Circ-ZNF609 Is a Circular RNA that Can Be Translated and Functions in Myogenesis

Keywords: circRNA, circular RNA, myogenesis, muscle differentiation, translation, cap independent, DMD, proliferation, non-coding RNA

CircRNAs Are Abundant, Conserved, and Highly Expressed

Total RNA was collected from two replicates of human and mouse (C₂C₁₂) myoblasts cultured in growth medium (GM) or induced to differentiate into myotubes (differentiation medium, DM). Paired-end ribominus RNA sequencing (RNA-seq) was performed, and the FindCirc computational pipeline (Memczak et al., 2013) was applied in order to detect circRNAs. RNA-seq data were first processed for gene expression analysis: reads were mapped with TopHat and the transcriptome was reconstituted with Cufflinks (data summarized in Figure S1A). Differential gene expression analysis was performed in order to check the quality of the in vitro differentiation experiments. While fragments per kilobase of transcript per million mapped reads (FPKMs) of genes in technical replicates were almost perfectly correlated (>98% correlation; Figure S1B, upper panels), gene expression in myotubes compared to myoblasts was highly diverse, with >3,000 genes significantly upregulated or downregulated in one of the two conditions, in both human and mouse systems (Figure S1B, lower panels). When looking at the enrichment of gene products upregulated in myotubes versus myoblasts in terms of gene ontology (GO) biological process keywords, we found a consistent and significant enrichment for genes related to muscle differentiation and function (Figure S1C). Moreover, the expression of well-known specific markers of muscle differentiation was assessed by qRT-PCR, which confirmed the data obtained by RNA-seq (Figure S1D).

RNA-seq reads were then realigned to the reference genomes, the ones contiguously aligned were discarded, and the remaining reads were used as input for FindCirc (Figure 1A) to detect head-to-tail splicing sites. As described in Figure 1B, thousands of circular splicing events were found in both human and mouse samples, with almost 90% deriving from internal exons of protein-coding genes (Figure 1C). Almost 10% of circRNAs was expressed at similar or higher levels with respect to the linear counterpart (by comparing the number of reads mapping on circular versus linear splice junctions). Interestingly, almost 600 of these circRNAs were conserved between species of the ∼2,100 and 1,600 circRNAs identified in human and mouse, respectively (Figure 1D, full list in Table S1). Our criterion for conservation was any circRNA in which the genomic location in human overlapped with the syntenic region in mouse. Considering that low-abundance species might have been missed by this analysis, the actual species overlap may be even higher. Indeed, when filtering human circRNAs for high expression level (more than five reads), the overlap with mouse circRNAs increased from ∼25% to 40%.

CircRNA Expression Is Modulated in Muscle Differentiation and Disease

To perform quantitative analyses on circRNA expression and regulation, we took human samples and reduced further our list to a set of highly expressed molecules by requiring at least five unique reads mapping to the head-to-tail junction. With this threshold, while the technical replicates had an overall very good correlation (Figure 2A), the comparison between myoblasts and myotubes revealed a global change in circRNA expression (∼45% circRNAs having ≥2-fold variation in one of the two conditions, Figure 2B), meaning that these molecules were modulated in response to cell differentiation. RNA-seq data from human myoblasts derived from DMD patients were also analyzed. Hierarchical clustering analysis of normal and dystrophic myoblasts and myotubes revealed that indeed DMD cells have a unique signature in terms of circRNA expression levels (Figure 2B): specific subsets of transcripts were differently abundant in DMD with respect to controls both in GM and in DM conditions. This is in agreement with the notion that Duchenne cultures have a much slower progression into the differentiation process (Cazzella et al., 2012). Notably, circRNA abundance tends in general to increase during differentiation (Figure 1B), as described for neuronal differentiation (Rybak-Wolf et al., 2015). The circular/linear ratio also followed this trend (Figures 2C and 2D). This is possibly related to the high stability of these molecules: during differentiation, induction of circRNAs at the transcriptional level, combined with a slow turnover, could lead to their accumulation over time.

Then we addressed the question of whether modulation of circRNA expression in myogenesis was due to transcriptional regulation of the host gene or to post-transcriptional control, such as competitive biogenesis between the linear and the circular isoforms within the same gene (Ashwal-Fluss et al., 2014). Therefore, we analyzed the relationship between the fold change ratio of circular versus linear expression level in the two conditions tested (GM versus DM), and we found a positive correlation (Figure 2E). Additionally, according to FPKMs calculated with Cuffdiff, the abundance of circRNAs produced from upregulated, stable, or downregulated genes indicated that the induction of circRNAs coming from upregulated genes was significantly higher than that of stable and downregulated genes (Figure 2F). However, exceptions were detected, such as the BNC2 gene that produces mainly the linear mRNA in human and mouse myoblasts in growth conditions but predominantly the circRNA in differentiated cells. Figure 2G shows circ-BNC2 in comparison with circ-CDYL, which is an example of a concomitant increase in both circular and linear isoforms during myogenesis, probably because of transcriptional activation of the locus.

RNAi-Based CircRNA Functional Screening

Specific criteria were applied in order to restrict the number of candidates for further characterization: (1) conservation, (2) expression level, (3) modulation during differentiation, and (4) circular/linear ratio (see the STAR Methods). This filtering yielded 31 circRNAs, which are shown in Table 1. The selected species were initially validated by RT-PCR (summarized in Table 1, full results in Figures S2A and S2B, list of primers in Table S2). Of 31 candidates, only one, circ-MYL4, was not detected as a band of the expected size in human samples. Circ-TTTY16 was instead not detected in mouse samples, because it is located in the Y chromosome, which is not present in C₂C₁₂ cells. For almost every circRNA, RT-PCR produced a band of the expected size and one or more larger products, possibly corresponding to concatemers generated by rolling circle retro-transcription. For a few circRNAs (CDYL, QKI, and ZNF609), we gel-extracted and sequenced those bands, confirming that they contained the head-to-tail junction and that they were concatemers (Figure S2C). The qRT-PCR analysis of a subset of six human circRNAs did not reveal a major second PCR product for any of them (Figure S2D), possibly because of the different enzyme and cycling conditions used with respect to non-quantitative PCR and opening to the possibility of using such technique for accurate measurement of relative circRNA expression (Figure S2E). Indeed, in all cases, the normalized RNA quantities measured by qRT-PCR reflected the levels observed by RNA-seq and RT-PCR (Figure S2E). For the same candidates, purification of total RNA with oligo dT revealed that they were preferentially recovered in the non-polyadenylated fraction with respect to their linear counterparts (Figure S2F). Similarly, comparison of dT- versus random examer-primed cDNA synthesis shows that circRNAs were retro-transcribed more efficiently with random examers than with dT with respect to linear RNAs (Figure S2G).

Resistance to the RNase R exonuclease was used to test whether the selected molecules were circular (Suzuki et al., 2006). Of 30 putative circRNAs, 29 were confirmed as non-accessible to the exonuclease. Only one, circ-NEB, was digested by RNase R as efficiently as its linear counterpart (summarized in Table 1, full results in Figures S2H and S2I). Moreover, nuclear/cytoplasmic fractionation indicated that all circular species were almost exclusively located in the cytoplasm, both in mouse and human cells (Figures S2J and S2K).

We then designed an image-based functional genetic screen to further characterize these molecules (summarized in Figure 3A). We tried to design at least two small interfering RNAs (siRNAs) targeting each of the 29 circRNAs, according to the following features: (1) binding site at the head-to-tail junction; (2) 7-mer seed not present in the linear host gene; and (3) chemical modifications within the seed region, in order to reduce the chance of an miRNA-like effect and passenger strand inactivation to direct strand-specific Ago loading. These modifications and their applications have been the objective of extensive studies (Jackson et al., 2006, Chen et al., 2008), and they have been here designed for customized targets (On-Target-plus by Dharmacon). Following these rules, it was possible to design two siRNAs for 20 circRNAs, one for five circRNAs, and none for the four remaining ones (Figure S3A; siRNAs listed in Table S3). Due to the poor transfectability of human primary myoblasts, we developed a reverse transfection protocol that allowed high-efficiency transfection of siRNAs in these cells (see the STAR Methods). The 45 siRNAs plus a negative control were applied in triplicate to myoblast cultures in a 96-well format, and, 24 hr post-transfection, cells were switched to DM for 48 and 96 hr. The relative abundance of the circular versus linear forms was tested in order to confirm the specific downregulation of the circRNA. This revealed that at least one siRNA for 17 different genes specifically downregulated the circular form without affecting the linear mRNA (Figure S3B).

Immunofluorescence analysis was performed for two common myogenic markers, Myogenin (MYOG) and Myosin Heavy Chain (MHC), together with DAPI staining (Figure S3C). Several readouts were analyzed in order to obtain a detailed picture of how knockdown of each circRNA affected cell differentiation and morphology. We included in this analysis 11 different phenotypes, including total cell number, the fraction of MYOG- or MHC-positive cells, and additional morphological parameters, such as the fusion index. From this screening, two strong and opposite phenotypes associated with siRNAs targeting circ-QKI and BNC2 emerged. The two siRNAs designed for circ-QKI (siRNA #1 and siRNA #2) both inhibited myoblast differentiation (Figure 3B). While siRNA #1 had a specific effect on circ-QKI and none on QKI mRNA, siRNA #2 did not alter circ-QKI levels but downregulated QKI mRNA (Figure S3B). This supports the hypothesis that both circ-QKI and QKI mRNA participate in the same process. To confirm that hypothesis, we raised a third siRNA specifically targeting QKI mRNA (si-QKI-L; Figure S3D). si-QKI-L, and additional experimental replicates with si-QKI #1 (Figures 3C, S3B, S3D, and S3E), had again a specific anti-myogenic effect, thus confirming that both the circRNA and the mRNA could be involved in promoting differentiation (Figure S3E, as in Li et al., 2003).

In the case of circ-BNC2, si-BNC2 #1 efficiently downregulated circ-BNC2 but had also a small effect on BNC2 mRNA, while si-BNC2 #2 targeted specifically circ-BNC2 and had no effect on the phenotype (Figure S3B). These data suggested that the observed phenotype was likely associated with BNC2 mRNA knockdown. We confirmed this by using a third siRNA, designed specifically for BNC2 mRNA (si-BNC2-L; Figures S3D and S3E), and we observed the same effect as with additional replicates of si-BNC2 #1 (Figures 3C, S3D, and S3E). Notably, during differentiation, a strong increase of the circular RNA paralleled the decrease of the linear BNC2 mRNA (Figures 2G, S2A, and S2B). Therefore, it is likely that biogenesis of circ-BNC2 is a mechanism for competing with the production of the anti-myogenic BNC2 mRNA when differentiation starts.

Interestingly, both circRNAs, upregulated during in vitro differentiation of control myoblasts, were downregulated in DMD conditions (Figure 3D), well correlating with the notion that dystrophic cells have altered progression into the differentiation process (Cazzella et al., 2012). For a different set of circRNAs highly expressed in myoblasts, we investigated the relationship between their abundance and proliferation capacity through a bromodeoxyuridine (BrdU)-labeling assay (Figure S3C). Among the circRNAs tested, we found that the knockdown of circ-ZNF609 (successfully accomplished with siRNA #1) reduced BrdU incorporation by ∼80% (Figure 4A). In line with this, also markers of proliferation, such as CDK1 and cyclin A2, were significantly reduced after circRNA knockdown (Figure 4E), indicating a specific role of circ-ZNF609 in myoblast proliferation. Similar effects, even if at lower extents, were observed in murine cells (Figures S4A and S4B). The initial screening was subsequently validated by comparing the activity of three distinct siRNAs: si-circ (corresponding to siRNA #1 used in the screening), which targets the circ-ZNF609 head-to-tail junction; si-ex2, targeting both the circRNA and its linear counterpart; and si-mRNA targeting only the linear mRNA (Figure 4B). Both si-circ and si-ex2 reduced myoblast growth rate by 80% while si-mRNA, targeting only the linear mRNA, did not affect proliferation (Figures 4C and 4D). These results allowed us to exclude that the observed phenotype was due to an off-target effect, and they indicated that the decrease in proliferation was solely due to the circRNA activity. Both circular and linear ZNF609 RNA levels were measured after treatments by qRT-PCR, confirming the specificity of the three siRNAs (Figure 4E). Moreover, northern blot analysis confirmed that circ-ZNF609 was susceptible to si-circ treatment and resistant to RNase R (Figure 4F), while the linear ZNF609 mRNA was resistant to si-circ and susceptible to RNase R (Figure 4F). Notably, the circular form co-migrated with the product derived from an artificial construct, p-circ (see next paragraph), overexpressing circ-ZNF609 RNA (Figure 4F).

Also interestingly, circ-ZNF609, which is downregulated during myogenesis in control myoblasts, was instead found at elevated levels in DMD conditions (Figure 4G). This is consistent with the delayed differentiation phenotype of dystrophic primary myoblasts and with the persistence of a high proportion of proliferating, non-fusing myoblasts (Cazzella et al., 2012). These data reinforce further the connection of circ-ZNF609 and myoblast proliferation.

Circ-ZNF609 Encodes a Protein

Circ-ZNF609 originates from the circularization of the second exon of its host gene. A 753-nt open reading frame (ORF) is present in circ-ZNF609, spanning from the putative AUG of the host gene to a STOP codon created 3 nt after the splice junction (Figure 5A). To determine whether this ORF (circORF) is functional, we first used sucrose gradient fractionation to test circ-ZNF609 loading onto polysomes. Figure 5B shows that a small but significant proportion of circ-ZNF609 sedimented with heavy polysome fractions while the rest remained in the non-bound fraction. The circular conformation of the polysome-bound circ-ZNF609 RNA was confirmed by RNase R resistance (Figures S5A and S5B). Moreover, we excluded the presence of possible linear concatemers by northern blot (Figure 4F; see also RT-PCR with primers in exons 1 and 3 of the ZNF609 gene in Figure S5C). In puromycin-treated myoblasts, circ-ZNF609 shifted to lighter polysomes, similarly to the behavior of the linear ZNF609 and HPRT mRNAs, suggesting that its sedimentation pattern was due to active translation. As a control, a circRNA with no ORF, circ-PMS1, remained on free RNA fractions. The association of circ-ZNF609 with heavy polysomes was also confirmed in mouse (Figure S5D).

To test the protein-coding ability of circ-ZNF609, we took advantage of an expression vector that was able to produce circular transcripts (Kramer et al., 2015). P-circ contains the second exon of the ZNF609 gene and is able to express circ-ZNF609 RNA at high levels (Figure 4F). Sequencing of the RNA produced from this construct revealed its perfect correspondence with the endogenous circ-ZNF609 RNA (data not shown). A construct was derived from p-circ that contained a 3×FLAG-coding sequence immediately upstream of the STOP codon, such that a flagged protein could be produced only upon formation of a circular template (p-circ3xF; Figure 6A). Due to the poor transfection efficiency of plasmid DNA in human and murine myoblasts, the following experiments were carried out in HeLa and N2A cells. The results indicate that in both cell types, in comparison to mock-treated cells, p-circ3xF gave rise to two flagged proteins (Figure 6B). Inspection of the sequence indicated that a second ATG codon was present 150 nt downstream of the first one and in the same frame. Mutants lacking either one of the two initiation codons (p-circΔ1 and p-circΔ2) showed that two bands observed by western blot corresponded to two isoforms: the upper band (ATG1) derived from translation from the first ATG, while the lower one (ATG2) originated from the second start codon (Figure 6B). Deletion of both ATGs (p-circΔ1-2) abolished both products. An additional mutant, with a TGA Stop codon replacing ATG2 (Figure 6A; STOP2), confirmed that translation of the flagged proteins required initiation at those precise sites (Figure S6C).

An additional construct, p-lin3xF, was raised carrying the same ORF present in circ-ZNF609 in a linear conformation. When transfected in HeLa and N2A cells, this plasmid induced the expression of the same two proteins observed with the circular substrate, even if with a much higher efficiency (Figures S6A and S6B). Moreover, the flagged protein resulted in nuclear localization (Figure S6C). Titration of the proteins produced by the linear versus the circRNAs, normalized for the amount of RNA produced, indicated that circRNA was translated at almost two orders of magnitude lower efficiency than the linear form (Figure S6B). These results are in line with previous observations showing that re-initiation and internal initiation of translation can be as low as 1%–10% the efficiency of cap-dependent initiation (Merrick, 2004). Moreover, while the circular template yielded approximately the same amount of the two isoforms, translation of the linear one was strongly biased in favor of the first ATG, indicating a substantial difference in the translation initiation mechanism between the circular and linear transcripts.

In cells expressing p-circ3xF and the different ATG mutant derivatives, we confirmed the production of circular transcripts by northern analysis (Figure S6D), with a specific control of RNase R resistance (Figure S6E). We also verified the integrity of the ORF by Sanger sequencing of the RT-PCR products (data not shown), and we further excluded the production of linear concatemers through RT-PCR (Figures S6F and S6G).

To further prove the protein-coding ability of the circ-ZNF609 RNA derived from the chromosomal gene, we designed a genome-editing approach for inserting the 3×FLAG-coding sequence in the endogenous ZNF609 locus. We used the CRISPR/Cas9 system in mouse embryonic stem cells in combination with a DNA donor designed for inserting the 3×FLAG-coding sequence in the same position as in p-circ3xF, so that a flagged protein could be produced only upon circularization of the second exon of ZNF609 (Figure 6D). After transfection, clonal selection, and genotypization (Figures S6H–S6J), we obtained one positive clone carrying two modified alleles: the first one contained the expected flagged allele, while the second carried a small deletion across the 3′ splice site of the first intron, which prevented circularization of the second exon (Figure S6J; F/D clone). Therefore, from this cell line only the flagged allele would be able to produce the circ-ZNF609 RNA, and the amount of circRNA produced would be half of that produced by a wild-type (WT) line. Since circ-ZNF609 is highly expressed in neural tissues (Rybak-Wolf et al., 2015), we applied an established neuronal differentiation protocol (Wichterle and Peljto, 2008), and we checked for the expression of the FLAG-tagged circ-ZNF609 RNA by northern blot in WT cells and in the F/D clone. As expected for their genomic context, F/D cells produced roughly half the circRNA of WT cells (Figure S6K); moreover, the circRNA was slightly larger than in the WT because of the inserted tag. RT-PCR and Sanger sequencing confirmed that the circ-ZNF609 RNA produced by F/D cells was identical to that made by the p-circ3xF construct. Due to the low translation efficiency and the high background in western analysis, we performed immunoprecipitation of F/D cells lysates with an anti-FLAG antibody, and then we subjected proteins (size selected between 30 and 40 kDa) to mass spectrometry on a polyacrylamide gel. As positive controls, we immunoprecipitated proteins from cells transfected with the constructs overexpressing either the linear or the circular flagged ORF. Figure 6E shows that numerous peptides, mapping to the circORF, were present in overexpression conditions with the linear construct (p-lin3xF), fewer with overexpression of the p-circ3xF construct, and one in the F/D sample. This is in agreement with the lower levels of RNA produced by the chromosomal gene, with respect to those obtained with the overexpression of both p-circ3xF (Figure 4F) and p-lin3xF, and to the lower translational efficiency from circular templates (Figures S6A and S6B). No ZNF609 peptides were instead detected in control WT cells.

As low initiation efficiency is commonly associated with cap-independent translation, which instead serves as a regulatory mechanism responding to various external stimuli, such as heat shock stress (Holcik and Sonenberg, 2005), we tested protein production in such condition. As shown in Figure 6F, heat shock induces a strong and significant increase in translation of the flagged circ-ZNF609 transcript.

IRES Activity of Circ-ZNF609 UTR

Since circRNAs are devoid of cap structure, translation should occur through sequences able to act as internal ribosome entry sites (IRESs). Notably, phylogenetic analysis of the 5′ and 3′ UTRs of ZNF609 in vertebrates indicated that the portion of the 5′ UTR present in circ-ZNF609 was significantly more conserved than the other UTR sequences situated in the linear ZNF609 mRNA (Figure S6L). To test whether it possessed IRES-like activity, the circ-ZNF609 UTR was inserted between the Firefly and Renilla luciferases of a bicistronic construct (UTR; Figure 6G). With respect to a vector containing a viral IRES sequence (EMCV), UTR displayed very little luciferase activity (Figure 6H). Remarkably, Renilla luciferase raised to levels even higher than those of EMCV when introns from ZNF609 (UTR-inZNF), HPRT (UTR-inHPRT), and β-globin (UTR-inβglob) genes were inserted into the UTR construct, reconstituting the original splice junction of circ-ZNF609. Furthermore, deletion of the 5′ splice site in UTR-inβglob (UTR-Δss) completely abolished luciferase levels (Figure 6H), indicating that Renilla luciferase (Rluc) activity did not originate from cryptic splice sites and ultimately proving the relevance of splicing in the induction of IRES activity. Finally, the circ-ZNF609 UTR was replaced with an unrelated sequence of similar length (MCS-inβglob). Notably, this construct was not able to trigger Rluc activity (Figures 6G and 6H), thus pointing out that IRES activity also relies on specific features of the UTR sequence. RT-PCR analysis indicated that correct splicing, where applicable, was obtained for all constructs (Figure S6M). Altogether, these data show that the UTR of circ-ZNF609 is able to work as an IRES in a splicing-dependent manner.

Moreover, synthetic FLAG-tagged circ-ZNF609 made by in vitro ligation (Figure S6N) was not translated upon transfection in comparison to the same molecule produced through back-splicing from plasmid p-circ3xF, further demonstrating the requirement of a splicing-dependent process for efficient circRNA translation (Figures S6N and S6O).

Key Resources Table

Contact for Reagents and Resource Sharing

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Irene Bozzoni (irene.bozzoni@uniroma1.it).

Experimental Model and Subject Details

Method Details

Quantification and Statistical Analysis

Data and Software Availability

All software used in this manuscript is listed in the Key Resources Table. Additional dedicated scripts developed for this work are available upon request. The accession number for the sequences reported in this paper is GEO: GSE70389.

Document S1. Figures S1–S6

Table S1. The CircRNAs Detected by RNA-Seq, Related to Figure 1

Table S2. Oligonucleotides Used in This Work, Related to STAR Methods

Table S3. The siRNAs Used in This Work, Related to Figure 3

Document S2. Article plus Supplemental Information

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