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. 2025 May 26;104(8):105339. doi: 10.1016/j.psj.2025.105339

Chicken CircZNF609 encodes a protein induced by IRES-like region that inhibits the proliferation and promotes the differentiation of myoblasts

Lei Li a,b,1, Zihao Zhang a,b,1, Haidong Xu a,b, Qiying Leng a,b, Wenjin Shen a,b, Shudai Lin a,b, Lilong An a,b, Li Zhang a,b,⁎
PMCID: PMC12179659  PMID: 40482626

Abstract

Skeletal muscle is comprised of multinucleated myofibers that arise from myogenesis, a highly coordinated process encompassing the proliferation and differentiation of myoblasts. During this process, circular RNAs (circRNAs) are recognized as functional non-coding transcripts that play important roles in the regulation of skeletal myogenesis. Chicken ZNF609 (Zinc Finger Protein 609) is a member of the zinc finger proteins (ZNFs) family which serve as versatile regulators in cell proliferation, differentiation and metabolism. Analyze of circRNA sequencing data in chicken from our previous work revealed a circRNA originating from the ZNF609 gene, circZNF609. Here, we verified circZNF609 by sanger sequencing and RNase R treatment. And we found that chicken circZNF609 was upregulated in thigh muscles with the growth of chickens. Then we demonstrated that circZNF609 inhibited myoblast proliferation while promoting myogenic differentiation. Furthermore, we revealed a novel circ-ZNF609-encoded protein circZNF609-232aa that was responsible for the downregulation of myoblast proliferation and upregulation of differentiation. The translation of circ-ZNF609 was induced by its IRES-like regions. Our study not only presented a critical role of circ-ZNF609 in the regulation of myogenesis, but also identified a novel protein circZNF609-232aa which exerted its key effects, contributing to the understanding of complex skeletal muscle development.

Keywords: Chicken, CircZNF609, CircRNA-encoded proteins, Myoblasts, Proliferation and differentiation

Introduction

Skeletal muscle consists of multinucleated myofibers formed through the myogenesis, which is a highly orchestrated process composed of the proliferation, differentiation of mononucleated myoblasts, as well as their fusion with existing myofibers to augment the pool of myonuclei (Chal and Pourquié, 2017; Yin et al., 2013). Proliferation and differentiation of myoblasts to produce multinucleated myofibers are crucial for the muscle development, postnatal growth and repair. These highly dynamic and sophisticated processes involve coordination among cell cycle, muscle-specific transcriptional program and cell fusion, which are regulated by multiple myogenic regulatory factors (Biressi et al., 2007).

Non-coding RNAs, such as long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs), are emerging as important regulators of myoblasts development and skeletal muscle formation (Das et al., 2020; Huang et al., 2024). Circular RNAs are endogenous non-coding RNAs covalently closed by a non-canonical splicing and single-stranded RNA molecules (Das et al., 2020). Nowadays, owing to the advent of high-throughput RNA sequencing and specific bioinformatics tools, thousands of circRNAs have been identified (Vo et al., 2019). Mounting evidence suggests that circRNAs can act directly to influence the proliferation and differentiation of myoblasts (Wang et al., 2023; Yan et al., 2020), or they can regulate the skeletal muscle-specific gene expression via serving as miRNA sponges, interfering with RNA stability and translation (Li et al., 2023; Wang et al., 2019a). Most circular RNAs (circRNAs) are derived from exons and predominantly localized in the cytoplasm. Additionally, the presence of internal ribosome entry site (IRES) structures within circRNAs suggests their potential for translation into proteins (Chen et al., 2020). Currently, studies indicate that circRNAs can be translated into polypeptides in a splicing-dependent and cap-independent manner to control myoblast proliferation and differentiation (Lin et al., 2024; Shi et al., 2022). Despite these findings, the dynamic expression and polypeptides translation of circular RNAs during skeletal muscle development remains largely unexplored, and their roles in myogenesis and muscle development necessitate further investigation.

The chicken ZNF609 (Zinc Finger Protein 609, NM_001397242.1) located on chromosome 10 with 9 exons, is a member of the zinc finger proteins (ZNFs) family. Zinc finger proteins are abundant in eukaryotic cells and serve as versatile regulators in numerous biological processes, including cell proliferation, differentiation and metabolism (Jen and Wang, 2016). Several zinc finger proteins play critical roles in the regulation of skeletal muscle development. For instance, Zfp593 was found to be expressed in differentiated myotubes and repressed the myoblasts differentiation via attenuation of ERK1/2 and p38 phosphorylation (Lynch et al., 2019), knockout of Zfp422 in skeletal muscle impaired the formation of embryonic muscle (Nie et al., 2020). Moreover, muscle cell differentiation was hindered with interference of zinc finger E-box-binding homeobox 1 (ZEB1) in mouse skeletal myoblast (C2C12) (Jia et al., 2024), indicating a critical role of zinc finger proteins family in myoblasts myogenesis.

Analyze of circRNA sequencing data in chicken from our previous work revealed a circRNA originating from the ZNF609 gene, circZNF609. Chicken circZNF609 is a covalently closed circular RNA, which originates from the primary transcript of exon1 of ZNF609 and contains 867 bp nucleotides. CircZNF609 (circ-ZNF609) is typically formed by back-splicing, in which a downstream splice donor site is joined to an upstream splice acceptor site, resulting in a loop structure that contains a specific junction site (Wang et al., 2022). In hepatocellular carcinoma, circ-ZNF609 elevated the expression of GLI2 to promote its proliferation and metastasis (He et al., 2020). CircZNF609 was also found to abnormally upregulate in multiple tumor tissues and cell lines, promoting the tumor proliferation, migration and invasion (Wang et al., 2022). However, in murine myoblasts, knockdown of circZNF609 significantly reduced the proliferation rate of myoblasts (Legnini et al., 2017). Additionally, recent investigations showed that circZNF609 contains open reading frame in common with the linear transcript, and it is translated into a peptide in a splicing-dependent and cap-independent manner (Sun et al., 2023). Since circRNAs are always of high stability and evolutionarily conserved (Liu and Chen, 2022), circZNF609 might also perform an important role in chicken skeletal muscle development via translating into a peptide.

In this study, we found that chicken circZNF609 was upregulated in thigh muscles with the chickens growth. Functional investigations showed that overexpression of circZNF609 inhibited myoblast proliferation while promoting myogenic differentiation. Furthermore, we identified a novel protein circZNF609-232aa encoded by circZNF609 via the IRES-like regions induction that was responsible for the regulation of myogenesis. In summary, this study suggests that circZNF609 functions as a critical regulator in skeletal myogenesis, providing new insights into the development of chicken skeletal muscle development.

Materials and methods

Animals and cells

For the collection of chicken thigh muscle and pectoral muscle tissues, hatching eggs for 1 to 12-week-old Yuexi frizzled feather chicken and dwarf chicken were provided by the chicken farm of Guangdong Ocean University. All chickens were obtained from a single hatch and raised indoors. All animal studies complied with the guidelines of “The Instructive Notions with Respect to Caring for Laboratory Animals” issued by the Ministry of Science and were approved by the Animal Protection Council of Guangdong Ocean University (SYXK-2021-0154).

Chicken primary myoblasts were isolated from the leg muscle of 10-day-old chick embryo as previous described (Guo et al., 2024). Cells were cultured in growth medium (GM) containing RPMI-1640 (Gibco, C11875500BT, Grand Island, NY, USA) with high glucose, 15 % fetal bovine serum (FBS, Gibco, A3160802, USA), 100 U/mL penicillin and 100 μg/mL streptomycin (final concentration, Gibco, 15140122, USA) at 37°C with 100 % humidity under 5 % CO2. Upon reaching 90 % confluence, cells were induced to differentiate for 48 h using the differentiation medium (DM) containing RPMI-1640 with high glucose, no fetal bovine serum, 2 % horse serum (Gibco, 26050088, USA), 100 U/mL penicillin and 100 μg/mL streptomycin.

Plasmid construction and cell transfection

CircRNA overexpression vectors: pCD2.1-circZNF609 for circZNF609 overexpression, pCD2.1-circZNF609-flag for verifying the encoding capacity of circZNF609, pCD2.1-HA-circZNF609-232aa-flag for validation of the protein encoded by circZNF609. The full length sequences of circZNF609 and circZNF609-232aa were amplified from chicken primary myoblasts by PCR (Supplementary file 1: Table S1). The PCR products were isolated from agarose gel and cloned into the pCD2.1-ciR vector (Jisai, GS0102, Guangzhou, China). The successful overexpression plasmid was confirmed by DNA sequencing.

Linear transcript overexpression vectors: pCMV-circZNF609-232aa for circZNF609-232aa overexpression, pCMV-circZNF609-232aa-MT for circZNF609-232aa overexpression with initiation codon mutation. The full length sequences of circZNF609-232aa and circZNF609-232aa with initiation codon mutation were amplified from chicken primary myoblasts by PCR (Supplementary file 1: Table S1). The PCR products were isolated from agarose gel and cloned into the pCMV-Myc vector (Huayueyang, VECT6183, Beijing, China). The successful overexpression plasmid was confirmed by DNA sequencing.

IRES-like region validation reporter vectors: IRES-like fragment 1, 2 or its combinations of circZNF609 were amplified from chicken primary myoblasts by PCR (Supplementary file 1: Table S1). The PCR products were isolated from agarose gel and cloned into the Luc2-IRES-Report vector (Jisai, Guangzhou, China) and pCD2.1-ciR-GFP vector with mCherry and Flag tags inserted. The successful overexpression plasmid was confirmed by DNA sequencing.

Cell transfection was carried out using Lipofectamine 3000 reagent (Invitrogen, L3000015, MA, USA) according to the manufacturer’s protocols. Lipofectamine 3000 reagents and nucleic acids were diluted in OPTI-MEM with Reduced Serum Medium (Gibco, 31985070, MA, USA).

RNA extraction, cDNA synthesis and quantitative real-time PCR

Total RNA was extracted from cells and tissues using HiPure Total RNA Kit (R4130-03, Magen Biotechnology Co, Ltd, Guangzhou, China) according to the manufacturer's protocol. Total RNA was reverse-transcribed into cDNA using the HiScript III RT SuperMix for qRT-PCR (+gDNA wiper) (R323-01, Vazyme Biotech Co., Ltd., Nanjing, China) according to the manufacturer's protocol. Quantitative real-time PCR assays were conducted on CFX Connect Real-Time System (BIO-RAD, Singapore) using ChamQ Universal SYBR qPCR Master Mix (Q711-02, Vazyme Biotech Co., Ltd, Nanjing, China), with all samples run in triplicate. Nuclear and cytoplasmic RNA extractions were conducted using the purification kit (21000, Norgenbiotek, Canada) according to the manufacturer's protocol. qPCR primers were listed in Supplementary file 1: Table S2 and Table S3. Chicken U6 and GAPDH were used as nuclear internal control and cytoplasmic internal control in nuclear and cytoplasmic separation experiments. Chicken β-actin were used as internal control in normal qPCR, and the relative expression level was calculated using the 2-△△Ct relative quantitative method, as previously described (Kenneth and Thomas, 2001).

Validation of circRNA

Back-splicing junction and full length of circZNF609 were amplified and validated by sanger sequencing (Supplementary file 1: Table S1). To verify the basic properties of circZNF609, total RNA was incubated with or without RNase R (R0301, Geneseed, China), followed by synthesis of cDNA utilizing the PrimeScript RT reagent kit (RR047Q, TaKaRa, Tokyo, Japan) and qRT-PCR analysis (Supplementary file 1: Table S2).

Immunofluorescence

For immunofluorescence, cells were fixed in 4 % PFA (P0099, Beyotime, Shanghai, China) for 30 min at RT and then washed three times for 5 min per wash with PBS. Cells were then permeabilized with 0.2 % Triton X-100 for 10 min and blocked in goat serum for 1 h. After blocking, the cells were incubated with anti- MyHC (B103, DSHB, USA) overnight at 4°C. After incubating with the secondary antibody conjugated to FITC for 1 h at RT, the cells were incubated with DAPI (E607303-0020, Beyotime, China) for 10 min at RT. Fluorescence was visualized using the FSX100 fluorescence microscope (6E07981,OLYMPUS, Tokyo, Japan).

Western blot

Total protein from cultured myotubes was extracted using RIPA lysis buffer (P0013, Beyotime, China) with PMSF protease inhibitor (ST506, Beyotime, China), after incubation for 15 min on ice and centrifugation at 13,000× g for 10 min at 4°C, the supernatant was collected. After measuring the protein concentrations, the same amount of protein from each sample was separated by SDS-PAGE and then transferred to polyvinylidene fluoride membranes (ISEQ00010, Millipore, China). The membranes were blocked in TBS containing 5 % (w/v) skimmed milk powder at RT for 1 h. Subsequently, the membranes were incubated against anti-PCNA (AF0261, Beyotime, China), anti-CCND1 (TA6234, Abmart, China), anti-CDK2 (AF1063, Beyotime, China), anti-MYHC (B103, DSHB), anti-MYOD (Abbkine, ABP53067, China) and anti-β-actin (AF003, Beyotime, China), at 4°C overnight. Blots were developed using Femto Light Chemiluminescence Kit (SQ201, EpiZyme, China), and detection was performed using the Chemiluminescence detection system (1600, Tanon, China).

CCK-8 assay

Cells were seeded into 96-well plates and transfected with different vectors when the cell density reaching 80 %. The proliferation of cells were assessed by the Cell Counting Kit-8 (GlpBio, CA, USA) every 12 h according to the manufacturer's protocol, and the absorbance was measured at 450 nm using a Multiskan FC microplate photometer (1425955, ThermoFisher, USA).

EDU assay and flow cytometry

Cell-Light EdU Apollo In Vitro Kit (C10310, RiboBio, Guangzhou, China) was used for Edu assay according to the manufacturer’s protocols. Cell nuclei were stained by DAPI. EdU-stained cells were visualized under the FSX100 fluorescence microscope (6E07981,OLYMPUS, Tokyo, Japan). Flow cytometry assays were performed using PI/RNase Staining Solution (SL7092, Coolaber, Beijing, China) according to the manufacturer’s protocols and analyzed by BD Accuri C6 flow cytometer (BD Biosciences, San Jose, CA, USA).

Dual-luciferase reporter assay

For IRES-like region validation, the DF-1 cells were transfected with a series of the reporter plasmids described above. At 48 h after transfection, the luciferase activities and Renilla luciferase of the cells were measured using the Dual-Glo® Luciferase Assay System (Promega, USA) and Synergy™ Neo 2 Multi-Mode Microplate Reader (Biotek, Winooski, VT, USA) with Gen5 software (Biotek, Winooski, VT, USA). The levels of firefly luciferase activity were normalized to Renilla luciferase activity.

Fluorescence in situ hybridization assay

Cy3-labeled circZNF609 probes were designed and synthesized by GenePharma (Shanghai, China). Hybridization was performed overnight with circZNF609 probes according to the manufacturer’s instructions. The images were acquired on Zeiss LSM710 Laser Scanning Confocal Microscope (Zeiss Instrument Inc., Germany). The sequences of circZNF609 probe for FISH were listed in Supplementary file 1: Table S4.

Mass spectrometry identification

Protein bands from the coomassie brilliant blue staining gels corresponding to the molecular weight of circZNF609 were excised for in-gel trypsin digestion and MS (Mass spectrum) analysis. Selected bands were sent to MS facility (PTM-biolab, Hangzhou, China) for target identification using LC-MS/MS on Thermo Q Exactive HF or Thermo Orbitrap-Velos Pro. Mascot distiller (version 2.6, Matrix Science, UK) was used to convert raw data to mgf or mzML format for downstream analysis (confidence ≥ 95 % and Unique peptides ≥ 1 as the peptide search condition).

Statistical analysis

Statistical analyses were performed on SPSS 19.0 analytics package (SPSS, Inc., Chicago, IL, USA) using the two-tailed Student's t-test for comparing two groups, or the one-way analysis of variance with Tukey's multiple comparison test for comparing among multiple groups. All data were presented as mean ± standard error mean (SEM). Probability of < 5 % (p < 0.05) was considered statistically significant, * p < 0.05; ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Results

The characteristics of CircZNF609

Analysis of circRNA sequencing data in chicken (GenBank accession number: PRJNA554754) from our previous work (Jiao et al., 2024), revealing a circRNA (circZNF609) originating from the primary transcript of exon1 and its upstream of ZNF609 (Fig. 1A,B). Before the functional study of circZNF609, we primarily testified the characteristics of the circZNF609. After examined by PCR and sanger sequencing, the circZNF609 was verified to be formed by the reverse splicing of the 5′ upstream of exon 1 and the 3′ end of exon 1 in ZNF609 gene, with a full length of 867 nt (Fig. 1C,D). Total RNA was reverse-transcribed into cDNA using oligo d(T)18 or random primers, and qPCR results showed that the expression levels of circZNF609 using oligo d(T)18 was significantly lower than that by random primers (Fig. 1E). While the expression levels of ZNF609 linear transcript showed no significant difference between the oligo d(T)18 group and the random primers group (Fig. 1E), suggesting that circZNF609 was a circular RNA with a head-to-tail junction and the absence of a poly(A) tail. Comparing to the linear transcript of ZNF609, the resistance to digestion by RNase R exonuclease further verified the circular RNA structure of circZNF609 (Fig. 1F).

Fig. 1.

Fig 1

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Characterization of the existence and subcellular distribution of chicken circZNF609. (A) Schematic illustration of the genomic localization and splicing pattern of circZNF609. (B) Schematic diagram of the formation of circRNA609. (C) Sanger sequencing results of the head-to-tail junction in circZNF609. (D) RT-PCR revealed the full length of circRNA609. (E) The relative expression of circZNF609 and ZNF609 in chicken liver was detected by qPCR under the reverse-transcription using oligo d(T)18 or random primers. (F) The relative expression of circZNF609 and ZNF609 in chicken liver was detected by qPCR in the presence or absence of RNase R. (G) FISH assay was used to detect the expression of circZNF609 in the nuclear and cytoplasm of DF-1. (H) Nuclear and cytoplasmic separation and qPCR were used to detect the expression of circZNF609 in the nuclear and cytoplasm of DF-1. (I) The relative expression levels of circZNF609 in thigh muscle and pectoral muscle of 1 week chicken were quantified by qPCR. (J) The relative expression levels of ZNF609 in thigh muscle of different weeks of chicken were quantified by qPCR. (K) The relative expression levels of circZNF609 in thigh muscle of different weeks of chicken were quantified by qPCR. (L) The relative expression levels of circZNF609 in pectoral and thigh muscle of normal chicken and dwarf chicken were quantified by qPCR. All data represent three independent replications. In panels (E-F, H-I, L), data are presented as mean ± SEM, n = 3 per group, * p < 0.05; ** p < 0.01, *** p < 0.001. In panels (J, K), data are presented as mean ± SEM, n = 3 per group, different lowercase superscripts denote significant differences (p < 0.05).

We then evaluated the localization of circZNF609. FISH assay demonstrated that circZNF609 was present in both the nuclear and cytoplasm (Fig. 1G). Further investigation by nuclear and cytoplasmic separation experiments showed that circZNF609 was predominately distributed in the cytoplasm (Fig. 1H). In addition, qPCR results showed that the expression level of circZNF609 in thigh muscle was significantly higher than that in pectoral muscle (Fig. 1I). According to the qPCR results, the expression level of ZNF609 in the thigh muscles increased rapidly in the second week, while the expression levels in the third, fourth and fifth weeks were lower than that in the second week, and then began a second round of high expression in the sixth week. (Fig. 1J). And the expression level of circZNF609 in the thigh muscles also showed a similar trend (Fig. 1K), implying a comparable function between circZNF609 and ZNF609. Also, the expression level of circZNF609 is significantly higher in normal chickens than in dwarf chickens (Fig. 1L), indicating an important role of circZNF609 in muscle development.

Overexpression of CircZNF609 inhibits myoblasts proliferation and promotes myogenic differentiation

Given the significant upregulation of circZNF609 in thigh muscles with the growth of the chickens, and zinc finger proteins play critical roles in the regulation of skeletal muscle development, we further investigated the underlying function of circZNF609 on chicken primary myoblasts. To explore the impact of circZNF609 on the proliferation of myoblasts, we overexpressed circZNF609 in myoblasts. As expected, comparing to the control group, the expression level of circZNF609 was significantly upregulated in pCD2.1-circZNF609-transfected cells (Fig. 2A). qPCR results showed that overexpression of circZNF609 in myoblasts significantly decreased the expression of PCNA (Proliferating Cell Nuclear Antigen), CCND1 (Cyclin D1), CCNE1 (Cyclin E1) and CDK2 (Cyclin-Dependent Kinase 2), which regulate the myogenic cell proliferation (Fig. 2A). Similar to the qPCR results, western blotting also demonstrated decreased PCNA, CCND1 and CDK2 protein levels in circZNF609-overexpressing myoblasts compared to control (Fig. 2B).

Fig.2.

Fig2

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Chicken circZNF609 inhibits myoblasts proliferation. (A) The relative expression levels of myoblast proliferation marker genes in circZNF609-overexpressing myoblasts were quantified by qPCR. (B) The relative protein expression levels of PCNA, CCND1 and CDK2 in circZNF609-overexpressing myoblasts and control groups. The figures on the left were folds of band intensities relative to the control groups, and the band intensities were quantified by ImageJ. (C) Cell proliferation of circZNF609-overexpressing myoblasts was assessed by CCK-8 assay (mean ± SEM, n = 8 per group, * p < 0.05; ** p < 0.01, *** p < 0.001). (D) Cell proliferation of circZNF609-overexpressing myoblasts was assessed by EdU assay. (E) Cell cycle analysis of circZNF609-overexpressing myoblasts. All data represent three independent replications. In panels (A-B, d-E), data are presented as mean ± SEM, n = 3 per group, * p < 0.05; ** p < 0.01, *** p < 0.001, **** p < 0.0001.

To obtain a further validation, proliferation assay and cell cycle analysis were conducted. CCK-8 assay showed that the proliferation capacity of myoblasts was significantly decreased with the overexpression of circZNF609 comparing to the control (Fig. 2C). EdU assay also demonstrated that circZNF609 overexpression significantly downregulated the myoblasts proliferation (Fig. 2D). Flow cytometry analysis revealed that overexpression of circZNF609 significantly increased the cell population in G0/G1 phase and decreased the cell population in S phase and G2/M phase, leading to cell arrest in the G0/G1 phase (Fig. 2E).

After inducing myoblasts differentiation, the expression levels of the myoblast differentiation marker genes, MYHC and MYOD were significantly increased in circZNF609 overexpression myoblasts (Fig. 3A). Correspondingly, the protein expression levels of MYHC and MYOD were increased with circZNF609 overexpression (Fig. 3B). To further investigate, we performed immunofluorescence staining with MYHC antibody on myoblasts following differentiation, and overexpression of circZNF609 significantly improved the myotube formation (Fig. 3C). Collectively, these results indicated that chicken circZNF609 could inhibit the myoblasts proliferation and promote the myogenic differentiation.

Fig.3.

Fig3

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Chicken circZNF609 promotes myogenic differentiation. (A) The relative expression levels of myoblast differentiation marker genes in circZNF609-overexpressing myoblasts were quantified by qPCR. (B) The relative protein expression levels of MYHC and MYOD in circZNF609-overexpressing myoblasts and control groups. The figures on the left were folds of band intensities relative to the control groups, and the band intensities were quantified by ImageJ. (C) Differentiation index of myoblasts treated with overexpression of circZNF609 was assessed by immunofluorescence staining. Cells were differentiated for 48 h after transfection. All data represent three independent replications. In all panels, data are presented as mean ± SEM, n = 3 per group, * p < 0.05; ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Chicken Circ-ZNF609 encodes a novel protein

Emerging evidence suggested that circRNAs have the potential to encode proteins, which could be the participants in the regulation of myogenesis (Chen et al., 2023; You et al., 2025). Using the ORFfinder (Sayers et al., 2010), open reading frame (ORF) and relative amino acid sequence were predicted, revealing the potential of circZNF609 to encode a 232 amino acids peptide (circZNF609-232aa) with a molecular weight of 23.39 kDa (Fig. 4A). The predicted protein circZNF609-232aa shares 200 amino acids with ZNF609 protein (Fig. 4A). To confirm the encoding capacity of circZNF609, we constructed a overexpression plasmids of circZNF609 with a flag tag inserted upstream of the stop codon (pCD2.1-circZNF609-flag) (Fig. 4B), which could generate a flagged protein when the circular template is formed. After transfected pCD2.1-circZNF609-flag, circZNF609-232aa was observed via flag antibody with a molecular weight of about 24 kDa (Fig. 4C). To get on a further validation, the protein band of the putative molecular weight was collected for LC-MS/MS (Fig. 4D). LC-MS/MS analysis revealed three peptides that matched the amino acid sequence of circZNF609-232aa with flag tag inserted at the end of the sequence (Fig. 4E). Moreover, we further constructed a overexpression plasmids of circZNF609-232aa with a HA tag inserted upstream and a flag tag inserted downstream (pCD2.1-HA-circZNF609-232aa-flag) (Fig. 4B). Western blot using HA antibody, we confirmed the protein production of circZNF609 (Fig. 4F). Therefore, these results revealed that chicken circ-ZNF609 could encode a novel protein circZNF609-232aa.

Fig.4.

Fig4

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Validation of the protein encoded by chicken circZNF609. (A) Left: Schematic illustration of the sequence structure of circZNF609. Right: Amino acid sequence alignment of the proteins encoded by circZNF609-232aa and ZNF609. (B) Schematic illustration of the plasmid construction for pCD2.1-circZNF609-flag and pCD2.1-HA-circZNF609-232aa-flag. (C) Representative western blots of the proteins derived from pCD2.1-circZNF609-flag. (D) Total cell lysates of myoblasts transfected with pCD2.1-circZNF609-flag were separated by SDS-PAGE. Bands representing proteins between 26 kDa were excised manually and submitted for identification by LC-MS/MS. (E) LC-MS/MS-identified amino acid sequences of the circZNF609-232aa-flag (red, upper panels), the specific charge of corresponding peptide (lower panels). (F) Representative western blots of the proteins derived from pCD2.1-HA-circZNF609-232aa-flag.

Internal ribosome entry site (IRES)-like regions induce CircZNF609 translation

Due to the absence of cap structure in circRNAs, the translation of circRNA is expected to occur through sequences which could be serve as internal ribosome entry sites (IRESs) (Fan et al., 2022). To determine the protein coding mechanism underlying circZNF609, IRES-like region analysis of circZNF609 was predicted using IRESfinder (Zhao et al., 2018). According to the IRESfinder results, two highest score IRES-like fragments (+632 to 1r+9 for fragment1 and +82 to +276 for fragment2) were selected for further investigation (Supplementary file 1: Table S5). Dual-luciferase assays were performed on DF-1 cells transfected with Luc2-IRES-reporter vectors containing different IRES-like fragments or its combinations (Fig. 5A). The luciferase activities of Luc2-Fragment1-report (p < 0.05) and Luc2-Fragment2-report were upregulated compared to the control (Fig. 5B), implying that circZNF609 contains two IRES-like regions which could induce 5′ cap-independent translation. Furthermore, the Luc2-Fragment1-report exhibited a greater effect than Luc2-Fragment2-report (Fig. 5B), indicating that fragment1 (+632 to 1r+9) might be the essential IRES-like region to promote the translation of circZNF609. In addition, using the DNA Secondary Structure Predictor (https://www.vectorbuilder.cn/tool/dna-secondary-structure.html), preliminary secondary structure models for IRES-like fragments of circZNF609 were constructed (Fig. S1). The results showed that Fragment1 contained higher GC content and lower minimum free energy (MFE) than Fragment2 (Fig. S1), indicating a more stable structure which was consistent with its higher promotion induction effect.

Fig.5.

Fig5

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Translation of circZNF609 was driven in a IRES-dependent way. (A) Schematic diagram of the dual-luciferase reporter (Luc2-IRES-Report vector) containing different fragments of IRES-like region sequence in circZNF609, which was used for IRES verification. (B) Ratio of Firefly luciferase to Renilla luciferase activity of DF-1 cells after transfection with different Luc2-IRES-Report vectors shown in (A). Data are presented as mean ± SEM, n = 3 per group, * p < 0.05; ** p < 0.01. (C) Schematic diagram of the pCD2.1-ciR-GFP plasmids containing mCherry tag, flag tag and different fragments of IRES-like region sequence in circZNF609, which was used for IRES verification. (D) Representative western blots of the proteins in DF-1 cells after transfection with different pCD2.1-ciR-GFP plasmids shown in (C). (E) Immunofluorescence of DF-1 cells after transfection with different pCD2.1-ciR-GFP plasmids shown in (C).

To further elucidate the IRES potentials of these IRES-like fragments, pCD2.1-ciR-GFP plasmids with mCherry tag, flag tag and different IRES-like fragments inserted were constructed and transfected into DF-1 (Fig. 5C). Western blotting demonstrated the expected proteins in pCD2.1-Frag1-mCherry-Flag and pCD2.1-Frag2-mCherry-Flag (Fig. 5D). Moreover, immunofluorescence of mCherry also showed that the mCherry proteins were expressed under the regulation of IRES-like fragments of circZNF609 (Fig. 5E). Collectively, these results revealed that the translation of circZNF609 was induced by its IRES-like regions.

Chicken CircZNF609 inhibits the myoblasts proliferation and promotes the differentiation via translation into CircZNF609-232aa

To explore the effect of circZNF609-232aa on primary myoblasts, the pCMV-circZNF609-232aa linear overexpression vector were constructed and transfected into primary myoblasts (Fig. 6A). qPCR results showed that overexpression of circZNF609-232aa in myoblasts significantly decreased the expression of PCNA, CCND1, CDK2 and CCNE1, which regulate the myogenic cell proliferation (Fig. 6A). Western blotting also demonstrated decreased protein levels of PCNA, CCND1 and CDK2 in myoblasts overexpressing circZNF609-232aa compared to control (Fig. 6B). Proliferation assay and cell cycle analysis revealed that overexpression of circZNF609-232aa downregulated the proliferation of myoblasts (Fig. 6C,D).

Fig.6.

Fig6

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Chicken circZNF609-232aa protein inhibits the myoblasts proliferation. (A) The relative expression levels of myoblast proliferation marker genes in circZNF609-232aa-overexpressing myoblasts were quantified by qPCR. (B) The relative protein expression levels of PCNA, CCND1 and CDK2 in circZNF609-232aa-overexpressing myoblasts and control groups. The figures on the left were folds of band intensities relative to the control groups, and the band intensities were quantified by ImageJ. (C) Cell proliferation of circZNF609-232aa-overexpressing myoblasts was assessed by EdU assay. (D) Cell cycle analysis of circZNF609-232aa-overexpressing myoblasts. (E) The relative expression levels of circZNF609-232aa in myoblasts transfected with pCMV-circZNF609-232aa linear overexpression vector (pCMV-circZNF609-232aa) or circZNF609-232aa-promoter-mutant linear overexpression vector (pCMV-circZNF609-232aa-MT) were quantified by qPCR. (F) Western blotting assessed the protein production of myoblasts transfected with pCMV-circZNF609-232aa or pCMV-circZNF609-232aa-MT. (G) The relative expression levels of myoblast proliferation marker genes between myoblasts transfected with pCMV-circZNF609-232aa and pCMV-circZNF609-232aa-MT. (H) The relative protein expression levels of PCNA, CCND1 and CDK2 in myoblast proliferation marker genes between myoblasts transfected with pCMV-circZNF609-232aa and pCMV-circZNF609-232aa-MT. The figures on the left were folds of band intensities in pCMV-circZNF609-232aa relative to that in pCMV-circZNF609-232aa-MT groups, and the band intensities were quantified by ImageJ. (I) Cell proliferations of myoblasts transfected with pCMV-circZNF609-232aa or pCMV-circZNF609-232aa-MT were assessed by EdU assay. (J) Cell cycle analysis of myoblasts transfected with pCMV-circZNF609-232aa or pCMV-circZNF609-232aa-MT. All data represent three independent replications. In panels (A-D,G-J), data are presented as mean ± SEM, n = 3 per group, * p < 0.05; ** p < 0.01, *** p < 0.001, **** p < 0.0001. In panel (E), data are presented as mean ± SEM, n = 3 per group, different lowercase superscripts denote significant differences (p < 0.05).

To further verify that it is the circZNF609-232aa protein, rather than its linear transcript, that exerts its roles, the circZNF609-232aa-promoter-mutant linear overexpression vector (pCMV-circZNF609-232aa-MT) were constructed and transfected into myoblasts (Fig. 6E). Western blotting results showed that only pCMV-circZNF609-232aa could express circZNF609-232aa protein (Fig. 6F), indicating a successful promoter mutant of circZNF609-232aa in pCMV-circZNF609-232aa-MT. Comparing to pCMV-circZNF609-232aa-MT, qPCR results revealed that overexpression of pCMV-circZNF609-232aa significantly downregulated the expression of PCNA, CCND1, CDK2 and CCNE1 (Fig. 6G). Consistent with the qPCR results, western blotting also demonstrated reduced protein levels of PCNA, CCND1 and CDK2 in myoblasts transfected with pCMV-circZNF609-232aa (Fig. 6H). EdU assay demonstrated that circZNF609-232aa overexpression significantly hindered the myoblasts proliferation compared to the overexpression of circZNF609-232aa-MT (Fig. 6I). Comparing to pCMV-circZNF609-232aa-MT, flow cytometry analysis revealed that overexpression of circZNF609-232aa significantly increased the cell population in G0/G1 phase and decreased the cell population in S phase and G2/M phase (Fig. 6J).

After inducing myoblasts differentiation, the expression levels of the myoblast differentiation marker genes, MYHC and MYOD were significantly elevated in circZNF609-232aa overexpression myoblasts (Fig. 7A). Correspondingly, the protein expression levels of MYHC and MYOD were increased with circZNF609-232aa overexpression (Fig. 7B). Immunofluorescence staining with MYHC antibody on myoblasts following differentiation showed that the overexpression of circZNF609-232aa significantly promoted the formation of myotube (Fig. 7C).

Fig.7.

Fig7

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Chicken circZNF609-232aa protein promotes the myoblasts differentiation. (A) The relative expression levels of myoblast differentiation marker genes in myoblasts transfected with pCMV-circZNF609-232aa after inducing differentiation were quantified by qPCR. (B) The relative protein expression levels of MYHC and MYOD in circZNF609-232aa-overexpressing myoblasts and control groups after inducing differentiation. The figures on the left were folds of band intensities relative to the control groups, and the band intensities were quantified by ImageJ. (C) Differentiation index of myoblasts treated with overexpression of circZNF609-232aa was assessed by immunofluorescence staining. Cells were differentiated for 48 h after transfection. (D) The relative expression levels of myoblast differentiation marker genes in myoblasts transfected with pCMV-circZNF609-232aa and pCMV-circZNF609-232aa-MT after inducing differentiation were quantified by qPCR. (E) The relative protein expression levels of MYHC and MYOD in myoblasts transfected with pCMV-circZNF609-232aa and pCMV-circZNF609-232aa-MT after inducing differentiation. The figures on the left were folds of band intensities in pCMV-circZNF609-232aa relative to that in pCMV-circZNF609-232aa-MT groups, and the band intensities were quantified by ImageJ. (F) Differentiation indexes of myoblasts transfected with pCMV-circZNF609-232aa and pCMV-circZNF609-232aa-MT were assessed by immunofluorescence staining. Cells were differentiated for 48 h after transfection. All data represent three independent replications. In all panels, data are presented as mean ± SEM, n = 3 per group, * p < 0.05; ** p < 0.01.

Furthermore, transfecting the wild type and pCMV-circZNF609-232aa-MT into the myoblasts, qPCR results revealed that overexpression of circZNF609-232aa significantly upregulated the expression of MYHC and MYOD comparing to overexpression of pCMV-circZNF609-232aa-MT (Fig. 7D). The protein expression levels of MYHC and MYOD were also increased in myoblasts transfected with pCMV-circZNF609-232aa comparing to pCMV-circZNF609-232aa-MT (Fig. 7E). Further investigation by immunofluorescence staining with MYHC antibody demonstrated the increased myotube formation with the transfection of pCMV-circZNF609-232aa comparing to pCMV-circZNF609-232aa-MT (Fig. 7F). Taken together, these results indicated that chicken circZNF609 inhibited the myoblasts proliferation and promoted the myogenic differentiation via translation into a novel protein circZNF609-232aa driven by IRES-like regions.

Discussion

The regulation processes through myogenic proliferation and differentiation were extraordinarily complex, involving coordination among cell cycle, muscle-specific transcriptional program, cell elongation, and fusion (Zhang et al., 2024b, 2022). A thorough understanding of the molecular basis of skeletal myogenesis is important for the skeletal muscle-related molecular breeding and development of poultry industry. Besides the well-established roles of transcription factors in myogenic regulations, accumulating researches suggested that circRNAs are novel regulators during myogenesis and involving in muscle-related diseases (Huang and Choo, 2025). Analysis of circRNA sequencing data in chicken from our previous work (Jiao et al., 2024), revealing a circRNA (circZNF609) originating from the primary transcript of exon1 of ZNF609.

It was reported that BTB-zinc-finger protein served as a muscle-specific transcriptional regulator, and its mutant in muscles resulted in uncontrolled contractile forces causing muscle rupture and degeneration during development (Zhang et al., 2024a). Knockdown of zinc finger-containing transcription factor KLF4 promoted myoblast proliferation and inhibited myoblast fusion, which impaired the muscle formation further affecting physical activity and skeletal muscle regeneration (Cai et al., 2023). Therefore, zinc finger protein plays an important role in myogenesis. With regard to the circRNA derived from zinc finger protein ZNF609, circZNF609 has been identified as a promoter of myoblast proliferation in murine (Legnini et al., 2017). The expression of myogenic transcription factors negatively correlates with circZNF609 in C2C12 myoblast cells, with circZNF609 acting as a decoy for miR-194-5p to promote the BCLAF1 expression, thereby suppressing the differentiation of myoblasts (Wang et al., 2019b). Here, we found that the expression level of ZNF609 in the thigh muscles gradually increased with the chickens growth, and the expression level of circZNF609 also showed a similar trend. Given the high conservation of circRNAs across species (Arcinas et al., 2019), and the similar expression trend between ZNF609 and circZNF609, it is highly plausible that chicken circZNF609 also plays a critical role in muscle development. This study aimed to reveal the function of circZNF609 in chicken skeletal muscle development. Here, we found that overexpression of circZNF609 could inhibit myoblasts proliferation and promote myogenic differentiation, suggesting that circZNF609 may have different effects on myoblast proliferation in mammals and chickens.

The majority of circRNAs have been found to localize in the cytoplasm (Han et al., 2018), which is consistent with our results. Most circRNAs are derived from exons and predominantly localized in the cytoplasm, implying their potential capability for translation into proteins. Emerging evidence has demonstrated their capacity to encode peptides, which also have relevant biological functions (Kong et al., 2020), offering a novel perspective for investigating the functional roles of circRNAs in skeletal muscle development. For instance, it was found that circRBBP7 encodes a 13 kDa protein to promote the proliferation, differentiation of myoblasts and regeneration of myofibers in mice (Yang et al., 2024). The circNEB-peptide, which is consistent with our findings to be localized in the nucleus and cytoplasm, has been found to promote myoblasts proliferation and differentiation in vitro, and induce muscle regeneration in vivo (Huang et al., 2024). In this study, our sequence analysis showed that the ORF length of circZNF609 is 699 bp and the rolling loop is translated into a protein with 232 amino acids. Further identification of the circZNF609-233aa peptide by mass spectrometry demonstrated the authenticity of circZNF609 encoding.

Currently, several translation mechanisms of circRNAs have been identified, including rolling circle amplification translation, translation mediated by UTR translation activation elements, and translation mediated by m6A modification (Abe et al., 2015; Di Timoteo et al., 2020; Komar et al., 2012; Shi et al., 2020). Especially, the average frequencies of the IRES-like elements were similar to random elements in the linear mRNAs, while the average frequencies of the IRES-like elements in circRNAs were significantly higher compared to the linear mRNAs (Fan et al., 2022; Gao et al., 2016), indicating that endogenous circRNAs are enriched with short IRES-like elements to be translated. Given that a sequence of length N can be fragmented into N-5 consecutive hexamers, and considering that over 99 % of circRNAs exceed 100 nt in length (Glažar et al., 2014), the majority of circRNAs are likely to harbor internal IRES-like short elements by chance (Fan et al., 2022). Consequently, nearly all open reading frames (ORFs) within circRNAs possess the potential to be translated through IRES-like short elements (Fan et al., 2022). In the present study, the results from dual-luciferase assays, western blotting and immunofluorescence staining indicated that chicken circZNF609 was translated in an IRES-like regions-dependent way. And we further revealed that circZNF609 was translated into a novel protein circZNF609-232aa to exert its regulatory effect in myogenesis. Collectively, these findings illustrated that circZNF609 encodes a novel protein circZNF609-233aa which exerts the downregulation role of myoblast proliferation and upregulation role of differentiation.

Conclusion

In summary, we revealed that cricZNF609 is a critical regulator of myogenesis, which is of benefit to the understanding of skeletal muscle regulation mechanisms and molecular breeding in poultry industry. In this study, we demonstrated that cricZNF609 contributed to the myoblasts development and contained a 699 bp open reading frame. Subsequent mechanistic exploration revealed that this open reading frame could be translated into a novel 232 amino acids peptide induced by IRES-like regions. And we found that circZNF609-232aa inhibited proliferation and promoted myogenic differentiation in chicken primary myoblasts. The present findings suggest that circZNF609 is a critical regulatory factor in myogenesis and exert its regulatory effect via translation into circZNF609-232aa (Fig. 8). Nevertheless, although the role and mechanism of circZNF609 in promoting myogenesis is established in this study, its application in poultry industry still has a long way to go. Our findings expand the current understanding of circRNAs and highlight significant role of the coding potential in circRNAs. The circZNF609 and its derived protein may provide novel promising targets for the molecular breeding in poultry industry.

Fig.8.

Fig8

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Model of chicken circZNF609 encodes a protein that inhibits the proliferation and promotes the differentiation of myoblasts.

Availability of data and materials

All data generated or analyzed during this study are included in this published article/Supplementary Materials.

Declaration of generative AI and AI-assisted technologies in the writing process

During the preparation of this work the authors did not use any Al and Al-assisted technologies

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This study was supported by the Key Project of Guangdong-Guangxi Joint Fund of the Natural Science Foundation of Guangdong Province (2020B1515420008), the National Natural Science Foundation of China (32402741) and the Guangdong Province Basic and Applied Basic Research Regional Joint Fund-Youth Fund Project (2023A1515110023).

Footnotes

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.psj.2025.105339.

Appendix. Supplementary materials

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mmc2.docx (17.5KB, docx)

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Supplementary Materials

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Data Availability Statement

All data generated or analyzed during this study are included in this published article/Supplementary Materials.

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