A Circular RNA Generated from Nebulin (NEB) Gene Splicing Promotes Skeletal Muscle Myogenesis in Cattle as Detected by a Multi-Omics Approach

Abstract

Cattle and the draught force provided by its skeletal muscle have been integral to agro-ecosystems of agricultural civilization for millennia. However, relatively little is known about the cattle muscle functional genomics (including protein coding genes, non-coding RNA, etc.). Circular RNAs (circRNAs), as a new class of non-coding RNAs, can be effectively translated into detectable peptides, which enlightened us on the importance of circRNAs in cattle muscle physiology function. Here, RNA-seq, Ribosome profiling (Ribo-seq), and peptidome data are integrated from cattle skeletal muscle, and detected five encoded peptides from circRNAs. It is further identified and functionally characterize a 907-amino acids muscle-specific peptide that is named circNEB-peptide because derived by the splicing of Nebulin (NEB) gene. This peptide localizes to the nucleus and cytoplasm and directly interacts with SKP1 and TPM1, key factors regulating physiological activities of myoblasts, via ubiquitination and myoblast fusion, respectively. The circNEB-peptide is found to promote myoblasts proliferation and differentiation in vitro, and induce muscle regeneration in vivo. These findings suggest circNEB-peptide is an important regulator of skeletal muscle regeneration and underscore the possibility that more encoding polypeptides derived by RNAs currently annotated as non-coding exist.

Keywords: Cattle; Ribo-seq; SCF complex; circNEB; myogenesis; skeletal muscle.

Figure 1

Transcriptome and translatome identification of translatable transcripts, qPCR validation, and bos‐circNEB encoded polypeptide detection. A) Annotation, classification, and distribution ratio of circRNA sequences. B) Library construction and sequencing workflow of Ribo‐seq. C) Total RNA after RNase R digestion: five candidate coding circRNAs in cattle muscle were analyzed by qPCR. As the linear control mRNA, the expression of actin decreased significantly after digestion (n = 3). D) Digestion of the linear transcript, circRNAs were translated in vitro and bos‐circNEB polypeptide was detected by western blotting. E) Both cattle muscle tissue and fetal cattle myoblast expressed bos‐circNEB peptide as detected by western blotting. The band size of the bos‐circNEB polypeptide was 99 kDa. F) Immunofluorescence were used to detect the expression of bos‐circNEB encoded peptides during the proliferation and 5 days after differentiation of myoblasts (n = 6). Data are represented as the mean ± SEM and analyzed by Student's t‐test. ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001 for groups connected by horizontal lines. p‐values < 0.05 were considered statistically significant.

Figure 2

CircNEB promotes the proliferation of cattle fetal myoblasts. A) The proliferation of myoblasts after over‐expression of bos‐circNEB was measured by CCK8 assay. The cell proliferation rate of circNEB group was significantly higher than that of pCD2.1 control group (n = 6). B) EdU experiments detected the efficiency of myoblast proliferation. Hoechst‐stained nuclei are blue, EdU incorporated in replicated nuclei are red, and merged shows the proportion of replicating nuclei. Statistics of EdU data at 10× magnification, the percentage of red fluorescent nuclei within fields were counted. Each dot represents a different field of view (n = 10). C) Detection of bos‐circNEB over‐expression in cattle myoblasts by qPCR (n = 3). D) Gene expression of cattle myoblast in proliferation stage overexpressing bos‐circNEB detected by qPCR (n = 3). CDK2, CyclinD1 and PCNA transcripts were significantly increased. E) Expressions of CDK2, CyclinD1 and PCNA proteins in cattle myoblasts overexpressing bos‐circNEB were detected by Western blotting. F) Flow cytometry analysis of the effect of bos‐circNEB on the cell cycle of cattle myoblasts (n = 3). The ratio of S and G2/M phase were higher in the circNEB group than control group, and the proportion of cells in the proliferative phase increased. Data are represented as the mean ± SEM and analyzed by Student's t‐test. ^*, ^**, ^*** represent p < 0.05, < 0.01, < 0.001, respectively. p‐values < 0.05 were considered statistically significant.

Figure 3

CircNEB promotes the differentiation of cattle fetal myoblasts and myotube formation. A) The bright field pictures of myoblasts induced to differentiate for 5 days were taken under 10× and 4× microscope magnification, respectively. Myotubes from the circNEB group differentiated better than those from the pCD2.1 control group. B) The number of myotubes formed in 4× microscope fields were counted separately, and each dot in the figure represents the statistical number of a visual field (n = 20). After statistics, the myotubes in circNEB group were significantly increased than that of control group. C) Immunofluorescence staining of bos‐circNEB encoded peptide and DAPI to visualize myotubes and nuclei, respectively. D) Statistical results of cell number of myotube fusion (pCD2.1, n = 36; circNEB, n = 27). The average number of myotube fusion cells in the circNEB group was significantly higher than that in the pCD2.1 control group. E) qPCR was used to detect the expression of muscle differentiation genes in differentiated myoblasts. The transcriptional expressions of MyoG and Myhc were significantly up‐regulated (n = 3). F) The expression of muscle differentiation protein was detected by western blotting. MyoD, MyoG, and Myhc proteins were significantly up‐regulated. Data are represented as the mean ± SEM and analyzed by Student's t‐test. ns, ^**, ^*** represent p > 0.05, < 0.01, < 0.001, respectively. p‐values < 0.05 were considered statistically significant.

Figure 4

CircNEB promotes C2C12 cell proliferation and differentiation. A) Detection of overexpression efficiency of mus‐circNEB in C2C12 cells. CircNEB expression was significantly upregulated after transfection (n = 3). B) The expression of mus‐circNEB peptide was detected by western blotting. Overexpression of mus‐circNEB increased the expression of circNEB peptide. C) C2C12 cells were induced to differentiate for 1, 3, and 5 days of myotube differentiation after plasmid transfection (n = 3). D) The expression of proliferation‐related genes in C2C12 cells was detected by qPCR. Results showed that the expression of CDK2 and CyclinD1 were significantly up‐regulated (n = 3). E) The expression of cell proliferation‐related proteins were detected by western blotting, and the expressions of CDK2, CyclinD1, and PCNA were up‐regulated. F) The expression of C2C12 differentiation‐related genes were detected by qPCR assay (n = 3). G) Western blotting assay of differentiation related proteins in C2C12 cells. Data are represented as the mean ± SEM and analyzed by Student's t‐test. ^*, ^** represent p < 0.05, < 0.01, respectively. p‐values < 0.05 were considered statistically significant.

Figure 5

Co‐IP analysis of the circNEB peptide interacting proteins, cell fluorescence co‐localization and detection of myocyte protein ubiquitination. A) Mass spectrometry analysis of Co‐IP proteins, bos‐circNEB peptide specifically pulled down 20 proteins. The quenched group was the negative control and the NC group was the blank control. B) Co‐IP pulled down proteins were separated by SDS‐PAGE and revealed differential proteins at 19, 23, 43, and 51 kDa that were identified by mass spectrometry as SKP1, MYL6B, TPM1, and NDUFV1. C) In the figure, green is the fusion expression of EGFP‐circNEB peptide, red is the signal of Cy3 secondary antibody binding, and blue is the nucleus stained by hoechest. The co‐localization of SKP1, TPM1 and NDUFV1 proteins with EGFP‐circNEB peptide were detected. D) qPCR detected SKP1, p27, and p57 expression (n = 3). E) The protein expression of p27, p57, and SKP1 was assessed by Western blotting. Bos‐circNEB was transfected into mature myocytes and empty pCD2.1 vector was transfected into the control group. CHX and MG132 were added to the medium 24 h after transfection, with “‐” indicating no addition and “+” indicating the addition of additives. F) K11 ubiquitination modified western blotting detection. p27‐Kip1 (27 kDa) and p57‐Kip2 (57 kDa) bands were detected, respectively. G) The protein expression of p27, p57, cyclinD1, and CDK2 was assessed by Western blotting. SKP1 protein expression was manipulated in mature myocytes using siRNA and overexpression techniques, with si‐NC and pCDNA3.1 serving as control groups. MG132 was added to the medium 24 h after transfection, with “‐” indicating no addition and “+” indicating the addition of additives. Data are represented as the mean ± SEM and analyzed by Student's t‐test. ns, ^* represent p > 0.05, < 0.05, respectively. p‐values < 0.05 were considered statistically significant.

Figure 6

Mechanism of circNEB‐peptide regulating myoblasts. In the figure, circNEB was created by the reverse splicing of exons 67–70 of the NEB gene, circularized and transported into cytoplasm for rolling circle translation. The molecular weight of circNEB polypeptide was 99 kDa. The polypeptide was located in the nucleus and cytoplasm. On the one hand, the circNEB polypeptide in the nucleus promotes the formation of SCF ubiquitinase III, and SCF ubiquitinase modifies the ubiquitination of KIP complex (negative regulator of cell cycle) and degrades it through proteasome. The inhibition of KIP complex on CDK and cyclin was relieved, and then the proliferation of myoblasts was promoted. On the other hand, circNEB polypeptides localized in the cytoplasm was targeted to TPM1 in the myosin class 2 complex, which is an important structure of myofilaments and promotes myoblasts differentiation to functional muscle structures. In addition, the downstream PI3K‐Akt signal pathway was activated. The expression of PI3CA and Akt1 transcription was significantly up‐regulated, which jointly regulated the myogenic differentiation of myoblasts.

Figure 7

CircNEB promotes regeneration after mice tibialis anterior muscle injury. A) CTX were injected into the tibialis anterior muscle of mice to construct the muscle injury model. After 48 h of CTX injection, white deposits between muscles were visible. B) HE staining after sectioning showed that muscle fibers dissolved 48 h after CTX injection, accompanied by inflammatory cell infiltration. C) After CTX muscle injury, mus‐circNEB was overexpressed at the injury site. Staining of tibialis anterior muscle sections at 14th and 21st days, we could observe that the recovery phenomenon was evident in the circNEB group, while muscle damage were still visible in the pCD2.1 control group. D) The efficiency of mus‐circNEB overexpression in vivo was detected by qPCR (n = 5). E) HE staining were performed with analysis of muscle tissue sections, and muscle fiber recovery was continuously observed at 7, 14, 21, and 28 days. The circNEB group recovered faster than the control group, and the recovery effect was evident at 14 days. F) qPCR was performed to analyze the relative expression pattern of genes in tibialis anterior muscle 14 and 21 days after overexpression of mus‐circNEB, respectively (n = 5). The internal reference gene was actin and normalized with the gene expression of pCD2.1 control group as the reference. G) Muscle tissues were cryosectioned and analyzed by immunofluorescence. H) Western blotting analysis of differentially expressed proteins for 14 days after muscle injury repair (n = 3). Data are represented as the mean ± SEM and analyzed by Student's t‐test. ^*, ^** represent p < 0.05, < 0.01, respectively. p‐values < 0.05 were considered statistically significant.

Figure 8

CircNEB improves aging muscle fiber atrophy. A) Overexpression of pCD2.1 and bos‐circNEB in the left and right hind legs of 3.5‐year‐old rabbits, respectively. The blue arrow in the figure is the site of the biceps femoris injection, and the red arrow is the site of the quadriceps femoris injection. Two injections were made at multiple sites per site. B) Muscle fiber cross‐sections were observed by HE staining of muscles from the two groups at 10 days after injection, respectively. C) There was no significant difference in the number of muscle fibers per unit area between the two groups (n = 7). D) The total area of muscle fibers per unit area in circNEB group (n = 9) was significantly higher than that in control group (n = 11). E) The mean cross‐sectional area of muscle fibers in circNEB group (n = 7) was significantly higher than that in control group (n = 9). F) Immunofluorescence analysis of in situ expression of muscle tissue proteins. G) The expression of muscle development protein was detected by western blotting. H) Statistical analysis of grayscale values in Western blotting assay (n = 3). Data are represented as the mean ± SEM and analyzed by Student's t‐test. ns, ^*, ^** represent p > 0.05, < 0.05, < 0.01, respectively. p‐values < 0.05 were considered statistically significant.