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Guanine-rich RNA binding protein GRSF1 inhibits myoblast differentiation through repressing mitochondrial ROS production
Experimental Cell Research 381 (2019) 139–149
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Guanine-rich RNA binding protein GRSF1 inhibits myoblast differentiation through repressing mitochondrial ROS production
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Wenxin Yin1, Lin Yang1, Delin Kong, Yuzhe Nie, Yang Liang, Chun-Bo Teng∗,[email protected] College of Life Science, Northeast Forestry University, Harbin, China
A R T I C LE I N FO
A B S T R A C T
Keywords: GRSF1 Myoblast/ myo-differentiation Differentiation GPX4 ROS
Guanine-rich RNA sequence binding factor 1 (GRSF1) is a member of the RNA-binding protein (RBP) family. GRSF1 regulates RNA metabolism through RNA processing, transport and translation in the cytoplasm and mitochondria. However, its role in myogenesis has not been investigated. Here, we demonstrated that the expression of mitochondrial GRSF1 was negatively related to the differentiation of mouse skeletal myoblasts. Interference with GRSF1 promotes myogenesis both in vitro and in vivo without affecting MyoD expression or cell proliferation. Further studies illustrated that GRSF1 regulated myogenic differentiation through direct targeting of mitochondrial GPX4, a key regulator of the cellular redox status, leading to the modulation of ROS levels, which is important for myogenesis. Our findings underscore a critical function for GRSF1 during skeletal myogenesis linked to its regulation of muscle redox homeostasis.
1. Introduction Skeletal myogenesis depends on the myogenic differentiation process whereby satellite cell-derived mononuclear myoblasts exit the cell cycle and fuse into multinucleated myotubes/myofibers [1,2]. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are essential mediators of muscle differentiation [3], and they have long been associated with skeletal muscle physiology [4,5]. It has been found that NF-κB (nuclear factor kappa B) activation by PI3K (phosphoinositide-3kinase) stimulates the myogenic process by increasing the expression of inducible nitric oxide synthase (iNOS) [6–8], while the activated NFκB/iNOS pathway promotes muscle differentiation by increasing ROS levels mediated through NADPH (nicotinamide adenine dinucleotide phosphate)oxidase [9]. Further, mitochondrial ROS (mtROS) induces PTEN oxidation to stimulate the PI3K/AKT/mTOR signaling pathway and promote autophagy, which is required for muscle differentiation and regeneration [10]. Moreover, conditional mutants of the redoxstate regulator PITX2/3 have shown that moderate overproduction of ROS is required for satellite cells to exit the cell cycle and initiate differentiation through the redox activation of p38α MAP kinase, while high levels lead to senescence and regenerative failure in satellite cells [11,12]. Therefore, there must be a tightly controlled mechanism to regulate intracellular levels of ROS as well as antioxidant levels in skeletal muscle cells. Guanine-rich RNA sequence binding factor 1 (GRSF1) is a
∗ 1
ubiquitously occurring RNA-binding protein (RBP) and contains three quasi-RNA-recognition motifs (qRRM) [13–17]. GRSF1belongs to the heterogeneous nuclear ribonucleoprotein F/H (hnRNP F/H) family but differs from other members of this family [13]. hnRNP F/H proteins primarily function in the nucleus and participate in splicing regulation [18,19], while GRSF1 frequently functions outside the nucleus. In the cytoplasm, GRSF1 regulates translation of eukaryotic and viral mRNA transcripts mainly though binding to the 5′ untranslated region (5′ UTR) containing G-repeats [16,17,20–24]. In mitochondria, GRSF1 has been detected in dynamic mitochondrial RNA granules (MRGs) and regulates mitochondrial gene expression at the post-transcription level [25,26]. GRSF1 plays a role in cellular senescence by regulating mitochondrial homeostasis [27]. GRSF1 has high affinity to G-rich RNA sequences, and such G-rich elements are theoretically prone to form special four-stranded structures called G-quadruplexes (G4s) [28]. Several studies have highlighted the key role of GRSF1 in G4-mediated posttranscriptional control of nuclear-encoded RNAs and mitochondrial RNAs [29,30]. Although GRSF1 has not been linked to any specific human disease, it apparently plays a role in murine embryo development as a component downstream of the Wnt/beta-catenin signaling pathway [31]. GRSF1 knockdown in mouse embryos led to developmental retardation of the brain [23]. However, whether GRSF1 participates in the muscle differentiation is unknown. In the present investigation, we studied the role of GRSF1 in myogenesis using in vitro and in vivo models. We found that GRSF1 suppresses myoblast
Corresponding author. Hexing Road 26, Harbin, 150040, China. Wenxin Yin and Lin Yang contributed equally to this work.
https://doi.org/10.1016/j.yexcr.2019.05.004 Received 6 February 2019; Received in revised form 2 May 2019; Accepted 3 May 2019 Available online 11 May 2019 0014-4827/ © 2019 Elsevier Inc. All rights reserved.
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differentiation via post-transcriptional modification of GPX4, a key regulator of the cellular redox status. We further demonstrated that GRSF1 regulates myogenesis through modulating ROS levels and at least partly by promoting the expression of GPX4.
MYOG MYOD1 GAPDH
2. Materials and methods 2.1. Cell culture
2.4. Lentivirus package
The mouse myoblast C2C12 cell line (ATCC, Manassas, VA, USA) was maintained in growth medium (GM) which contained DMEM (Hyclone, Logan, UT) supplemented with 10% (v/v) fetal bovine serum (Gibco, Grand Island, NY, USA) and 2 mM GlutaMAX™ (Gibco, Grand Island, NY, USA) at 37 °C with a 5% CO2 concentration for two days. C2C12 cells were induced to differentiate by replacing the media with differentiation medium (DM) containing DMEM supplemented with 2% (v/v) horse serum (Gibco, Grand Island, NY, USA) and 2 mM GlutaMAX™. Complete differentiation was achieved after 3 days in DM.
The 293 T cells (ATCC, Manassas, VA, USA) were seeded in a culture flask at a density of 2 × 104 cells/cm2 in DMEM (Hyclone, Logan, UT) supplemented with 15% (v/v) fetal bovine serum (Gibco, Grand Island, NY, USA) and 2 mM GlutaMAX™ (Gibco, Grand Island, NY, USA) at 37 °C with the CO2 concentration 5%. 293 T cells were replaced in a serum-free medium when they fused at a density of approximately 70%–80%, and the cells were transfected with purified viral plasmids (SL4-TRIBE and LV3-shGRSF1 or corresponding control) for packaging in a Polyethylenimine-Based method. 293 T cells were added to fetal bovine serum 6–8 h later. Medium containing the virus was collected and centrifuged by ultracentrifugation after 72 h. Lentiviruses were dissolved in PBS and stored at −80 °C. shGRSF1-sense: 5′-AAA GCA CAG GGA AGAAAT TGG TA-3’. shGRSF1-antisense: 5′-TA CCA ATT TCT TCC CTG TGC TTT-3’. (Corresponding to bases 982-1004 of the murine Grsf1 gene, NCBI accession no.: NM_178700.5)
2.2. Transient transfection Plasmid vector pcDNA3.1 containing flag-tagged CDS of GRSF1 (NM_178700.5) or m-GPX4 (NM_008162.4) constructs were transfected into C2C12 cells at a density of approximately 30%–40% by Lipofectamine™ 2000 Transfection Reagent (Thermo Fisher Scientific) according to the manufacturer's instructions. The efficiency of transfection was monitored by detecting of each mRNA level using qPCR and immunoblot analysis. Empty plasmid vectors were used as a negative control. SiRNAs for GRSF1 or GPX4 were purchased from GenePharma. The siRNAs (50 nM/24-well dish) were transfected into C2C12 cells at a density of approximately 30%–40% by Lipofectamine™ RNAiMAX Transfection Reagent (Thermo Fisher Scientific) according to the manufacturer's instructions. The efficiency of knockdown was monitored by qPCR and immunoblot analysis. A non-silencing siRNA was as a control.
siGPX4#1 siGPX4#2 siGRSF1#1 siGRSF1#2
2.5. TRIBE method TRIBE method was based on the report before [32]. Briefly, C2C12 was infected with lentivirus expressing TRIBE-GRSF1 fusion protein or control in the presence of 8 μg/mL of Polybrene (Santa Cruz Biotechnology). 72 h later, they were treated with puromycin (5 μg/mL) (Sigma Aldrich) to select for cells stably expressing integrated vectors. The 5′UTR of m-GPX4 was PCR-amplified using cDNA obtained from pooled TRIBE-GRSF1 overexpressing or control populations, and cloned by pEASY-T5 Zero Cloning Kit (cat# CT501-01, Transgene Biotech, Beijing, China). Sequence was analyzed using at least six positive clones of each sample to evaluate the edit sites and the efficiency of edit events.
sense 5′- CCGGAGGAAGGUCCAGAGG -3′, antisense 5′- CCUCUGGACCUUCCUCCGG-3’; sense 5′- CTATCTCTAGCTAGCCCTA-3′, antisense 5′- TAGGGCTAGCTAGAGATAG-3’; sense 5′- AAAGCACAGGGAAGAAAUUGGUA-3′, antisense 5′- UACCAAUUUCUUCCCUGUGCUUU-3’; sense 5′- GCGGUAUGUGGAAGUGUAUGAAAUA-3′, antisense 5′- UAUUUCAUACACUUCCACAUACCGC-3’;
2.6. Muscle injury The protocol for animal use was approved by the Animal Care and Ethics Committee of Northeast Forestry University, and all the procedures were carried out in accordance with the approved guidelines. C57BL/6 female mice were purchased from the Second Affiliated Hospital of Harbin Medical University (Harbin, China) and were injured in the tibialis anterior muscles (TA) of both legs by injection with 50 μL of 1.5% (v/v) barium chloride (BaCl2) in PBS supplemented with 50 μL of lentivirus (shNC or shGRSF1) per leg. Before the procedure, the mice were anesthetized. The right TA muscles were injured with lentivirus expressing shGRSF1, and the left TA muscles were injured with lentivirus expressing shNC as a control. The injured muscles were dissected on day 0, day 1, day 3 and day 7 after injury. Immediately after isolation, the muscles were dissected for hematoxylin and eosin (H&E) staining. In each experiment, at least three animals were analyzed for each time point after injection.
2.3. Quantitative RT-PCR Total RNA was extracted using TRIzol™ Reagent (Thermo Fisher Scientific) according to the manufacturer's instructions. The mRNA levels were measured using a SYBR Green-based RT-PCR Master Mix (Cat# 04913914001, Roche). The thermal cycler conditions were as follows: 10 min at 95 °C followed by 40 cycles of 95 °C for 30 s and 60 °C for 30 s. Finally, the samples were held at 72 °C and melting curves were conducted from 72 °C to 95.1 °C. All tests were performed in triplicate and all experiments were repeated three times in a Light-Cycler 480 System (Roche, Basel, Switzerland). Expression data were normalized to the geometric mean of housekeeping gene GAPDH to control the variability in expression levels and were analyzed using the 2−ΔΔCT method. The primer sequences designed by Primer 5 software were as follows:
GRSF1 GPX4 MYH3
reverse, 5′- CACCTCTTTGATTTTGGCTTCC-3′ forward,5′- ACAATCTGCACTCCCTTACG-3′ reverse,5′- GTGATGGCTTTTGACACCAAC-3′ forward, 5′- CCAATGCGATTTATCAGGTGC-3′ reverse, 5′- CGAAAGGACAGTTGGGAAGAG-3′ forward, 5′-TGACCACAGTCCATGCCAT-3′ reverse, 5′-TTCTAGACGGCAGGTCAGGT-3′
2.7. Flow cytometry analysis The intracellular ROS level and mitochondrial superoxide were measured by 2′,7′-dichlorofluorescin diacetate (DCFH-DA; Beyotime Biotechnology, Nanjing, China) and MitoSOX (Invitrogen) staining using a flow cytometer (BD C6 Biosciences, San Jose, CA, USA), respectively. C2C12 cells were trypsinization and washed twice with PBS
forward, 5′-CCTAAATAGAGATGGGAAACGGAG-3′ reverse, 5′-AGGCATCCACATCTTCGTTG-3′ forward, 5′- GCAATGAGGCAAAACTGACG-3′ reverse, 5′- CTTGATTACTTCCTGGCTCCTG-3′ forward, 5′- AAGCTCGTCACTTTGGTACAG-3′
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Fig. 1. GRSF1 plays an important role in myoblast differentiation. (A) Upper: Schematic diagram of the differentiation process of mouse myoblasts through a time course following proliferation. Cells were cultured in growth media (GM) for 48 h to reach full confluence (D0). Cells entered the differentiated state from the proliferative state by switching from high-serum growth media to low-serum differentiation media (DM). C2C12 differentiated for 3 days. Lower: Relative levels of endogenous GRSF1 mRNA and protein levels in mouse myoblasts (C2C12) measured over time. Error bars using the standard deviation (SD), *p < 0.05, **p < 0.01, ***p < 0.001, ns, not significant. (B) Immunoblot of the GRSF1 protein from C2C12 cells following GRSF1 knockdown (KD) or the control. C2C12 cells were transfected with two different siRNAs against GRSF1, as well as a non-targeting siRNAs as controls (day −2). At 2 days post-transfection (D0), equal amounts of protein were loaded based on the bicinchoninic acid (BCA) assay; β-actin (ACTB) was a loading control. Bottom: Relative levels of MYH3 mRNA following transfection with GRSF1-targeting or control siRNAs for 48 h measured at the indicated time points of differentiation (D0, D1 and D3). Error bars using the Standard Error of the Mean (SEM), *p < 0.05, **p < 0.01, ***p < 0.001. ns, not significant. (C) Immunoblot of the FLAG protein from C2C12 cells following control or GRSF1 overexpression (tagged with FLAG) 48 h post-transfection (D0). β-tubulin (TUBULIN) was used as a loading control. Bottom: Relative levels of MYH3 mRNA following ectopic expression of GRSF1 during differentiation (D0, D1 and D3). Error bars using the Standard Error of the Mean, *p < 0.05, **p < 0.01, ***p < 0.001. (D) Immunofluorescence labeling with antibodies against the myosin heavy chain (MYHC, green) 3 days after induction of differentiation (D3). Scale bars, 100 μM. Fusion index was determined by the percentages of nuclei contained in the myotubes (a MYHC+ cell with at least two nuclei). 6–9 randomly chosen fields were analysed per sample for each experiment. Error bars using the Standard Error of the Mean (SEM), **p < 0.01. (E) Immunofluorescence labeling with antibodies against the myosin heavy chain (MYHC, green) 3 days after induction of differentiation following control or Flag-tagged GRSF1 overexpression. And C2C12 cells were stained with Mitotracker (Red) and incubated with antibodies against FLAG (green) for GRSF1 subcellular localization. Fusion index was determined by the percentages of nuclei contained in the myotubes (a MYHC+ cell with at least two nuclei). 6–9 randomly chosen fields were analysed per sample for each experiment. Error bars using the Standard Error of the Mean (SEM), **p < 0.01.
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Fig. 2. GRSF1 knock-down or overexpression does not affect MYOD expression or cell proliferation. (A) Relative levels of MYOD mRNA following transfection at the indicated time of differentiation (D0, D1 and D3). Error bars using the Standard Error of the Mean; ns, not significant. (B) Upper: Immunoblot for MYOD protein following transfection immediately prior to induction of differentiation (D0). ACTB: β-actin, loading control. Lower: quantification of the MYOD protein levels relative to the loading control. Error bars using the Standard Error of the Mean; ns, not significant. (C, D) Immunofluorescence of C2C12 cells incorporated with EdU (red) 24 h after GRSF1 KD or the control. Cells were fixed after 3 h of EdU labeling. Boxplot, percentage of EdU+ nuclei measured over ten fields; whiskers, maximum and minimum over the fields. Scale bars, 100 μM. (E, F) The same as (C, D), but with cells after GRSF1 overexpression or the control. Scale bars, 100 μM.
and then incubated with 5 μM DCFDA or 5 μM MitoSOX at 37 °C for 30 min. Then, the cells were washed twice and resuspended in PBS. The mean DCFH-DA fluorescence intensity was measured at an excitation of 488 nm and emission of 525 nm, while the mean fluorescence intensity of MitoSOX was measured at 510 nm and emission at 580 nm. In all experiments, 10,000 viable cells were analyzed, and data analysis was performed using FlowJo X software.
overnight at 4 °C. The secondary antibodies, anti-mouse or anti-rabbit IgG (H + L) conjugated to horseradish peroxidase (HRP) (Proteintech), were incubated for 1 h at 37 °C. High-sig ECL Western Blotting Substrate (cat# 1805001, Tanon) was used for visualization of the signal. Western blots were analyzed using ImageLab (Tanon, Shanghai, China). 2.9. Immunofluorescence staining
2.8. Western blot/immunoblot analysis For fusion assay, cells were seeded onto 24-well dishes for the experiment. After washing with PBS, cells were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.3% Triton X-100 for 15 min, and blocked with 10% horse serum for 50 min at 37 °C. The cells were then incubated with the primary antibody, MYHC (cat# bs10905 R, Bioss), in 10% horse serum for 1 h at 37 °C, followed by incubation with the secondary antibody (cat# ab150077, Abcam) in 1% BSA for 1 h at 37 °C. Hoechst 33342 (cat# C1022, Beyotime, Shanghai, China) was applied for nuclear staining for 5 min at room temperature. Localization of MYHC and Hoechst were determined by fluorescence microscopy. For subcellular localization, C2C12 was transfected with
Cell lysates were collected in cell lysis buffer (cat# P0013J, Beyotime Biotechnology) with protease inhibitor (cat# 36978, Thermo Fisher Scientific). Denatured proteins were electrophoresed on SDSPAGE gels and transferred onto nitrocellulose membranes. Membranes were blocked with 5% skim milk powder solution at 37 °C for 1 h and then hybridized with the primary antibodies GRSF1 (cat# D264133, Sangon Biotech), FLAG (cat# MA1-91878, Thermo Fisher Scientific), GPX4 (cat#bs-3384 R, Bioss), MYOG (cat# 19249-I-AP, Proteintech), MYOD1 (cat# 18943-1-AP, Proteintech), β-tubulin (cat.# MA5-11732, Thermo Fisher Scientific) or β-actin (cat# A5441, Sigma Aldrich) 142
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Fig. 3. A moderate change in ROS levels is related to GRSF1-mediated regulation of myoblast differentiation. (A) ROS detection by flow cytometry in C2C12 cells following knockdown of GRSF1 or a control using the fluorescent dye DCFH-DA. The histogram indicates the fluorescence intensity measured in at least three independent experiments. Error bars using the Standard Error the Mean, *p < 0.05, **p < 0.01, ***p < 0.001. (B) ROS detection by flow cytometry in C2C12 cells following overexpression of GRSF1 or the control using the fluorescent dye DCFH-DA. The histogram indicates the fluorescence intensity measured in at least three independent experiments. Error bars using the Standard Error the Mean, *p < 0.05, **p < 0.01, ***p < 0.001. (C) Mitochondrial superoxide staining by MitoSOX following GRSF1 silencing or overexpression for 48 h in C2C12 cells. Scale bars, 50 μM. (D) Mitochondrial superoxide levels determined with MitoSOX by flow cytometry following GRSF1 silencing or overexpression for 48 h in C2C12 cells. Error bars using the Standard Error the Mean of three independent experiments, *p < 0.05, **p < 0.01. (E) ATP detection by a luminescence detector in C2C12 cells according to the manufacturer's instructions of ATP Assay Kit (Beyotime). All of the results are presented as the means ± SEM of values obtained in three independent experiments, *P < 0.05, **P < 0.01, ***P < 0.001. (F) Immunofluorescence labeling with antibodies against MYHC (green) 3 days after induction of differentiation (D3). NAC was added or not added at 24 h after siRNA transfection at a concentration of 3 mM. At 48 h post-transfection, cells were induced for differentiation. Scale bars, 100 μM. The percentages of nuclei contained in the myotubes (fusion index) are shown on the right. 6–9 randomly chosen fields were analysed per sample for each experiment. Error bars using the Standard Error of the Mean (SEM), **p < 0.01.
GRSF1-FLAG plasmid. 24 h later, 5 μg/mL of CHX (cycloheximide, Sigma) was added for 2 h to block GRSF1-FLAG translation in endoplasmic reticulum. After incubation with Mito-Tracker Red CMXRos (200 nM) (Beyotime) for 30 min, cells were fixed and permeabilized as described above, and stained with anti-FLAG antibody (cat# MA191878, Thermo Fisher Scientific) followed by Alexa Fluor 488-conjugated anti-mouse IgG (Abcam). Immunofluorescent imaging was
performed using DeltaVision OMX (General Electric Company, Fairfield, Connecticut, USA). 2.10. 5-Ethynyl-2′-deoxyuridine (EdU) fluorescence stain C2C12 cells were cultured in GM with EdU (1:1000) at 37 °C with a 5% CO2 atmosphere for 3 h. Next, cells were treated according to the 143
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Fig. 4. GPX4 mRNA is a target of GRSF1 in C2C12. (A) Schematic representation of three types of GPX4 transcripts, the mitochondrial (m-GPx4), nuclear (n-GPx4) and cytoplasmic (c-GPX4) isoforms. Filled rectangle, coding sequence region (CDS); outlined rectangle, untranslated region (UTR); fold lines, intron. Only m-GPx4 contains a GRSF1 binding site in the 5′UTR. (B) Schematic diagram of TRIBE: Native Drosophila ADAR (dADAR) is composed of two double-stranded RNA-binding domains (dsRBDs) and a deaminase domain (catalytic domain) that catalyzes an adenosine-to-inosine (A to I) conversion. TRIBE-fusion protein: the dsRBDs of ADAR were replaced with GRSF1, and the catalytic domain of ADAR could edit the target transcripts of the GRSF1 protein. (C) Relative levels of GRSF1 mRNA from C2C12 cells stably expressing the TRIBE-fusion protein compared with the control immediately prior to day 0 induction of differentiation. *p < 0.05, **p < 0.01, ***p < 0.001. (D) Sequencing analysis of m-GPX4 mRNA from C2C12 cells of a stable expression cell strain or control cell lines immediately prior to day 0 induction of differentiation. Mutated sites are indicated in red. (E) Immunoblot of the GPX4 protein from C2C12 cells following transfection with a GRSF1-targeting or control siRNA immediately prior to day 0 induction of differentiation. β-actin (ACTB) was used as a loading control.
manufacturer's protocol for the Cell-Light™ EdUTP Apollo®567 TUNEL In Situ Detection Kit (20 T) (cat# C10810-1, Ribobio, China). The fluorescence signals were visualized by fluorescence microscopy.
(Fig. 1E) of MYH3 during myoblast differentiation. While ectopic expression of isoform 2 (NM_001098476.2) showed little impact on this process (Fig. S1F). We also observed a similar regulation pattern for MYOG (a marker of myogenic commitment) in siGRSF1-transfected and GRSF1-overexpressing cells (Figs. S1B–E). These results indicated that GRSF1 played a negative role in myogenesis.
2.11. Statistical analysis The results are expressed as the mean ± standard error of the mean (SEM) of at least three independent experiments. The results were analyzed by Student's t-test for two comparisons or two-way ANOVA for multiple comparisons to calculate the P values. *P < 0.05, **P < 0.01 and ***P < 0.001 were considered statistically significant.
3.2. GRSF1 suppresses myogenesis in a MYOD- and cell cycle-independent manner We next sought to determine the molecular mechanism of how GRSF1 represses myogenesis. MYOD is an earlier myogenic determination factor than MYOG. MYOD initiates skeletal myogenesis, and its forced expression alone is sufficient to influence myogenic progress [36,37]. We therefore wondered whether GRSF1 suppressed myogenesis via regulating MYOD expression. However, when we interfered with GRSF1 or overexpressed GRSF1, MYOD did not change significantly either at the mRNA or protein level during the differentiation process (Fig. 2A and B). Myoblasts need increased MYOD expression and cell cycle withdrawal to initiate differentiation [38]. Under proliferative conditions, we detected the cell division of C2C12 through an EdU incorporation assay 24 h after interference or overexpression of GRSF1. The results showed no obvious effect of the knockdown of GRSF1 on the proliferation of C2C12 (Fig. 2C and D). In addition, excessive expression of GRSF1 did not significantly promote proliferation (Fig. 2E and F). These results show that GRSF1 regulates myoblast differentiation independently of controlling MYOD expression and cell cycle regulation.
3. Results 3.1. GRSF1 knockdown promotes myoblast differentiation in C2C12 cells Among the RNA binding proteins, HuR [33], FXR1P [34] and AUF1 [35] have been implicated in myogenesis. We hypothesized that other RNA binding proteins may also have a functional role in myoblast differentiation. The online microarray data revealed that GRSF1 expression was decreased during C2C12 differentiation (GEO profiles: GDS587, Supplementary Fig. S1A). Through quantitative analysis, we confirmed that the mRNA level of GRSF1 increased during proliferation but steadily decreased from the beginning of differentiation in the mouse myoblast cell line C2C12 (Fig. 1A). We hypothesized that GRSF1 plays a role in myofiber differentiation. To explore the functional role of GRSF1 in myogenesis, we knocked down the expression of GRSF1 by transient transfection of two different small interfering RNAs (siRNAs) against GRSF1 into C2C12 cells cultured in growth media (GM) for 48 h before differentiation (D0) (Fig. 1B). We found that knockdown of GRSF1 can significantly accelerate the expression of MYH3 (myosin heavy chain 3), a terminal differentiation marker of myoblasts, both at the mRNA and protein level, throughout the myogenic process. (Fig. 1B and D). Overexpression of Flag-tagged GRSF1 (isoform 1, NM_178700.5) delayed the transcription (Fig. 1C) and translation
3.3. GRSF1 regulates muscle cell differentiation through mitochondrial ROS GRSF1 was identified as a cytoplasmic protein in the early stages of research [13], but it was recently found that GRSF1 also has important functions in human mitochondria [25,26]. In this study, we confirmed that the contributing GRSF1 (isoform 1) was mostly co-localized with mitochondria in C2C12 cells (Fig. 1E). And, when GRSF1 was knocked 144
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(caption on next page)
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Fig. 5. GPX4 influences the cellular ROS levels in C2C12 and is related to the siGRSF1-induced promotion of myoblast differentiation. (A) Immunoblot of the GPX4 protein from C2C12 cells following GPX4 knockdown or overexpression immediately prior to day 0 induction of differentiation. *p < 0.05, **p < 0.01, ***p < 0.001. (B) Total intracellular ROS detection by flow cytometry in C2C12 cells following GPX4 overexpression or the control 48 h post-transfection using the fluorescent dye DCFH-DA. The histogram indicates the fluorescence intensity measured in at least three independent experiments. Error bars using the Standard Error of the Mean, *p < 0.05, **p < 0.01, ***p < 0.001. (C) is the same as (A), except that it includes cells after transfection with GPX4-targeting or control siRNAs. (D) Mitochondrial superoxide staining by MitoSOX following GPX4 silencing or overexpression for 48 h in C2C12 cells. Scale bars, 50 μM. Right: Mitochondrial superoxide levels determined with MitoSOX by flow cytometry following GPX4 silencing or overexpression for 48 h in C2C12 cells. Error bars using the Standard Error the Mean of three independent experiments, *p < 0.05, **p < 0.01. (E) Immunofluorescence labeling with antibodies against MHC (green) 3 days after induction of differentiation (D3). NAC was added or not added 24 h after siRNA transfection at a concentration of 3 mM. At 48 h post-transfection, cells were induced for differentiation. The percentages of nuclei contained in the myotubes (fusion index) was analysed with 6–9 randomly chosen fields per sample for each experiment. Error bars using the Standard Error of the Mean (SEM), *p < 0.05, **p < 0.01. (F) Immunofluorescence labeling with antibodies against MYHC (green) 3 days after induction of differentiation (D3). The NC and GPX4 overexpression plasmid were co-transfected with siGPX4 or siNC, respectively. At 48 h post-transfection, cells were induced for differentiation. Scale bars, 100 μM. Fusion index are shown by analyzing 6–9 randomly chosen fields per sample for each experiment. Error bars using the Standard Error of the Mean (SEM). *p < 0.05, **p < 0.01.
interference with GRSF1 (Fig. S2A). Moreover, after adding CHX (cyclohexane) to abolish the translation program, GRSF1 silencing does not accelerate the degradation of GPX4 protein (Figs. S2B and S2C). We hypothesized that GRSF1 regulated m-GPX4 translation.
down, the ATP levels were affected in C2C12 (Fig. 3E), which confirmed that GRSF1 was required for mitochondrial function of myogenic cells. It has been reported that an increase of mitochondrial ROS (mtROS) will promote the differentiation of C2C12 muscle cells [10,39]. We hypothesized that siGRSF1-mediated enhancement of muscle differentiation was achieved by increasing mtROS levels. Thus, we tested the role of GRSF1 in ROS production. We first observed that total intracellular ROS levels, determined by diacetate form of DCFH (DCF-DA) fluorescence intensity, rose during interference with GRSF1 (Fig. 3A), while overexpression of GRSF1, which contained a mitochondrial targeting signal (Fig. 1E), decreased ROS levels (Fig. 3B). Under the same condition, the fluorescence intensity of MitoSOX, a Mitochondria-specific superoxide fluorescent probe, indicating mitochondrial superoxide content, was changed in a similar pattern (Fig. 3C and D). Moreover, NAC (N-Acetyl-L-cysteine), an antioxidant that can remove the ROS induced by GRSF1 silencing, abrogated the promotion of differentiation after siGRSF1 treatment (Fig. 3F). These results suggested that GRSF1 modulated the differentiation of C2C12 in a manner dependent on its effect on mitochondrial ROS levels.
3.5. GPX4 is involved in GRSF1-mediated inhibition of myogenesis via its role in ROS elimination As an antioxidant, GPX4 overexpression was negatively correlated with total intracellular and mitochondrial specific ROS accumulation [41] (Fig. 5A, B, 5D). To verify that GRSF1 affects the differentiation of C2C12 through the regulation of GPX4, we used two GPX4 siRNAs to test the effect on ROS levels and the myogenic program. We found that interference with GPX4 (Fig. 5A) can increase both intracellular and mitochondrial specific ROS levels (Fig. 5C and D) and promote C2C12 muscle differentiation, but this promotion was inhibited by adding NAC (Fig. 5E). We further confirmed that m-GPX4 overexpression attenuated the promotion of siGRSF1 on myoblast differentiation (Fig. 5F). These results demonstrated that the inhibitory role of GRSF1 in myogenesis is at least partially ascribed to its regulation of m-GPX4 during translation.
3.4. Mitochondrial GPX4 is regulated by GRSF1 in C2C12
3.6. Lentivirus-mediated GRSF1 knockdown accelerates muscle regeneration in a mouse model
Mitochondrial ROS are derived from the electron transport chain through a leakage of electrons during ATP production or an imbalance in cellular oxidant/antioxidant systems [40]. GRSF1-overexpression decreased ROS levels without affecting ATP production (Fig. 3B–E), so we hypothesize that GRSF1 regulates cellular redox status by decreasing oxidants or increasing antioxidants. Among the reported GRSF1 targets, GPX4 is an antioxidant belonging to the glutathione peroxidase family [23]. The GPx4 gene transcribes three transcripts via alternative start codons, generating mitochondrial (m-GPx4), nuclear (n-GPx4) and cytoplasm (c-GPX4) isoforms according to the UniProt database. GRSF1 has been reported to bind the m-GPX4 5′UTR AGGGGA site (Fig. 4A) [23]. To further determine whether GRSF1 targeted m-GPX4 mRNA in muscle cells, we adopted an in-vivo detection method, TRIBE (targets of RNA-binding proteins identified by editing) [32]. In brief, the GRSF1 protein was fused with the ADAR catalytic structure domain (ADARcd). ADAR is an RNA editing enzyme that consists of two parts, a double-stranded RNA binding domain and a catalytic domain which can catalyze adenosine ammonia into inosine. When the fusion protein was expressed, ADARcd was able to randomly mutate adenosine to inosine in the target mRNA of GRSF1. Then, inosine was mistakenly read as guanosine via in-vitro sequencing (Fig. 4B). In C2C12 stably expressing the GRSF1-ADARcd fusion protein (Fig. 4C), we found that m-GPX4 mRNA was mutated as we expected (Fig. 4D), while n-GPX4 and c-GPX4 mRNA were not mutated (data not shown), indicating that GRSF1 was able to target the mRNA of m-GPX4 in the C2C12 cells. We also confirmed that GRSF1 silencing induced the down regulation of m-GPX4 protein levels (Fig. 4E). These results showed that GRSF1 was able to positively regulate m-GPX4. However, there was no significant decrease in the mRNA levels of GPX4 following
To test the potential usage of GRSF1 knockdown in muscular damage repair, we used a mechanical muscle damage mouse model. To apply siGRSF1 in vivo, we generated lentiviruses expressing shGRSF1 according to the siGRSF1#1 sequence and we confirmed that lentivirusshGRSF1 did work similarly to siGRSF1 in the C2C12 cell line (Fig. 6A and B). The anterior tibial muscle of mice was injected with barium chloride to generate the mouse muscle injury model, and then lentiviruses-shNC or shGRSF1 were injected to test their role in muscle regeneration. Muscle repair was detected at Day 1, Day 3, and Day 7 postinjury. We demonstrated that at 3 d post-injury, almost no muscle fiber regeneration was observed in shNC group, whereas shGRSF1 group already displayed newly formed muscle fibers with central located nuclei. At 7 d post-injury, control group had regenerated muscle fibers with centrally located nuclei, whereas the diameters of the newly formed muscle fibers were smaller than that in GRSF1-knockdown group (Fig. 6C). According to the quantifications, 7 days after injury, the average cross-sectional area (CSA) of muscle fibers increased in GRSF1-knockdown group compared to the control group. In addition, the numbers of myofibers containing two or more centrally located nuclei were also significantly increased in GRSF1-knockdown group. We calculated the average muscle fiber areas and found that there were a large number of muscle fibers with an area greater than 1000 μm2 in GRSF1-knockdown group, and muscle fibers with an area less than 700 μm2 were almost nonexistent in GRSF1-knockdown group (Fig. 6D). These results demonstrated that the GRSF1-knockdown promoted regeneration of adult skeletal muscle. 146
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Fig. 6. Knockdown of GRSF1 promotes skeletal muscle regeneration in a mouse muscle injury model. (A) Immunoblot of the GRSF1 protein from C2C12 cells infected with lentivirus-shGRSF1 or the control immediately prior to induction of differentiation (D0). β-actin (ACTB) was used as a loading control. (B) Immunofluorescence labeling with antibodies against MYHC (green) from C2C12 cells infected with lentivirus-shGRSF1 or the control 3 days after induction of differentiation (D3). Scale bars, 100 μM. Fusion index are shown by analyzing 6–9 randomly chosen fields per sample for each experiment. Error bars using the Standard Error of the Mean (SEM). *p < 0.05, **p < 0.01. (C) Representative images of H&E staining of tibialis anterior muscle cross-sections in mice after lentiviral infection at 1, 3 and 7 days post-BaCl2 injury. Scale bars, 100 μM. (D) Left: average cross-sectional area (CSA) of regenerating myofibers (with centrally located nuclei) per field at 7 days post injury. Center: the number of myofibers containing centralized nuclei per field at 7 d post-injury. Right: Distribution of newly formed myofiber CSAs at 7 d postinjury. N = 3 biological repeats in each group. Error bars using the Standard Error of the Mean (SEM). *p < 0.05, **p < 0.01.
4. Discussion
MYOD expression could enhance MYHC expression during myotube formation [42,43]. However, we did not find the conserved G-rich binding site of GRSF1 in MYOG mRNAs. Additionally, the EdU results indicated that GRSF1 did not affect the proliferation of myoblasts (Fig. 2). The expression of cell cycle-related genes was minorly changed both in the GRSF1 knockdown and overexpression studies (data not show). Thus, unlike other RBPs related in myogenesis, GRSF1-mediated prevention of the myogenic process may not be through direct targeting of key myogenic factors or regulation of the cell cycle program. Studies have been reported that an isoform of GRSF1 was localized in human mitochondria, and decreases in GRSF1 can lead to abnormal
Here we reported that RNA binding protein GRSF1 has a negative role in muscle differentiation. In past decades, several other specialized RBPs have been investigated for their effect on myogenesis, such as HuR and FXR1P. They target MYOD, MYOG and/or cell cycle regulator P21 to cause a premature cell cycle exit of myoblasts and promote terminal differentiation [33,34]. In our study, both GRSF1 silencing and overexpression influenced the level of the key myogenic transcription factor MYOG (Fig. S1), but did not affect that of MYOD (Fig. 2A and B). It has been reported that an increase in MYOG without changes in 147
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2572016EAJ3).
mitochondrial structure and function [25,26]. According to the Gene database, mouse GRSF1 gene encodes proteins with two isoforms that are highly similar to human GRSF1. We cloned the mitochondrial-localized isoform, which contains the same mitochondrial targeting signal on the N-terminal peptide as the human GRSF isoform 1, and we found that its overexpression delayed myocyte differentiation (Fig. 1C and E), while another isoform without mitochondrial targeting signal, had no effect during differentiation (Fig. S1F). Immunofluorescence staining confirmed that this functional GRSF1 mostly overlapped with mitochondria (Fig. 1E). RNAi-mediated GRSF1 knockdown also affected mitochondrial function in C2C12, as we found a mild decrease in ATP production (Fig. 3E). Therefore, we speculated that GRSF1 may participate in myogenesis depending on its mitochondrial function. Previous studies reported that mitochondria-derived ROS stimulated muscle differentiation [10,39]. Our results showed that an increase in the ROS level was necessary for GRSF1 decrease-mediated promotion of C2C12 differentiation (Fig. 3F). Mitochondrial GRSF1 overexpression also played a negative role in mitochondrial ROS accumulation (Fig. 3C and D). Thus, GRSF1 functioned in the mitochondrial redox status to regulate myoblast differentiation. GPX4 is a multifunctional selenoprotein that has been implicated in functions such as apoptosis, gene regulation, and anti-oxidative defense [41,44,45]. A previous study has been reported that a murine GRSF1 isoform binds to a defined target sequence of mitochondrial GPX4 mRNA, which was determined by in-vitro and in-vivo system such as RNA mobility gel shift assays, luciferase-based reporter driven by 5′UTR and RNA immunoprecipitation. Moreover, polysomal fractions assay displayed that GRSF1 up-regulates m-GPx4 expression at the translational level [23]. In our study, we used a genetic tool (TRIBEGRSF1) [41] to identify GRSF1 in vivo targets, which is comparatively simple to perform and allows to study in small numbers of specific cells. We further confirmed that GRSF1 could bind to 5′UTR of m-GPX4 mRNA as it marked by novel RNA-editing events near the AGGGGA site (Fig. 4), while we did not observe the editing events in other transcripts of GPX4 such as n-GPx4 and c-GPx4 (data not show), which was consistent with the fact that there was no G rich region in these mRNAs. We also demonstrated that the absence of GRSF1 led to reduced GPX4 protein levels (Fig. 4E). This process did not result from down-regulation of GPX4 mRNA levels or increased protein degradation (Fig. S2), via cycloheximide pulse-chase assay, consistent with previous study that polysomal fractions assay displayed GRSF1 up-regulates m-GPx4 expression at the translational level [23]. Moreover, we found that silencing GPX4 in C2C12 enhanced the ability of myoblasts to fuse into myotubes via its effect on mitochondrial ROS accumulation (Fig. 5). This result suggested that GPX4 may participate in ensuring ROS homeostasis in myoblasts and prevent their premature commitment to the myogenic process. It was unclear whether the inhibition of myogenesis by GRSF1 was completely dependent on GPX4 function. The precise mechanism will be investigated in future studies. Overall, our results demonstrate that in skeletal muscle, GRSF1 is emerging as a cellular redox status regulator in mitochondria and plays a negative role in myogenesis, partly through targeting GPX4, a modifier of ROS levels. A comprehensive understanding of the function of GRSF1 in myogenesis will allow the development of applications for muscle regeneration in human disease.
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Conflicts of interest The authors declare no conflicts of interest. Acknowledgements This work was supported by the Natural Science Foundation of Heilongjiang Province (ZD2017001), the National Natural Science Foundation of China (No. 31472159 and 31272520), and the Fundamental Research Funds for the Central Universities of China (No. 148
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Guanine-rich RNA binding protein GRSF1 inhibits myoblast differentiation through repressing mitochondrial ROS production
T
Wenxin Yin1, Lin Yang1, Delin Kong, Yuzhe Nie, Yang Liang, Chun-Bo Teng∗,[email protected] College of Life Science, Northeast Forestry University, Harbin, China
A R T I C LE I N FO
A B S T R A C T
Keywords: GRSF1 Myoblast/ myo-differentiation Differentiation GPX4 ROS
Guanine-rich RNA sequence binding factor 1 (GRSF1) is a member of the RNA-binding protein (RBP) family. GRSF1 regulates RNA metabolism through RNA processing, transport and translation in the cytoplasm and mitochondria. However, its role in myogenesis has not been investigated. Here, we demonstrated that the expression of mitochondrial GRSF1 was negatively related to the differentiation of mouse skeletal myoblasts. Interference with GRSF1 promotes myogenesis both in vitro and in vivo without affecting MyoD expression or cell proliferation. Further studies illustrated that GRSF1 regulated myogenic differentiation through direct targeting of mitochondrial GPX4, a key regulator of the cellular redox status, leading to the modulation of ROS levels, which is important for myogenesis. Our findings underscore a critical function for GRSF1 during skeletal myogenesis linked to its regulation of muscle redox homeostasis.
1. Introduction Skeletal myogenesis depends on the myogenic differentiation process whereby satellite cell-derived mononuclear myoblasts exit the cell cycle and fuse into multinucleated myotubes/myofibers [1,2]. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are essential mediators of muscle differentiation [3], and they have long been associated with skeletal muscle physiology [4,5]. It has been found that NF-κB (nuclear factor kappa B) activation by PI3K (phosphoinositide-3kinase) stimulates the myogenic process by increasing the expression of inducible nitric oxide synthase (iNOS) [6–8], while the activated NFκB/iNOS pathway promotes muscle differentiation by increasing ROS levels mediated through NADPH (nicotinamide adenine dinucleotide phosphate)oxidase [9]. Further, mitochondrial ROS (mtROS) induces PTEN oxidation to stimulate the PI3K/AKT/mTOR signaling pathway and promote autophagy, which is required for muscle differentiation and regeneration [10]. Moreover, conditional mutants of the redoxstate regulator PITX2/3 have shown that moderate overproduction of ROS is required for satellite cells to exit the cell cycle and initiate differentiation through the redox activation of p38α MAP kinase, while high levels lead to senescence and regenerative failure in satellite cells [11,12]. Therefore, there must be a tightly controlled mechanism to regulate intracellular levels of ROS as well as antioxidant levels in skeletal muscle cells. Guanine-rich RNA sequence binding factor 1 (GRSF1) is a
∗ 1
ubiquitously occurring RNA-binding protein (RBP) and contains three quasi-RNA-recognition motifs (qRRM) [13–17]. GRSF1belongs to the heterogeneous nuclear ribonucleoprotein F/H (hnRNP F/H) family but differs from other members of this family [13]. hnRNP F/H proteins primarily function in the nucleus and participate in splicing regulation [18,19], while GRSF1 frequently functions outside the nucleus. In the cytoplasm, GRSF1 regulates translation of eukaryotic and viral mRNA transcripts mainly though binding to the 5′ untranslated region (5′ UTR) containing G-repeats [16,17,20–24]. In mitochondria, GRSF1 has been detected in dynamic mitochondrial RNA granules (MRGs) and regulates mitochondrial gene expression at the post-transcription level [25,26]. GRSF1 plays a role in cellular senescence by regulating mitochondrial homeostasis [27]. GRSF1 has high affinity to G-rich RNA sequences, and such G-rich elements are theoretically prone to form special four-stranded structures called G-quadruplexes (G4s) [28]. Several studies have highlighted the key role of GRSF1 in G4-mediated posttranscriptional control of nuclear-encoded RNAs and mitochondrial RNAs [29,30]. Although GRSF1 has not been linked to any specific human disease, it apparently plays a role in murine embryo development as a component downstream of the Wnt/beta-catenin signaling pathway [31]. GRSF1 knockdown in mouse embryos led to developmental retardation of the brain [23]. However, whether GRSF1 participates in the muscle differentiation is unknown. In the present investigation, we studied the role of GRSF1 in myogenesis using in vitro and in vivo models. We found that GRSF1 suppresses myoblast
Corresponding author. Hexing Road 26, Harbin, 150040, China. Wenxin Yin and Lin Yang contributed equally to this work.
https://doi.org/10.1016/j.yexcr.2019.05.004 Received 6 February 2019; Received in revised form 2 May 2019; Accepted 3 May 2019 Available online 11 May 2019 0014-4827/ © 2019 Elsevier Inc. All rights reserved.
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differentiation via post-transcriptional modification of GPX4, a key regulator of the cellular redox status. We further demonstrated that GRSF1 regulates myogenesis through modulating ROS levels and at least partly by promoting the expression of GPX4.
MYOG MYOD1 GAPDH
2. Materials and methods 2.1. Cell culture
2.4. Lentivirus package
The mouse myoblast C2C12 cell line (ATCC, Manassas, VA, USA) was maintained in growth medium (GM) which contained DMEM (Hyclone, Logan, UT) supplemented with 10% (v/v) fetal bovine serum (Gibco, Grand Island, NY, USA) and 2 mM GlutaMAX™ (Gibco, Grand Island, NY, USA) at 37 °C with a 5% CO2 concentration for two days. C2C12 cells were induced to differentiate by replacing the media with differentiation medium (DM) containing DMEM supplemented with 2% (v/v) horse serum (Gibco, Grand Island, NY, USA) and 2 mM GlutaMAX™. Complete differentiation was achieved after 3 days in DM.
The 293 T cells (ATCC, Manassas, VA, USA) were seeded in a culture flask at a density of 2 × 104 cells/cm2 in DMEM (Hyclone, Logan, UT) supplemented with 15% (v/v) fetal bovine serum (Gibco, Grand Island, NY, USA) and 2 mM GlutaMAX™ (Gibco, Grand Island, NY, USA) at 37 °C with the CO2 concentration 5%. 293 T cells were replaced in a serum-free medium when they fused at a density of approximately 70%–80%, and the cells were transfected with purified viral plasmids (SL4-TRIBE and LV3-shGRSF1 or corresponding control) for packaging in a Polyethylenimine-Based method. 293 T cells were added to fetal bovine serum 6–8 h later. Medium containing the virus was collected and centrifuged by ultracentrifugation after 72 h. Lentiviruses were dissolved in PBS and stored at −80 °C. shGRSF1-sense: 5′-AAA GCA CAG GGA AGAAAT TGG TA-3’. shGRSF1-antisense: 5′-TA CCA ATT TCT TCC CTG TGC TTT-3’. (Corresponding to bases 982-1004 of the murine Grsf1 gene, NCBI accession no.: NM_178700.5)
2.2. Transient transfection Plasmid vector pcDNA3.1 containing flag-tagged CDS of GRSF1 (NM_178700.5) or m-GPX4 (NM_008162.4) constructs were transfected into C2C12 cells at a density of approximately 30%–40% by Lipofectamine™ 2000 Transfection Reagent (Thermo Fisher Scientific) according to the manufacturer's instructions. The efficiency of transfection was monitored by detecting of each mRNA level using qPCR and immunoblot analysis. Empty plasmid vectors were used as a negative control. SiRNAs for GRSF1 or GPX4 were purchased from GenePharma. The siRNAs (50 nM/24-well dish) were transfected into C2C12 cells at a density of approximately 30%–40% by Lipofectamine™ RNAiMAX Transfection Reagent (Thermo Fisher Scientific) according to the manufacturer's instructions. The efficiency of knockdown was monitored by qPCR and immunoblot analysis. A non-silencing siRNA was as a control.
siGPX4#1 siGPX4#2 siGRSF1#1 siGRSF1#2
2.5. TRIBE method TRIBE method was based on the report before [32]. Briefly, C2C12 was infected with lentivirus expressing TRIBE-GRSF1 fusion protein or control in the presence of 8 μg/mL of Polybrene (Santa Cruz Biotechnology). 72 h later, they were treated with puromycin (5 μg/mL) (Sigma Aldrich) to select for cells stably expressing integrated vectors. The 5′UTR of m-GPX4 was PCR-amplified using cDNA obtained from pooled TRIBE-GRSF1 overexpressing or control populations, and cloned by pEASY-T5 Zero Cloning Kit (cat# CT501-01, Transgene Biotech, Beijing, China). Sequence was analyzed using at least six positive clones of each sample to evaluate the edit sites and the efficiency of edit events.
sense 5′- CCGGAGGAAGGUCCAGAGG -3′, antisense 5′- CCUCUGGACCUUCCUCCGG-3’; sense 5′- CTATCTCTAGCTAGCCCTA-3′, antisense 5′- TAGGGCTAGCTAGAGATAG-3’; sense 5′- AAAGCACAGGGAAGAAAUUGGUA-3′, antisense 5′- UACCAAUUUCUUCCCUGUGCUUU-3’; sense 5′- GCGGUAUGUGGAAGUGUAUGAAAUA-3′, antisense 5′- UAUUUCAUACACUUCCACAUACCGC-3’;
2.6. Muscle injury The protocol for animal use was approved by the Animal Care and Ethics Committee of Northeast Forestry University, and all the procedures were carried out in accordance with the approved guidelines. C57BL/6 female mice were purchased from the Second Affiliated Hospital of Harbin Medical University (Harbin, China) and were injured in the tibialis anterior muscles (TA) of both legs by injection with 50 μL of 1.5% (v/v) barium chloride (BaCl2) in PBS supplemented with 50 μL of lentivirus (shNC or shGRSF1) per leg. Before the procedure, the mice were anesthetized. The right TA muscles were injured with lentivirus expressing shGRSF1, and the left TA muscles were injured with lentivirus expressing shNC as a control. The injured muscles were dissected on day 0, day 1, day 3 and day 7 after injury. Immediately after isolation, the muscles were dissected for hematoxylin and eosin (H&E) staining. In each experiment, at least three animals were analyzed for each time point after injection.
2.3. Quantitative RT-PCR Total RNA was extracted using TRIzol™ Reagent (Thermo Fisher Scientific) according to the manufacturer's instructions. The mRNA levels were measured using a SYBR Green-based RT-PCR Master Mix (Cat# 04913914001, Roche). The thermal cycler conditions were as follows: 10 min at 95 °C followed by 40 cycles of 95 °C for 30 s and 60 °C for 30 s. Finally, the samples were held at 72 °C and melting curves were conducted from 72 °C to 95.1 °C. All tests were performed in triplicate and all experiments were repeated three times in a Light-Cycler 480 System (Roche, Basel, Switzerland). Expression data were normalized to the geometric mean of housekeeping gene GAPDH to control the variability in expression levels and were analyzed using the 2−ΔΔCT method. The primer sequences designed by Primer 5 software were as follows:
GRSF1 GPX4 MYH3
reverse, 5′- CACCTCTTTGATTTTGGCTTCC-3′ forward,5′- ACAATCTGCACTCCCTTACG-3′ reverse,5′- GTGATGGCTTTTGACACCAAC-3′ forward, 5′- CCAATGCGATTTATCAGGTGC-3′ reverse, 5′- CGAAAGGACAGTTGGGAAGAG-3′ forward, 5′-TGACCACAGTCCATGCCAT-3′ reverse, 5′-TTCTAGACGGCAGGTCAGGT-3′
2.7. Flow cytometry analysis The intracellular ROS level and mitochondrial superoxide were measured by 2′,7′-dichlorofluorescin diacetate (DCFH-DA; Beyotime Biotechnology, Nanjing, China) and MitoSOX (Invitrogen) staining using a flow cytometer (BD C6 Biosciences, San Jose, CA, USA), respectively. C2C12 cells were trypsinization and washed twice with PBS
forward, 5′-CCTAAATAGAGATGGGAAACGGAG-3′ reverse, 5′-AGGCATCCACATCTTCGTTG-3′ forward, 5′- GCAATGAGGCAAAACTGACG-3′ reverse, 5′- CTTGATTACTTCCTGGCTCCTG-3′ forward, 5′- AAGCTCGTCACTTTGGTACAG-3′
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Fig. 1. GRSF1 plays an important role in myoblast differentiation. (A) Upper: Schematic diagram of the differentiation process of mouse myoblasts through a time course following proliferation. Cells were cultured in growth media (GM) for 48 h to reach full confluence (D0). Cells entered the differentiated state from the proliferative state by switching from high-serum growth media to low-serum differentiation media (DM). C2C12 differentiated for 3 days. Lower: Relative levels of endogenous GRSF1 mRNA and protein levels in mouse myoblasts (C2C12) measured over time. Error bars using the standard deviation (SD), *p < 0.05, **p < 0.01, ***p < 0.001, ns, not significant. (B) Immunoblot of the GRSF1 protein from C2C12 cells following GRSF1 knockdown (KD) or the control. C2C12 cells were transfected with two different siRNAs against GRSF1, as well as a non-targeting siRNAs as controls (day −2). At 2 days post-transfection (D0), equal amounts of protein were loaded based on the bicinchoninic acid (BCA) assay; β-actin (ACTB) was a loading control. Bottom: Relative levels of MYH3 mRNA following transfection with GRSF1-targeting or control siRNAs for 48 h measured at the indicated time points of differentiation (D0, D1 and D3). Error bars using the Standard Error of the Mean (SEM), *p < 0.05, **p < 0.01, ***p < 0.001. ns, not significant. (C) Immunoblot of the FLAG protein from C2C12 cells following control or GRSF1 overexpression (tagged with FLAG) 48 h post-transfection (D0). β-tubulin (TUBULIN) was used as a loading control. Bottom: Relative levels of MYH3 mRNA following ectopic expression of GRSF1 during differentiation (D0, D1 and D3). Error bars using the Standard Error of the Mean, *p < 0.05, **p < 0.01, ***p < 0.001. (D) Immunofluorescence labeling with antibodies against the myosin heavy chain (MYHC, green) 3 days after induction of differentiation (D3). Scale bars, 100 μM. Fusion index was determined by the percentages of nuclei contained in the myotubes (a MYHC+ cell with at least two nuclei). 6–9 randomly chosen fields were analysed per sample for each experiment. Error bars using the Standard Error of the Mean (SEM), **p < 0.01. (E) Immunofluorescence labeling with antibodies against the myosin heavy chain (MYHC, green) 3 days after induction of differentiation following control or Flag-tagged GRSF1 overexpression. And C2C12 cells were stained with Mitotracker (Red) and incubated with antibodies against FLAG (green) for GRSF1 subcellular localization. Fusion index was determined by the percentages of nuclei contained in the myotubes (a MYHC+ cell with at least two nuclei). 6–9 randomly chosen fields were analysed per sample for each experiment. Error bars using the Standard Error of the Mean (SEM), **p < 0.01.
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Fig. 2. GRSF1 knock-down or overexpression does not affect MYOD expression or cell proliferation. (A) Relative levels of MYOD mRNA following transfection at the indicated time of differentiation (D0, D1 and D3). Error bars using the Standard Error of the Mean; ns, not significant. (B) Upper: Immunoblot for MYOD protein following transfection immediately prior to induction of differentiation (D0). ACTB: β-actin, loading control. Lower: quantification of the MYOD protein levels relative to the loading control. Error bars using the Standard Error of the Mean; ns, not significant. (C, D) Immunofluorescence of C2C12 cells incorporated with EdU (red) 24 h after GRSF1 KD or the control. Cells were fixed after 3 h of EdU labeling. Boxplot, percentage of EdU+ nuclei measured over ten fields; whiskers, maximum and minimum over the fields. Scale bars, 100 μM. (E, F) The same as (C, D), but with cells after GRSF1 overexpression or the control. Scale bars, 100 μM.
and then incubated with 5 μM DCFDA or 5 μM MitoSOX at 37 °C for 30 min. Then, the cells were washed twice and resuspended in PBS. The mean DCFH-DA fluorescence intensity was measured at an excitation of 488 nm and emission of 525 nm, while the mean fluorescence intensity of MitoSOX was measured at 510 nm and emission at 580 nm. In all experiments, 10,000 viable cells were analyzed, and data analysis was performed using FlowJo X software.
overnight at 4 °C. The secondary antibodies, anti-mouse or anti-rabbit IgG (H + L) conjugated to horseradish peroxidase (HRP) (Proteintech), were incubated for 1 h at 37 °C. High-sig ECL Western Blotting Substrate (cat# 1805001, Tanon) was used for visualization of the signal. Western blots were analyzed using ImageLab (Tanon, Shanghai, China). 2.9. Immunofluorescence staining
2.8. Western blot/immunoblot analysis For fusion assay, cells were seeded onto 24-well dishes for the experiment. After washing with PBS, cells were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.3% Triton X-100 for 15 min, and blocked with 10% horse serum for 50 min at 37 °C. The cells were then incubated with the primary antibody, MYHC (cat# bs10905 R, Bioss), in 10% horse serum for 1 h at 37 °C, followed by incubation with the secondary antibody (cat# ab150077, Abcam) in 1% BSA for 1 h at 37 °C. Hoechst 33342 (cat# C1022, Beyotime, Shanghai, China) was applied for nuclear staining for 5 min at room temperature. Localization of MYHC and Hoechst were determined by fluorescence microscopy. For subcellular localization, C2C12 was transfected with
Cell lysates were collected in cell lysis buffer (cat# P0013J, Beyotime Biotechnology) with protease inhibitor (cat# 36978, Thermo Fisher Scientific). Denatured proteins were electrophoresed on SDSPAGE gels and transferred onto nitrocellulose membranes. Membranes were blocked with 5% skim milk powder solution at 37 °C for 1 h and then hybridized with the primary antibodies GRSF1 (cat# D264133, Sangon Biotech), FLAG (cat# MA1-91878, Thermo Fisher Scientific), GPX4 (cat#bs-3384 R, Bioss), MYOG (cat# 19249-I-AP, Proteintech), MYOD1 (cat# 18943-1-AP, Proteintech), β-tubulin (cat.# MA5-11732, Thermo Fisher Scientific) or β-actin (cat# A5441, Sigma Aldrich) 142
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Fig. 3. A moderate change in ROS levels is related to GRSF1-mediated regulation of myoblast differentiation. (A) ROS detection by flow cytometry in C2C12 cells following knockdown of GRSF1 or a control using the fluorescent dye DCFH-DA. The histogram indicates the fluorescence intensity measured in at least three independent experiments. Error bars using the Standard Error the Mean, *p < 0.05, **p < 0.01, ***p < 0.001. (B) ROS detection by flow cytometry in C2C12 cells following overexpression of GRSF1 or the control using the fluorescent dye DCFH-DA. The histogram indicates the fluorescence intensity measured in at least three independent experiments. Error bars using the Standard Error the Mean, *p < 0.05, **p < 0.01, ***p < 0.001. (C) Mitochondrial superoxide staining by MitoSOX following GRSF1 silencing or overexpression for 48 h in C2C12 cells. Scale bars, 50 μM. (D) Mitochondrial superoxide levels determined with MitoSOX by flow cytometry following GRSF1 silencing or overexpression for 48 h in C2C12 cells. Error bars using the Standard Error the Mean of three independent experiments, *p < 0.05, **p < 0.01. (E) ATP detection by a luminescence detector in C2C12 cells according to the manufacturer's instructions of ATP Assay Kit (Beyotime). All of the results are presented as the means ± SEM of values obtained in three independent experiments, *P < 0.05, **P < 0.01, ***P < 0.001. (F) Immunofluorescence labeling with antibodies against MYHC (green) 3 days after induction of differentiation (D3). NAC was added or not added at 24 h after siRNA transfection at a concentration of 3 mM. At 48 h post-transfection, cells were induced for differentiation. Scale bars, 100 μM. The percentages of nuclei contained in the myotubes (fusion index) are shown on the right. 6–9 randomly chosen fields were analysed per sample for each experiment. Error bars using the Standard Error of the Mean (SEM), **p < 0.01.
GRSF1-FLAG plasmid. 24 h later, 5 μg/mL of CHX (cycloheximide, Sigma) was added for 2 h to block GRSF1-FLAG translation in endoplasmic reticulum. After incubation with Mito-Tracker Red CMXRos (200 nM) (Beyotime) for 30 min, cells were fixed and permeabilized as described above, and stained with anti-FLAG antibody (cat# MA191878, Thermo Fisher Scientific) followed by Alexa Fluor 488-conjugated anti-mouse IgG (Abcam). Immunofluorescent imaging was
performed using DeltaVision OMX (General Electric Company, Fairfield, Connecticut, USA). 2.10. 5-Ethynyl-2′-deoxyuridine (EdU) fluorescence stain C2C12 cells were cultured in GM with EdU (1:1000) at 37 °C with a 5% CO2 atmosphere for 3 h. Next, cells were treated according to the 143
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Fig. 4. GPX4 mRNA is a target of GRSF1 in C2C12. (A) Schematic representation of three types of GPX4 transcripts, the mitochondrial (m-GPx4), nuclear (n-GPx4) and cytoplasmic (c-GPX4) isoforms. Filled rectangle, coding sequence region (CDS); outlined rectangle, untranslated region (UTR); fold lines, intron. Only m-GPx4 contains a GRSF1 binding site in the 5′UTR. (B) Schematic diagram of TRIBE: Native Drosophila ADAR (dADAR) is composed of two double-stranded RNA-binding domains (dsRBDs) and a deaminase domain (catalytic domain) that catalyzes an adenosine-to-inosine (A to I) conversion. TRIBE-fusion protein: the dsRBDs of ADAR were replaced with GRSF1, and the catalytic domain of ADAR could edit the target transcripts of the GRSF1 protein. (C) Relative levels of GRSF1 mRNA from C2C12 cells stably expressing the TRIBE-fusion protein compared with the control immediately prior to day 0 induction of differentiation. *p < 0.05, **p < 0.01, ***p < 0.001. (D) Sequencing analysis of m-GPX4 mRNA from C2C12 cells of a stable expression cell strain or control cell lines immediately prior to day 0 induction of differentiation. Mutated sites are indicated in red. (E) Immunoblot of the GPX4 protein from C2C12 cells following transfection with a GRSF1-targeting or control siRNA immediately prior to day 0 induction of differentiation. β-actin (ACTB) was used as a loading control.
manufacturer's protocol for the Cell-Light™ EdUTP Apollo®567 TUNEL In Situ Detection Kit (20 T) (cat# C10810-1, Ribobio, China). The fluorescence signals were visualized by fluorescence microscopy.
(Fig. 1E) of MYH3 during myoblast differentiation. While ectopic expression of isoform 2 (NM_001098476.2) showed little impact on this process (Fig. S1F). We also observed a similar regulation pattern for MYOG (a marker of myogenic commitment) in siGRSF1-transfected and GRSF1-overexpressing cells (Figs. S1B–E). These results indicated that GRSF1 played a negative role in myogenesis.
2.11. Statistical analysis The results are expressed as the mean ± standard error of the mean (SEM) of at least three independent experiments. The results were analyzed by Student's t-test for two comparisons or two-way ANOVA for multiple comparisons to calculate the P values. *P < 0.05, **P < 0.01 and ***P < 0.001 were considered statistically significant.
3.2. GRSF1 suppresses myogenesis in a MYOD- and cell cycle-independent manner We next sought to determine the molecular mechanism of how GRSF1 represses myogenesis. MYOD is an earlier myogenic determination factor than MYOG. MYOD initiates skeletal myogenesis, and its forced expression alone is sufficient to influence myogenic progress [36,37]. We therefore wondered whether GRSF1 suppressed myogenesis via regulating MYOD expression. However, when we interfered with GRSF1 or overexpressed GRSF1, MYOD did not change significantly either at the mRNA or protein level during the differentiation process (Fig. 2A and B). Myoblasts need increased MYOD expression and cell cycle withdrawal to initiate differentiation [38]. Under proliferative conditions, we detected the cell division of C2C12 through an EdU incorporation assay 24 h after interference or overexpression of GRSF1. The results showed no obvious effect of the knockdown of GRSF1 on the proliferation of C2C12 (Fig. 2C and D). In addition, excessive expression of GRSF1 did not significantly promote proliferation (Fig. 2E and F). These results show that GRSF1 regulates myoblast differentiation independently of controlling MYOD expression and cell cycle regulation.
3. Results 3.1. GRSF1 knockdown promotes myoblast differentiation in C2C12 cells Among the RNA binding proteins, HuR [33], FXR1P [34] and AUF1 [35] have been implicated in myogenesis. We hypothesized that other RNA binding proteins may also have a functional role in myoblast differentiation. The online microarray data revealed that GRSF1 expression was decreased during C2C12 differentiation (GEO profiles: GDS587, Supplementary Fig. S1A). Through quantitative analysis, we confirmed that the mRNA level of GRSF1 increased during proliferation but steadily decreased from the beginning of differentiation in the mouse myoblast cell line C2C12 (Fig. 1A). We hypothesized that GRSF1 plays a role in myofiber differentiation. To explore the functional role of GRSF1 in myogenesis, we knocked down the expression of GRSF1 by transient transfection of two different small interfering RNAs (siRNAs) against GRSF1 into C2C12 cells cultured in growth media (GM) for 48 h before differentiation (D0) (Fig. 1B). We found that knockdown of GRSF1 can significantly accelerate the expression of MYH3 (myosin heavy chain 3), a terminal differentiation marker of myoblasts, both at the mRNA and protein level, throughout the myogenic process. (Fig. 1B and D). Overexpression of Flag-tagged GRSF1 (isoform 1, NM_178700.5) delayed the transcription (Fig. 1C) and translation
3.3. GRSF1 regulates muscle cell differentiation through mitochondrial ROS GRSF1 was identified as a cytoplasmic protein in the early stages of research [13], but it was recently found that GRSF1 also has important functions in human mitochondria [25,26]. In this study, we confirmed that the contributing GRSF1 (isoform 1) was mostly co-localized with mitochondria in C2C12 cells (Fig. 1E). And, when GRSF1 was knocked 144
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Fig. 5. GPX4 influences the cellular ROS levels in C2C12 and is related to the siGRSF1-induced promotion of myoblast differentiation. (A) Immunoblot of the GPX4 protein from C2C12 cells following GPX4 knockdown or overexpression immediately prior to day 0 induction of differentiation. *p < 0.05, **p < 0.01, ***p < 0.001. (B) Total intracellular ROS detection by flow cytometry in C2C12 cells following GPX4 overexpression or the control 48 h post-transfection using the fluorescent dye DCFH-DA. The histogram indicates the fluorescence intensity measured in at least three independent experiments. Error bars using the Standard Error of the Mean, *p < 0.05, **p < 0.01, ***p < 0.001. (C) is the same as (A), except that it includes cells after transfection with GPX4-targeting or control siRNAs. (D) Mitochondrial superoxide staining by MitoSOX following GPX4 silencing or overexpression for 48 h in C2C12 cells. Scale bars, 50 μM. Right: Mitochondrial superoxide levels determined with MitoSOX by flow cytometry following GPX4 silencing or overexpression for 48 h in C2C12 cells. Error bars using the Standard Error the Mean of three independent experiments, *p < 0.05, **p < 0.01. (E) Immunofluorescence labeling with antibodies against MHC (green) 3 days after induction of differentiation (D3). NAC was added or not added 24 h after siRNA transfection at a concentration of 3 mM. At 48 h post-transfection, cells were induced for differentiation. The percentages of nuclei contained in the myotubes (fusion index) was analysed with 6–9 randomly chosen fields per sample for each experiment. Error bars using the Standard Error of the Mean (SEM), *p < 0.05, **p < 0.01. (F) Immunofluorescence labeling with antibodies against MYHC (green) 3 days after induction of differentiation (D3). The NC and GPX4 overexpression plasmid were co-transfected with siGPX4 or siNC, respectively. At 48 h post-transfection, cells were induced for differentiation. Scale bars, 100 μM. Fusion index are shown by analyzing 6–9 randomly chosen fields per sample for each experiment. Error bars using the Standard Error of the Mean (SEM). *p < 0.05, **p < 0.01.
interference with GRSF1 (Fig. S2A). Moreover, after adding CHX (cyclohexane) to abolish the translation program, GRSF1 silencing does not accelerate the degradation of GPX4 protein (Figs. S2B and S2C). We hypothesized that GRSF1 regulated m-GPX4 translation.
down, the ATP levels were affected in C2C12 (Fig. 3E), which confirmed that GRSF1 was required for mitochondrial function of myogenic cells. It has been reported that an increase of mitochondrial ROS (mtROS) will promote the differentiation of C2C12 muscle cells [10,39]. We hypothesized that siGRSF1-mediated enhancement of muscle differentiation was achieved by increasing mtROS levels. Thus, we tested the role of GRSF1 in ROS production. We first observed that total intracellular ROS levels, determined by diacetate form of DCFH (DCF-DA) fluorescence intensity, rose during interference with GRSF1 (Fig. 3A), while overexpression of GRSF1, which contained a mitochondrial targeting signal (Fig. 1E), decreased ROS levels (Fig. 3B). Under the same condition, the fluorescence intensity of MitoSOX, a Mitochondria-specific superoxide fluorescent probe, indicating mitochondrial superoxide content, was changed in a similar pattern (Fig. 3C and D). Moreover, NAC (N-Acetyl-L-cysteine), an antioxidant that can remove the ROS induced by GRSF1 silencing, abrogated the promotion of differentiation after siGRSF1 treatment (Fig. 3F). These results suggested that GRSF1 modulated the differentiation of C2C12 in a manner dependent on its effect on mitochondrial ROS levels.
3.5. GPX4 is involved in GRSF1-mediated inhibition of myogenesis via its role in ROS elimination As an antioxidant, GPX4 overexpression was negatively correlated with total intracellular and mitochondrial specific ROS accumulation [41] (Fig. 5A, B, 5D). To verify that GRSF1 affects the differentiation of C2C12 through the regulation of GPX4, we used two GPX4 siRNAs to test the effect on ROS levels and the myogenic program. We found that interference with GPX4 (Fig. 5A) can increase both intracellular and mitochondrial specific ROS levels (Fig. 5C and D) and promote C2C12 muscle differentiation, but this promotion was inhibited by adding NAC (Fig. 5E). We further confirmed that m-GPX4 overexpression attenuated the promotion of siGRSF1 on myoblast differentiation (Fig. 5F). These results demonstrated that the inhibitory role of GRSF1 in myogenesis is at least partially ascribed to its regulation of m-GPX4 during translation.
3.4. Mitochondrial GPX4 is regulated by GRSF1 in C2C12
3.6. Lentivirus-mediated GRSF1 knockdown accelerates muscle regeneration in a mouse model
Mitochondrial ROS are derived from the electron transport chain through a leakage of electrons during ATP production or an imbalance in cellular oxidant/antioxidant systems [40]. GRSF1-overexpression decreased ROS levels without affecting ATP production (Fig. 3B–E), so we hypothesize that GRSF1 regulates cellular redox status by decreasing oxidants or increasing antioxidants. Among the reported GRSF1 targets, GPX4 is an antioxidant belonging to the glutathione peroxidase family [23]. The GPx4 gene transcribes three transcripts via alternative start codons, generating mitochondrial (m-GPx4), nuclear (n-GPx4) and cytoplasm (c-GPX4) isoforms according to the UniProt database. GRSF1 has been reported to bind the m-GPX4 5′UTR AGGGGA site (Fig. 4A) [23]. To further determine whether GRSF1 targeted m-GPX4 mRNA in muscle cells, we adopted an in-vivo detection method, TRIBE (targets of RNA-binding proteins identified by editing) [32]. In brief, the GRSF1 protein was fused with the ADAR catalytic structure domain (ADARcd). ADAR is an RNA editing enzyme that consists of two parts, a double-stranded RNA binding domain and a catalytic domain which can catalyze adenosine ammonia into inosine. When the fusion protein was expressed, ADARcd was able to randomly mutate adenosine to inosine in the target mRNA of GRSF1. Then, inosine was mistakenly read as guanosine via in-vitro sequencing (Fig. 4B). In C2C12 stably expressing the GRSF1-ADARcd fusion protein (Fig. 4C), we found that m-GPX4 mRNA was mutated as we expected (Fig. 4D), while n-GPX4 and c-GPX4 mRNA were not mutated (data not shown), indicating that GRSF1 was able to target the mRNA of m-GPX4 in the C2C12 cells. We also confirmed that GRSF1 silencing induced the down regulation of m-GPX4 protein levels (Fig. 4E). These results showed that GRSF1 was able to positively regulate m-GPX4. However, there was no significant decrease in the mRNA levels of GPX4 following
To test the potential usage of GRSF1 knockdown in muscular damage repair, we used a mechanical muscle damage mouse model. To apply siGRSF1 in vivo, we generated lentiviruses expressing shGRSF1 according to the siGRSF1#1 sequence and we confirmed that lentivirusshGRSF1 did work similarly to siGRSF1 in the C2C12 cell line (Fig. 6A and B). The anterior tibial muscle of mice was injected with barium chloride to generate the mouse muscle injury model, and then lentiviruses-shNC or shGRSF1 were injected to test their role in muscle regeneration. Muscle repair was detected at Day 1, Day 3, and Day 7 postinjury. We demonstrated that at 3 d post-injury, almost no muscle fiber regeneration was observed in shNC group, whereas shGRSF1 group already displayed newly formed muscle fibers with central located nuclei. At 7 d post-injury, control group had regenerated muscle fibers with centrally located nuclei, whereas the diameters of the newly formed muscle fibers were smaller than that in GRSF1-knockdown group (Fig. 6C). According to the quantifications, 7 days after injury, the average cross-sectional area (CSA) of muscle fibers increased in GRSF1-knockdown group compared to the control group. In addition, the numbers of myofibers containing two or more centrally located nuclei were also significantly increased in GRSF1-knockdown group. We calculated the average muscle fiber areas and found that there were a large number of muscle fibers with an area greater than 1000 μm2 in GRSF1-knockdown group, and muscle fibers with an area less than 700 μm2 were almost nonexistent in GRSF1-knockdown group (Fig. 6D). These results demonstrated that the GRSF1-knockdown promoted regeneration of adult skeletal muscle. 146
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Fig. 6. Knockdown of GRSF1 promotes skeletal muscle regeneration in a mouse muscle injury model. (A) Immunoblot of the GRSF1 protein from C2C12 cells infected with lentivirus-shGRSF1 or the control immediately prior to induction of differentiation (D0). β-actin (ACTB) was used as a loading control. (B) Immunofluorescence labeling with antibodies against MYHC (green) from C2C12 cells infected with lentivirus-shGRSF1 or the control 3 days after induction of differentiation (D3). Scale bars, 100 μM. Fusion index are shown by analyzing 6–9 randomly chosen fields per sample for each experiment. Error bars using the Standard Error of the Mean (SEM). *p < 0.05, **p < 0.01. (C) Representative images of H&E staining of tibialis anterior muscle cross-sections in mice after lentiviral infection at 1, 3 and 7 days post-BaCl2 injury. Scale bars, 100 μM. (D) Left: average cross-sectional area (CSA) of regenerating myofibers (with centrally located nuclei) per field at 7 days post injury. Center: the number of myofibers containing centralized nuclei per field at 7 d post-injury. Right: Distribution of newly formed myofiber CSAs at 7 d postinjury. N = 3 biological repeats in each group. Error bars using the Standard Error of the Mean (SEM). *p < 0.05, **p < 0.01.
4. Discussion
MYOD expression could enhance MYHC expression during myotube formation [42,43]. However, we did not find the conserved G-rich binding site of GRSF1 in MYOG mRNAs. Additionally, the EdU results indicated that GRSF1 did not affect the proliferation of myoblasts (Fig. 2). The expression of cell cycle-related genes was minorly changed both in the GRSF1 knockdown and overexpression studies (data not show). Thus, unlike other RBPs related in myogenesis, GRSF1-mediated prevention of the myogenic process may not be through direct targeting of key myogenic factors or regulation of the cell cycle program. Studies have been reported that an isoform of GRSF1 was localized in human mitochondria, and decreases in GRSF1 can lead to abnormal
Here we reported that RNA binding protein GRSF1 has a negative role in muscle differentiation. In past decades, several other specialized RBPs have been investigated for their effect on myogenesis, such as HuR and FXR1P. They target MYOD, MYOG and/or cell cycle regulator P21 to cause a premature cell cycle exit of myoblasts and promote terminal differentiation [33,34]. In our study, both GRSF1 silencing and overexpression influenced the level of the key myogenic transcription factor MYOG (Fig. S1), but did not affect that of MYOD (Fig. 2A and B). It has been reported that an increase in MYOG without changes in 147
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2572016EAJ3).
mitochondrial structure and function [25,26]. According to the Gene database, mouse GRSF1 gene encodes proteins with two isoforms that are highly similar to human GRSF1. We cloned the mitochondrial-localized isoform, which contains the same mitochondrial targeting signal on the N-terminal peptide as the human GRSF isoform 1, and we found that its overexpression delayed myocyte differentiation (Fig. 1C and E), while another isoform without mitochondrial targeting signal, had no effect during differentiation (Fig. S1F). Immunofluorescence staining confirmed that this functional GRSF1 mostly overlapped with mitochondria (Fig. 1E). RNAi-mediated GRSF1 knockdown also affected mitochondrial function in C2C12, as we found a mild decrease in ATP production (Fig. 3E). Therefore, we speculated that GRSF1 may participate in myogenesis depending on its mitochondrial function. Previous studies reported that mitochondria-derived ROS stimulated muscle differentiation [10,39]. Our results showed that an increase in the ROS level was necessary for GRSF1 decrease-mediated promotion of C2C12 differentiation (Fig. 3F). Mitochondrial GRSF1 overexpression also played a negative role in mitochondrial ROS accumulation (Fig. 3C and D). Thus, GRSF1 functioned in the mitochondrial redox status to regulate myoblast differentiation. GPX4 is a multifunctional selenoprotein that has been implicated in functions such as apoptosis, gene regulation, and anti-oxidative defense [41,44,45]. A previous study has been reported that a murine GRSF1 isoform binds to a defined target sequence of mitochondrial GPX4 mRNA, which was determined by in-vitro and in-vivo system such as RNA mobility gel shift assays, luciferase-based reporter driven by 5′UTR and RNA immunoprecipitation. Moreover, polysomal fractions assay displayed that GRSF1 up-regulates m-GPx4 expression at the translational level [23]. In our study, we used a genetic tool (TRIBEGRSF1) [41] to identify GRSF1 in vivo targets, which is comparatively simple to perform and allows to study in small numbers of specific cells. We further confirmed that GRSF1 could bind to 5′UTR of m-GPX4 mRNA as it marked by novel RNA-editing events near the AGGGGA site (Fig. 4), while we did not observe the editing events in other transcripts of GPX4 such as n-GPx4 and c-GPx4 (data not show), which was consistent with the fact that there was no G rich region in these mRNAs. We also demonstrated that the absence of GRSF1 led to reduced GPX4 protein levels (Fig. 4E). This process did not result from down-regulation of GPX4 mRNA levels or increased protein degradation (Fig. S2), via cycloheximide pulse-chase assay, consistent with previous study that polysomal fractions assay displayed GRSF1 up-regulates m-GPx4 expression at the translational level [23]. Moreover, we found that silencing GPX4 in C2C12 enhanced the ability of myoblasts to fuse into myotubes via its effect on mitochondrial ROS accumulation (Fig. 5). This result suggested that GPX4 may participate in ensuring ROS homeostasis in myoblasts and prevent their premature commitment to the myogenic process. It was unclear whether the inhibition of myogenesis by GRSF1 was completely dependent on GPX4 function. The precise mechanism will be investigated in future studies. Overall, our results demonstrate that in skeletal muscle, GRSF1 is emerging as a cellular redox status regulator in mitochondria and plays a negative role in myogenesis, partly through targeting GPX4, a modifier of ROS levels. A comprehensive understanding of the function of GRSF1 in myogenesis will allow the development of applications for muscle regeneration in human disease.
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Conflicts of interest The authors declare no conflicts of interest. Acknowledgements This work was supported by the Natural Science Foundation of Heilongjiang Province (ZD2017001), the National Natural Science Foundation of China (No. 31472159 and 31272520), and the Fundamental Research Funds for the Central Universities of China (No. 148
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