRAFT
lation of open reading frames (ORF) encompassing the allows investigating complex regulatory networks involv-
backsplice junction, which are not present in linear tran- ing circRNAs acting in a concerted way, such as by decoy-
scripts, are circRNA-specific. CircRNAs can be also trans- ing the same miRNAs or RBP, or miRNAs sharing TG. This
lated using long ORFs continuing along the circle [19, 20] new tool facilitates the interpretation of circRNA biologi-
using it as a ‘Mobius strip’ [21] (‘rolling circle translation’). cal and pathogenetic roles, and can guide the experimen-
The same circRNA can carry multiple functional tal investigation of circRNA-involving mechanisms.
sequences, thus playing diverse functions also with
different mechanisms. For instance, for circZNF609 both
translation to a peptide and ceRNA activity have been Methods
described [17, 22]. Also for circFOXO3 both interaction CircRNA sequence reconstruction
with proteins and miRNA sponging activity is reported in CircRNA sequences to be used for the predictions are
literature [23, 24]. obtained using the CircExtractor module described
The function of most of the thousands of circRNAs dis- below. Bedtools v2.28.0 [39] was used for file manip-
covered so far is still undetermined. Therefore, the pre- ulation and sequence extraction, as described in the
diction of circRNA function is instrumental to generate Results section. The circRNA genomic coordinate file
specific hypotheses about their molecular interactions and the build genomic reference file are in BED format.
and biological roles to be experimentally investigated, The predicted circRNA sequences in FASTA format
and to interpret the possible impact of circRNA dysreg- are saved in the result directory. To run predictions,
ulation in disease. circRNA sequences are modified in order to also detect
Several databases have been released with the goal functional elements overlapping the backsplice region.
of collecting information regarding known circRNAs. A pseudocircular sequence is created repeating the first
CircBase reports circRNA expression in different bio- 20 nt of the sequence at the end of each circRNA for
logical samples [25], CircR2Disease [26] and Circ2Traits MRE and RRE prediction, whereas the whole sequence
[27] contain, respectively, experimentally validated and is repeated twice to detect ORF crossing the backsplice
potential circRNA association with diseases. CircNet [28] junction. All the prediction coordinates are relative to
is specific for circRNA-miRNA-mRNA interaction net- the circRNA sequence used.
works, whereas circ2GO [29] merges circRNA expression,
gene ontology and miRNA prediction. CircInteractome MiRNA-binding site prediction
[30] and CircAtlas [31] collect data about miRNA and MiRNA sequences downloaded from miRBase release
RBP binding to circRNAs. RiboCIRC [32] and TransCirc 22.1 are included in the tool. A filter is then applied
[33] provide information about circRNA coding ability. to retrieve miRNA of the species selected by the user.
Importantly, to our knowledge, no databases provide pre- MiRanda 3.3a [40] and PITA [41] tools are used to predict
dictions of the three above-described circRNA functions miRNA binding sites in circRNA sequences. MiRanda
together. In addition, all of them provide precomputed parameters set as default by CRAFT are -sc 80 -en -15;
data and do not allow custom predictions. Often, data PITA is run with default parameters.
browsing is difficult and the user has no control about AGO2 binding data were downloaded from the doRiNA
the correspondence between circRNA sequence and database [42], merging all 17 files with AGO2 binding
functional predictions. information, and converted in hg38 coordinates through
A few tools are available hitherto for circRNA func- UCSC liftOver.
tional prediction. Among them, Cerina R shiny web Only miRNA binding sites predicted both by miRanda
application [34] and ACT web server [35] predict circRNA and PITA, and overlapping with AGO2 binding sites, are
functions exclusively based on the ceRNA model. kept. The AGO2 binding site file is optional. A different
 CRAFT tool for circular RNA function prediction | 3

file with AGO2 binding data can be provided by the user Assessment and sample analyses
in BED format. For all the presented analyses, known circRNA back-
Validated miRNA-TG were retrieved from three dif- splice coordinates were retrieved from CircBase. CircRNA
ferent databases (miRecords, mirTarBase and Tarbase) binding with miRNAs or RBP, and the coding potential
using the multiMiR 1.8.0 R package [43], selecting the information, were collected from literature (Table 1). Cir-
‘strong’ validation category of each database. cRNA annotation and genomic sequence are based on the
Ensembl GRCh38 v.93 human genome. CRAFT was run
RBP binding site prediction with default parameters.
RBP binding sites were predicted through beRBP [44] tool, CRAFT sensitivity was calculated as true positive
modified to give in output the ‘voteFrac’ score for all predictions (TP, known elements correctly predicted)
predictions, and run in ‘general mode’ searching for all over the total number of predictions (TP + FN, where
the 143 RBP and 175 Position Weight Matrices of the tool FN stands for false negatives). The prediction ratio was
calculated as the mean number of predictions at a

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database.
Result tables are given in plain text tabular format, certain setting over the mean number of predictions
keeping the output similar to that of the tool’s original obtained at the less stringent condition tested.
output, to allow deep inspection to expert users.
Results
Functional enrichments Overview of the CRAFT workflow
The multiMiR package is used to perform enrichments of The CRAFT analysis workflow comprises four main steps
miRNA association with drugs and diseases. Functional (Figure 1). First, the circRNA sequence is retrieved by a
enrichments are performed on validated TG of miRNAs custom tool, as described in the Results section ‘The
with predicted binding sites in circRNA sequences. CircExtractor module’. Second, using the combination
DOSE 3.18.2 package [45] is used for over-representation of different methods for miRNA and RBP binding sites
analysis, clusterProfiler 4.0.5 package [46] for Gene detection, and ORF finding, complete circRNA functional
Ontology analysis and KEGG over-representation test, predictions are obtained. Afterwards, the postprocessing
ReactomePA 1.30.0 package [47] for Reactome pathway phase integrates the predictions obtained for each cir-
analysis, meshes 1.12.0 package [48] for MeSH enrichment cRNA with knowledge databases for functional enrich-
analysis. Functional enrichments on validated target ment analysis of miRNAs, RBP and known miRNA-TG
genes of miRNAs with predicted binding sites in circRNA associated with the circRNAs in the previous step. Tab-
sequences can be performed only for Homo sapiens (hsa), ular and graphical visualization of results is provided
Mus musculus (mmu) and Rattus norvegicus (rno) species. for each circRNA. In the last CRAFT step, the integration
Functional enrichments are performed also on RBP of predictions for all the circRNAs given in input let to
with predicted binding sites in circRNA sequences using identify miRNAs, TG and RBP in common between differ-
the UniprotR 2.1.0 R package [49]; Gene Ontology, KEGG ent circRNAs, and in turn, multiple circRNAs potentially
enrichment, Reactome pathway and RBP-disease asso- impacting the same biological processes.
ciation analyses are obtained. Protein expression, and CRAFT is a self-standing tool written in Bash and
functional and miscellaneous information associated R languages. The Docker [77] containerization makes
with RBP were also retrieved. it usable and portable on all systems. CRAFT accepts
circRNAs of any species: the genome sequence and the
Open reading frames prediction gene annotation files must be given in input in FASTA
and GTF formats, respectively. The user has full control
Putative ORF are predicted through ORFfinder tool,
of the parameters used for each prediction tool and can
searching for ORF with the canonical ‘ATG’ start codon,
choose to perform either all or only part of the available
with minimal ORF length of 30 nt, and detected on plus
predictions. Moreover, after the first run, the user can
strand. All four kinds of ORFfinder output formats are
adjust the result visualization by modulating the output
produced.
filtering parameters, without the need of computing the
predictions again, thus saving computational time.
Graphical visualization
The R packages ggplot2 3.3.5 [50], pheatmap 1.0.12, tidy- CRAFT assessment and parameters optimization
verse 1.3.1 [51], car 3.0–11 [52] and enrichplot 1.12.2 [53] are We assessed how the parameter tuning and different
used for result graphical visualization, whereas tables tool combinations affected the whole number of sites
are printed through the DT 0.19 R package. predicted by CRAFT and its capability to detect func-
For the default graphical visualization, CRAFT filters tional elements, in order to optimize the default setting
the results with more stringent parameters: miRanda of the tool. With a literature survey, we collected 20 circR-
score > 80 and free energy < −15 kcal/mol, PITA Gduplex NAs with experimentally determined functions (Table 1),
< −15 kcal/mol and Gopen > −15 kcal/mol are set, beRBP including 26 functional elements, that were used as a
voteFrac ≥ 0.15. test set. For each prediction type, we tested whether
 4

Table 1. CircRNAs used for the CRAFT assessment analysis, with previously validated functional elements and biological or pathogenetic roles
|

CircRNA Backsplice Validated Validated CircRNA binding validation Tissue Function Reference
functional interaction/peptide
element (aa)
circBCRC3 2:231075511– MRE miR-182-5p Biotin-labeled RNA pulldown assay, Bladder cancer Tumor suppressor [54]
231087181:+ FISH, transfection
circHIPK3∗ 11:33286413-33287511:+ MRE miR-124-3p Luciferase reporter assay, Liver cancer Oncogenic [55]
Dal Molin et al.

biotin-labeled miRNA pulldown assay

circHIPK3∗ 11:33286413-33287511:+ MRE miR-653-5p, RIP assay, luciferase reporter assay, Gastric cancer Oncogenic [56]
miR-338-3p biotin-labeled miRNA pulldown assay
circNR3C1 5:143399656–143400852:- MRE miR-27a-3p Biotin-labeled miRNA pulldown assay, Bladder cancer Tumor suppressor [57]
FISH
circNRIP1 21:15014344–15043574:- MRE miR-149-5p Biotin-labeled RNA pulldown assay, Gastric cancer Oncogenic [58]
biotin-labeled miRNA assay,
dual-luciferase reporter assay, FISH,
qRT-PCR
circRNF20 9:101540203– MRE miR-487a Luciferase reporter assay, FISH Breast cancer Oncogenic [59]
101540975:+
circSHKBP1 19:40583398–40583717:+ MRE miR-582-3p Luciferase reporter assay, Gastric cancer Oncogenic [60]
biotin-labeled RNA pulldown assay,
FISH, qRT-PCR
circZNF609∗ 15:64499292-64500166:+ MRE miR-15a-5p, RNA pulldown assay, luciferase Hepatocellular Oncogenic [61]
miR-15b-5p reporter assay carcinoma
circZNF609∗ 15:64499292-64500166:+ MRE miR-138-5p Luciferase reporter assay, RIP assay Renal carcinoma Oncogenic [62]
circDCUN1D4 4:51863437–51886638:+ RRE HuR(ELAVL1) CLIP, RIP assay, biotin RNA pulldown Lung Tumor suppressor [63]
assay, Western blot adenocarcinoma
circHIPK3 11:33340968–33341686:+ RRE FUS Western blot, correlation analysis, Cervical cancer Oncogenic [64]
knockdown, qRT-PCR
circMBNL1 3:152299405– RRE MBNL1 RIP assay Neuronal tissues Gene regulator, circRNA [65]
152300367:+ biogenesis
circNSUN2 5:6623214–6625669:- RRE IGF2BP2 RNA pulldown assay, RIP, IF-FISH Colorectal Oncogenic [66]
carcinoma
circPABPN1 14:23324138–23324289:+ RRE HuR(ELAVL1) RIP, CLIP, biotin RNA pulldown assay Cervical Negative regulation of [67]
carcinoma PABPN1 mRNA and
tanslation
inibition/endogenous
competing RNA
circRHOBTB3 5:95755396–95763620:+ RRE HuR(ELAVL1) RNA pulldown assay, RNA Colorectal Tumor suppressor [68]
immunoprecipitation assays cancer
circSMARCA5∗ 4:143543509- RRE SRSF1 eCLIP Glioblastoma Tumor suppressor [69]
143543972:+ multiforme
circXIAP X:123888619– RRE FUS Mass spectrometry analysis, RNA Prostate cancer Oncogenic [70]
123892773:+ pull-down assay
circZKSCAN1 7:100023419– RRE FMRP(FMR1) RIP assay Hepatocellular Tumor suppressor [71]
100024307:+ carcinoma
circ-E-Cad 16:68811684–68815759:+ ORF 254 Monoclonal antibody, MS Glioblastoma Oncogenic [72]
circFNDC3B 3:172247533– ORF 218 LC–MS/MS Colon cancer Tumor suppressor [73]
172251541:+
circPLCE1 10:94030683–94032252:+ ORF 411 LC–MS Colorectal Tumor suppressor [74]
carcinoma
circSHPRH 6:145888020–145894977:- ORF 146 Antibody, LC–MS/MS Glioblastoma Tumor suppressor [75]
circSMO 7:129205203– ORF 193 Ribosomes enrichment assay, Glioblastoma Oncogenic [76]
129206587:+ immunoblot (IB), MS
circZNF609 15:64499292–64500166:+ ORF 251 Polysome association, western blot Muscle Implied in myogenesis [16]
∗ CircRNAsused to illustrate CRAFT output. MRE, miRNA recognition element; RRE, RBP recognition element; ORF, open reading frame. Is near to the circRNAs used to illustrate CRAFT output. MRE, miRNA recognition
element; RRE, RBP recognition element; ORF, open reading frame.

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Figure 1. CRAFT workf low, schematizing the pipeline input, the main analysis steps and the produced output.

different strategies and settings can recover the known determined AGO2 binding sites was useful. Particularly in
functional elements in the test set (sensitivity), and how combination with the intersection of miRanda and PITA
the prediction ratio (Methods section) were affected. predictions, it kept it with a good sensitivity (≥0.82), while
The results based on the analysis of the known MRE reducing the amount of predictions to only 6%, respective
is presented in Figure 2A. The sensitivity of predictions to the total set of predictions obtained from the union of
obtained by miRanda, PITA and intersection thereof miRanda and PITA (Figure 2A). The starting number of
was constantly high (≥0.9), and the large number of predictions to be intersected and combined with AGO2
predictions provided by single methods can be controlled binding sites is obviously reduced when more stringent
by their intersection, as shown by the line plot. At our thresholds for the four parameters provided by miRanda
tests, the addition of information about experimentally and PITA are set, as shown in Supplementary Figures 1
6 | Dal Molin et al.

and 2. The scatterplot of prediction scores provided by CRAFT when only the exons from the canonical tran-
by miRanda (Supplementary Figure 1) and PITA (Sup- script (Ensembl id: ENST00000261769.10) were used to
plementary Figure 2), with indications of the score predict the circRNA sequence.
pairs associated with known MRE, can help the user to
understand the effect of stringency on MRE detection. The CircExtractor module
For instance, keeping only the predictions with both The circRNA nucleotide sequence is necessary to per-
scores over the first quartile for both methods, 7/11 form functional predictions. Therefore, we included in
known MRE were correctly detected (sensitivity 0.71). our pipeline a module that takes as input the circRNA
Using the median as threshold for the same four scores, backsplice coordinates, the genome sequence and the
the number of predictions decreased (prediction ratio gene annotation, and reconstructs the circRNA sequence
19%), still identifying 4/11 positives (sensitivity 0.36). (Figure 1). If circRNA sequences are available to the user,
In summary, considering that the intersection of this step can be skipped.
miRanda and PITA predictions and the overlapping The CRAFT CircExtractor module reconstructs the

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with AGO2 binding sites controlled for the prediction putative circRNA sequence by intersecting the reference
number, while keeping in with a good sensitivity, we annotation with the backsplice coordinates. Four main
kept a relatively low stringency on miRanda and PITA cases are considered: (i) if the backsplice joins the ends
parameters in CRAFT default setting (‘M&P’ in Figure 2A), of two annotated exons, the predicted circRNA sequence
to give the user a fairly complete set of predictions. contains all and only the exons within the backsplice
Nevertheless, the interactive filtering provided by CRAFT coordinates (Figure 3A); (ii) if one or both backsplice
allows the user to customize the number of predictions ends map within an exon, only the exon segment within
after inspecting the results. the backsplice coordinates is considered, along with all
The results obtained by tests on RRE predictions other entire exons (Figure 3B); (iii) if both backsplice ends
are shown in Figure 2B and C. All known RRE (9/9, map in an intronic or intergenic region, the intronic or
sensitivity = 1) were detected at the lowest stringency of intergenic part between the backsplice ends is extracted
beRBP predictions score (voteFrac > 0), with an average (Figure 3C); and (iv), if one backsplice end maps into
of 316 (range 301–326, median 319) predictions per an exon, whereas the other end maps into an intron
circRNA (Figure 2B). At a less permissive condition or an intergenic region, the sequence will contain all
(voteFrac ≥ 0.1), sensitivity remained high (0.89; with 8/9 the exons within the backsplice coordinates, plus the
detected) and 188 predictions per circRNA on average intronic/intergenic segment between the backsplice
were obtained (range 68–316, median 158). Further start/end and the first/last exon (Figure 3D).
increasing stringency (voteFrac ≥ 0.15 and ≥ 0.2), most of
the known functional elements were still detected (sen- CRAFT output
sitivity 0.78 and 0.56, respectively), while considerably To illustrate CRAFT output, post-processing analyses
reducing the number of predictions to 42% and 27% of and data display, we applied CRAFT to three previously
the total, respectively (Figure 2B). At the most stringent characterized circRNAs, circSMARCA5, circHIPK3 and
score tested, only 1/9 known RRE element (SRSF1 binding circZNF609, from the above-considered group.
circSMARCA5) was detected, being anyhow the unique CRAFT outputs multiple HTML pages with interactive
passing the filter for circSMARCA5, with an average tables and figures, describing the predicted miRNA and
number of 5 predictions per circRNA (range 0–36, median RBP binding sites, and the putative ORF (Figure 1). Pre-
0; Figure 2B). Figure 2C shows more detail about the diction result tables link miRNAs, genes and proteins
distribution of RBP binding site prediction scores for to several databases (miRBase, GeneCards, NCBI Entrez,
each circRNA, indicating the scores of known binding Ensembl, Uniprot and NCBI Pubmed). All result tables
sites. Plus, for each voteFrac threshold, the boxplot on the are downloadable in comma-separated value (CSV) plain
right shows the distribution of the number of predictions text, Excel, and PDF formats, can be copied to clipboard or
obtained over the analyzed circRNAs. In summary, the printed, allow filtering and ordering, and display colored
best balance between sensitivity and overall number of bars to highlight important features.
predictions was observed at a voteFrac ≥ 0.15, and this A summary of functional predictions obtained for cir-
optimal threshold was set as default in the CRAFT RBP cZNF609 was used to illustrate the main features of
analysis. CRAFT output (Figures 4 and 5). For each circRNA, a
Coding potential analysis predicted an ORF overlap- dedicated page is given with detailed predictions. At first,
ping the backsplice junction for all six tested circRNAs. a circular plot shows predicted miRNA and RBP binding
In five out of six cases (83.3%), the predicted peptide positions in the circRNA sequence, and the longest pre-
length exactly corresponded to that of the validated dicted ORF (Figure 4A). Next, three sections are dedicated
peptide (Figure 2D). The difference in the circ-E-Cad pre- to miRNA, RBP and ORF predictions, respectively.
dicted ORF was due to a slight discrepancy between the The miRNA section provides, after a circular plot
sequence predicted by CircExtractor based on genome focusing exclusively on miRNA binding sites, a table with
annotation and the sequence considered in the previous the start and end binding positions and the prediction
study [72]. The ORF and peptide were correctly predicted scores (Figure 4B). Then, the list of all validated TG
 CRAFT tool for circular RNA function prediction | 7

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Figure 2. Assessment analysis of circRNAs with known functions. (A) The upper panel shows the number of validated circRNA-miRNA interactions
detected by different methods and combinations thereof for each of the considered circRNAs, whereas the line plot below reports the sensitivity and
the prediction ratio in the same conditions (M, miRanda; P, PITA; M | P, union of M and P; M & P, intersection of M and P; the same with AGO2, predicted
sites overlapping with experimentally determined AGO2 binding sites); the right axis shows the mean number of predictions for circRNA; (B) the upper
panel shows the number of validated circRNA-RBP interactions detected by different beRBP voteFrac thresholds, whereas the line plot below reports
the sensitivity and the prediction ratio in the same conditions; the right axis shows the mean number of predictions for circRNA; (C) distribution of RBP
binding site prediction scores for each circRNA (known binding sites are shown as orange points, the name is shown for the known site with the highest
score; the percentage on the right indicates the prediction ratio of the corresponding voteFrac threshold); the notch boxplot on the right represents the
distribution of the number of predicted RBP per circRNA at a certain voteFrac score; (D) barplot of the amino acid (aa) length of known (in green) and
predicted (in red) peptides for each circRNA.
8 | Dal Molin et al.

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Figure 3. The CircExtractor module of the CRAFT pipeline reconstructs the circRNA sequence using the backsplice coordinates and the genome
annotation and reference. (A–D) show the four cases encompassed by the method, according to the position of the backplice ends (green and red
triangles represent the backsplice start and end, respectively), in exonic (A–B), intronic (C) and intergenic sequences, and combinations thereof (D).

is provided for each miRNA, along with different The user can tune the filter stringency and regener-
summarization tables, including the number of TG asso- ate the HTML pages with updated tables and figures
ciated with each miRNA (Figure 4C), the list of miRNAs with minimal computation time. Two filtering modes are
associated with each TG, and miRNA-TG interactions allowed: (i) a ‘direct mode’, by directly inputting each pre-
associated with drugs or diseases. Functional enrichment diction tool threshold parameter; (ii) a ‘quantile mode’, to
results on validated TG are displayed, including disease- filter a subset of predictions based on a selected quantile
associated genes (Figure 4D), Gene Ontology, KEGG (f.i. display and analyze only the top 25% high-scoring
analysis, Reactome pathways (Figure 4E) and MeSH predictions for each tool).
terms. Of note, miR-15a-5p and miR-15b-5p binding sites In principle, different circRNAs can act as sponge for
were detected for circZNF609, in line with literature data the same miRNA, reinforcing the desuppression of the
[17, 61, 62]. miRNA targets. Multiple circRNAs can regulate the same
The RBP section, after a circular plot with RBP gene also through different miRNAs (Figure 1). Thus, if
binding positions and the corresponding interactive table more than one circRNA is given in input, CRAFT inte-
(Figure 5A), displays results of functional enrichments grates altogether the predictions obtained for the dif-
performed on RBP, considering Gene Ontology, KEGG ferent circRNAs. The common predictions are displayed
analysis, Reactome pathways (Figure 5B) and RBP- through tables and figures, to provide valuable hints
disease association (Figure 5C). Additional tables provide about the possible cooperation of circRNAs to the same
protein expression, functional and miscellaneous infor- regulatory axes. The 13 miRNAs with binding sites in the
mation associated with RBP. top 45% high-scoring predictions for both tools and com-
In the last section, the longest predicted ORF overlap- mon to multiple circRNAs, are shown in Figure 6A. More-
ping to the backsplice junction is shown by a circular plot over, in the CRAFT output, circRNAs are connected to
(Figure 5D), while a table reports, for all the predicted miRNA-validated TG through their predicted interactions
ORF, the start and end positions, the distance from the with miRNAs. In addition to tables with TG potentially
backsplice junction, and the predicted coding and pep- shared by circRNAs, CRAFT provides a network visual-
tide sequences in FASTA format (Figure 5E). ization of predicted circRNA-miRNA-TG axes in which
A default set of high-scoring predictions is reported multiple circRNAs are connected to the same TG through
and visualized in the output HTML pages (see Methods). the same or different miRNAs. For instance, circHIPK3
 CRAFT tool for circular RNA function prediction | 9

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Figure 4. CRAFT miRNA prediction output based on circZNF609 analysis. (A) Summary plot with predicted miRNA and RBP binding sites, and the longest
ORF (with a stop codon), along the circRNA sequence; (B) table of CRAFT predicted miRNA binding sites in circZNF609 sequence, with start and end
positions, and scores (orange and green boxes highlight predicted miR-15a-5p and miR-15b-5p binding sites, respectively); (C) table summarizing the
number of TG per miRNA (box colors as in B); (D) plot of disease-miRNA TG association enrichment analysis; (E) network showing Reactome pathway
enrichment analysis results on miRNA TG. In panels (B) and (C) only a small part of the corresponding table is shown.

and circZNF609 can potentially control the expression including circSMARCA5 whose interaction with SRSF1
of a group of eleven genes by sponging miR-1207-5p has been previously demonstrated [69].
(circHIPK3) or miR-5581-3p and miR-611 (circZNF609) Finally a barplot (Figure 6D) and a detailed table
(Figure 6B). present the number of predicted ORF for each circRNA,
Similarly, CRAFT displays RBP associated with RRE distinguishing between ORF with and without a stop
predicted in multiple circRNAs. Considering all the pre- codon.
dictions obtained with CRAFT default stringency, 48 RBP
are commonly associated with all the three circRNAs.
Of note, an interaction with the FUS RBP is predicted by Discussion
CRAFT for circHIPK3, in line with literature data [78], as Robust evidence on circRNA functions, involvement in
well as for circSMARCA5. The RBP with binding sites in biological processes, and oncogenic potential make these
the top 10% high-scoring predictions and common for molecules extremely attractive for both fundamental
multiple of the three circRNAs considered in our sample and cancer research [79]. CircRNAs can be detected and
analysis are shown in Figure 6C. All the considered cir- quantified from RNA-seq data using appropriate soft-
cRNAs have strongly predicted binding sites for SRSF1, ware tools, including [80, 81]. Next, the challenge faced
10 | Dal Molin et al.

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Figure 5. CRAFT RBP and ORF prediction output based on circZNF609 analysis. (A) Table of CRAFT predicted RBP binding sites in circZNF609 sequence,
with start position, binding probability and prediction score; (B) plot of Reactome enrichment analyses on RBP potentially interacting with circZNF609,
according to CRAFT predictions; (C) barplot of RBP-disease associations; (D) plot of the longest predicted ORF with a stop codon in circZNF609 sequence;
(E) table with start and end position, length and frame of predicted ORF. In panels (A) and (E) only a small part of the corresponding table is shown.

by studies aiming at defining circRNA roles in disease and Of note, CRAFT correctly detected the known ORF of
cancer is the identification of circRNA-involving mecha- circSHPRH, which is formed by the circularization of
nisms. CircRNA screening by massive silencing or over- four exons of the SHPRH gene. In specific instances,
expression studies and experimental investigation can the sequence reconstructed by CircExtractor can be
be of great help. Nevertheless, the prediction of circRNA different from the sequence produced by the splicing
potential interactions and functions is a precondition for of the exons included in the canonical transcript. This
the prioritization of circRNAs for functional experimen- is exemplified by circ-E-Cad, whose backsplice ends
tal studies, for the interpretation of experiment results include a region overlapped by 11 different transcripts
and the design of targeted mechanistic investigations. that use alternative 5 - and 3 -exon ends in the linear
Indeed, even once a circRNA has been demonstrated splicing, while the validated circ-E-Cad follows the
to significantly impact cell features, such as prolifera- linear splicing pattern of the canonical longer coding
tion, apoptosis, metabolism or differentiation, there is transcript of the gene. In general, the uncertainty
the need to disclose the involved molecules and regula- associated with circRNA sequence reconstruction based
tory axes. on exon annotations tends to increase with the size
CRAFT has been developed to overcome the limita- of the genomic region within the backsplice ends and,
tions of the currently available circRNA function pre- in particular, with the complexity of the alternative
diction methods. It allows the user to explore putative linear splicing pattern in the region. In this regard,
regulatory networks involving one or more circRNAs of our recommendation is to use the CRAFT sequence
interest, facilitating the interpretation of their biological reconstruction module if the circRNA sequences are
and pathogenetic role. Moreover, CRAFT is also a highly not known, or for massive analyses of several circRNAs.
portable tool, thanks to the containerization of the soft- Nevertheless, in projects focusing on the functional
ware, easy to use, and it requires minimal input data. experimental investigation of one or a few specific
CRAFT presents four main advantages over the exist- circRNAs, we advise using experimentally determined
ing tools. First, it provides the putative circRNA sequence. circRNA sequences, that provide a firmer ground to build
The sequence extraction is based on annotated exons, predictions on.
following the observation that linear splicing patterns The second main CRAFT asset is that it allows predic-
of circRNAs mainly join canonical exons [82]. The tion of the three most important circRNAs functions,
CircExtractor module in CRAFT merges all the overlap- whose biological relevance is supported by robust
ping exons within the circRNA coordinates. This works literature [9–14, 16, 18–20]. The most investigated
perfectly well for most cases, as indirectly demonstrated function of circRNAs is the ability to bind miRNAs. One
by our test on circRNAs proved to encode for peptides. limitation of miRNA binding site prediction algorithms
 CRAFT tool for circular RNA function prediction | 11

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Figure 6. Predictions of different circRNAs are combined to facilitate hypothesis generation. (A) Heatmap of the miRNAs common to at least two circRNAs,
considering the top 45% high-scoring miRNA binding site predictions; (B) network showing the 11 common validated TG, targeted by miR-1207-5p, miR-
5581-3p and miR-611, respectively associated to circHIPK3 (the first) and circZNF609 (the last two); high-scoring predictions was obtained with miRanda
score > 122 and energy < −24, PITA Gduplex < −20 and Gopen > −11; (C) heatmap of the RBP common to at least two circRNAs, considering the top 10%
high-scoring RBP binding site predictions; (D) summary barplot of the number of ORF predicted for the group of circRNAs considered by CRAFT, binning
ORF by the presence or absence of a stop codon in the reading frame. Canberra distance-based clustering has been used for heatmaps in (A) and (C).

is the low specificity of the predictions. To face this inspecting the possible interactions between circRNAs
problem, we used a dual strategy: (i) the combination and RBP, and also the identification of the RBP possi-
of two algorithms, miRanda [40, 83] and PITA [41], bly bound by multiple circRNAs. Of note, beyond the
and (ii) the integration of predictions with experimentally beRBP database of position weight matrices embedded
determined AGO2 binding sites. The assessment of the in CRAFT, custom matrices can be provided by the user,
CRAFT predictions on a sizable set of circRNAs with extending the analysis.
known functions showed that this approach can control CRAFT predicts the ORF in the circularized sequence,
the number of predictions, while holding good sensitivity. allowing the user to obtain the peptides potentially
Also the interaction of circRNAs with RBP can be asso- encoded by the circRNA and not by the linear transcript.
ciated with important functions [23]. In principle, cir- To what extent circRNAs are translated it is still a
cRNAs acting as decoys can counteract RBP functions, matter of debate [84], but CEP have been identified
whereas in other cases circRNAs can serve as scaffolds in different studies and, in several cases, were also
for the assembly of molecular complexes. CRAFT allows proven to be biologically active [85–87]. CRAFT aims
12 | Dal Molin et al.

at the prediction of circRNA-specific peptides, thus it of interest. The generation of new hypotheses about the
focuses on those ORF overlapping the backplice site, potential impact of circRNAs in biological processes,
which are not contained in linear transcripts. To undergo pathways and diseases can help circRNA prioritization
translation without a 5 cap, circRNAs can require for further study and the interpretation of their biological
specific sequence elements (internal ribosome entry and pathogenetic role.
sites (IRES) [88] and N6 -methyladenosine modification
[18, 89]) whose prediction can be envisaged as an as
interesting CRAFT future development.
Key Points
The third CRAFT advantage for the user is that it pro-
• CRAFT is a self-standing tool for comprehensive circRNA
vides a rich output with graphical visualizations, leverag-
function prediction.
ing functional enrichments and results linking to several • CRAFT functions include circRNA sequence reconstruc-
knowledge databases. The output can be explored, fil- tion, microRNA and RNA-binding protein response ele-
tered and reanalyzed as long as the user prefers to gener- ments and coding potential prediction.

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ate hypotheses. For instance, the connection of circRNAs • Predictions for multiple circRNAs are connected to infer
with miRNAs, validated miRNA-target genes, and specific possible cooperation networks and illustrate the poten-
pathways, functions and diseases can be of great help tial impact of circRNAs on biological and disease pro-
to identify regulatory networks to be further scrutinized. cesses.
This can be pursued with experimental investigation,
as well as integrating CRAFT results with circRNA and
gene expression data. For instance, positive correlations Supplementary data
between a circRNA and the validated target genes of miR-
Supplementary data are available online at https://
NAs potentially sponged by the circRNA can be indicative
academic.oup.com/bib.
of functional regulatory axes [4, 5].
Finally, we believe that the combination of predictions
for different circRNAs provided by CRAFT is a highly Data availability
useful and original feature. In research projects, when CRAFT tool is available on GitHub at https://github.com/
circRNAs dysregulated in cancer or disease tissue com- annadalmolin/CRAFT and on DockerHub at https://hub.
pared to a normal counterpart are identified, subsequent docker.com/repository/docker/annadalmolin/craft .
experimental investigation often focuses only on the elu-
cidation of the functions of one specific circRNA. Instead,
Authors’ contributions
cooperative effects of multiple dysregulated circRNAs in
pathogenetic mechanisms can be hypothesized. Thus, AD and SBo did the conceptualization; AD performed
the potential interaction of different circRNAs with the data curation; AD and SBo did the formal analysis; EG,
same RBP can be inspected in CRAFT. Plus, CRAFT allows SBr and SBo did the funding acquisition; AD and EG
the exploration of regulatory networks in which multiple did the methodology; SBo did project administration;
circRNAs can act as sponge for the same miRNA, rein- SBo collected the resources; AD and VD operated the
forcing the desuppression of the miRNA-target genes, or software; EG and SBo did the supervision; AD and SBo did
sponge different miRNAs with a common target gene, the visualization; AD, EG and SBo had written the original
possibly coregulating its expression. draft of the manuscript; Writing − Review & Editing, AD,
In this study, we appraised CRAFT predictions by using, EG, AB, CTP, SBr and SBo reviewed and edited the original
as benchmarking, a set of circRNAs with known func- draft of the manuscript. All authors read and approved
tions, including the three types of functional elements the final manuscript.
predicted by CRAFT. CRAFT sensitivity was assessed in
relation to the total number of predictions to be handled Funding
by the user at different settings. Our sensitivity estimates
were highly conservative, based on a pejorative scenario The Fondazione AIRC per la Ricerca sul Cancro, Milan,
in which all the functional elements are known and Italy [Investigator Grant 2017 20052 to SBo.]; the Ital-
thus all the predictions not corresponding with known ian Ministry of Education, Universities, and Research
functional elements are counted as false positives, even if [PRIN 2017 2017PPS2X4_003 to SBo]; Fondazione Umberto
some of them can be functional still not known. The con- Veronesi, Milan, Italy [Fellowship 2020 to EG]; Depart-
ducted tests informed the effects of different parameters, ment of Molecular Medicine of The University of Padova
allowing us to optimize the CRAFT strategy and default [to SBo]; PhD in Biosciences of The University of Padova
settings and providing intelligence about the analysis [to AB], Fondazione Cariparo [to SBr and SBo].
customization and results filtering for CRAFT users.
In conclusion, we are handing over the researchers in References
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