LncRNA-TBP is a novel lncRNA associated with myogenesis
Our previous RNA-seq study found a muscle-related lncRNA (LncRNA-TBP) was highly expressed in SOL (Fig. 1A, B). 5’ and 3’ ends of LncRNA-TBP were identified by RACE analysis (Fig. 1C). The NCBI BLAST indicated that LncRNA-TBP located on Chromosome 3 and spanned from 82341588 to 82342330, and 82369736 to 82370049 with 1057 nt long, relatively conserved in Meleagris gallopavo, Apteryx mantelli mantelli, and Numida meleagris (Fig. S1 and Table S1). LncRNA-TBP highly expressed in polyadenylated RNA (Fig. 1D). LncRNA-TBP upregulated during myogenic differentiation, and enriched in leg muscles and breast muscles (Fig. 1E, F), implying that it may play an important role in skeletal muscle development. In addition, cell-fractionation assays demonstrated that LncRNA-TBP is mainly present in the nucleus of chicken primary myoblasts (CPMs) (Fig. 1G). To further prove the coding potential of LncRNA-TBP, we analyzed the twelve potential ORFs of LncRNA-TBP by western blot. The results show that LncRNA-TBP is a lncRNA without protein-encoding potential (Fig. 1H).
LncRNA-TBP inhibits myoblast proliferation and promotes myoblast differentiation
LncRNA-TBP was predominantly expressed in breast muscle and leg muscle (Fig. 1F), implying that LncRNA-TBP plays an important role in myogenesis. To assess the effect of LncRNA-TBP on proliferation and differentiation of myoblast, the overexpression vector and inhibitor of LncRNA-TBP were transfected into CPMs (Fig. 2A and Fig. S2A). Overexpression of LncRNA-TBP increased the expression of cell cycle-inhibiting genes like CDKN1A and CDKN1B while decreasing the expression level of cell cycle-promoting genes like PCNA. The opposite result was observed with LncRNA-TBP knockdown (Fig. 2B and Fig. S2B). The 5-ethynyl-2’-deoxyuridine (EdU) staining and cell counting kit-8 (CCK-8) assay demonstrated that LncRNA-TBP overexpression significantly inhibited myoblast proliferation and viability (Fig. 2C to E). Conversely, interference with LncRNA-TBP promoted EdU incorporation and myoblast proliferation (Fig. S2C-E). At the same time, overexpression of LncRNA-TBP significantly increased the number of G0/G1 cells, and the number of S phase cells was lower than the control group, whereas myoblast division was inhibited with LncRNA-TBP interference (Fig. 2F and Fig. S2F).
To further investigate the potential function of LncRNA-TBP in myoblast differentiation, we detected expressions of myoblast differentiation marker genes (MyoD, MyHC, and MyoG) by using qPCR and western blot. The results showed that LncRNA-TBP overexpression increased expressions of myoblast differentiation marker genes (Fig. 2G, H). Moreover, immunofluorescence staining showed that overexpression of LncRNA-TBP facilitated myotube formation (Fig. 2I, J). In contrast, LncRNA-TBP interference suppressed myoblast differentiation (Fig. S2G-J).
LncRNA-TBP accelerates fatty acid oxidation, and enhances TCA cycle flux in skeletal muscle
To verify whether LncRNA-TBP regulates skeletal muscle development in vivo, lentiviral-mediated LncRNA-TBP overexpression (LV-LncRNA-TBP) or cholesterol-modified antisense oligonucleotide (Chol-ASO-LncRNA-TBP) were injected to the gastrocnemius of Xinghua chicken (Fig. 3A and Fig. S3A). LncRNA-TBP overexpressed increased mitochondrial DNA content, which potentially contributed to the acceleration of fatty acid oxidation (FAO) and inhibited the accumulation of free fatty acid (FFA) and triglyceride (TG) (Fig. 3B-D). In contrast, mitochondrial DNA content and fatty acid β-oxidation were reduced after the LncRNA-TBP knockdown (Fig. S3B-D). Besides, the qPCR and western blotting analyses showed that knockdown of LncRNA-TBP downregulated FAO-related genes like CPT1 and upregulating key genes involved in fatty acid synthesis (such as FASN), while opposite results were shown with LncRNA-TBP overexpression (Fig. 3E, F and Fig. S3E, F).
Mitochondria switch between lipid and glucose oxidation through the TCA cycle to generate ATP, which is pivotal for maintaining systemic energy homeostasis [19, 20]. Given that overexpression of LncRNA-TBP promoted the content of mitochondrial DNA (Fig. 3B), we performed a comparative metabolome analysis to study whether LncRNA-TBP functions muscle metabolism. The result of hierarchical clustering analysis (HCA) separated controls and overexpression of LncRNA-TBP (Fig. 3G and Table S2). For example, compared with control, glycolytic metabolites such as fructose 6-phosphate and glucose 6-phosphate were significantly decreased with LncRNA-TBP overexpression (Fig. 3H and Table S2). In the meantime, metabolites of the TCA cycle, including malic acid, isocitric acid, and fumaric acid were significantly promoted (Fig. 3H and Table S2). Altogether, our results indicated that LncRNA-TBP decreases the end products of glycolysis and elevates metabolites of the TCA cycle by promoting mitochondrial function, leading to reduction of lipid accumulation.
LncRNA-TBP activates slow-twitch muscle phenotype and induces muscle hypertrophy
Skeletal muscle development is primarily regulated by fiber type composition and muscle fiber size. The composition of myofiber types is closely related to the way muscles are metabolized [23, 24]. Given that LncRNA-TBP is highly expressed in SOL and mediated the flux of glycolysis and TCA cycle, we further examined whether LncRNA-TBP could affect the conversion of skeletal muscle fiber types in vivo. As expected, the activity of lactate dehydrogenase (LDH) was suppressed, while the activity of succinate dehydrogenase (SDH) was enhanced with LncRNA-TBP overexpression (Fig. 4A). Meanwhile, glycogen content was increased and expression of glycogenolytic and glycolytic genes was downregulated with overexpression of LncRNA-TBP (Fig. 4B, C). The opposite results were shown with the knockdown of LncRNA-TBP (Fig. S4A-C). The expression levels of fast-twitch myofiber genes like SOX6 and slow-twitch myofiber genes (such as TNNC1, TNNI1 and TNNT1) were further tested. It was found that overexpression of LncRNA-TBP promoted expressions of slow-twitch myofiber genes (Fig. 4D). More importantly, results of immunohistochemistry showed that LncRNA-TBP overexpression promoted the expression level of MYH7B/slow-twitch protein and suppressed the expression level of MYH1A/fast-twitch protein (Fig. 4E, F). On the contrary, LncRNA-TBP knockdown upregulated the fast-twitch protein level and drove the transformation of slow-twitch to fast-twitch myofibers (Fig. S4D-F).
Recent evidences have revealed that remodeling of skeletal muscle fiber types can affect muscle mass, and induce muscle hypertrophy and muscle atrophy by anabolic and catabolic signaling pathways, respectively [25]. LncRNA-TBP overexpression leads to increased muscle mass and cross-sectional area (CSA), while the opposite result occurred upon LncRNA-TBP knockdown (Fig. 3G and h and Fig. S4G, H), suggesting that LncRNA-TBP regulates skeletal muscle hypertrophy. Autophagy is a highly conserved homeostatic process carrying out degradation of cytoplasmic components including damaged organelles, toxic protein aggregates, and intracellular pathogen [26]. Maintaining basal autophagy flux is essential to clear damaged organelles or recycle macromolecules in muscles during metabolic stress [27]. To further explore the regulatory mechanism of LncRNA-TBP in inducing muscle hypertrophy, we detected expressions of autophagy-related genes. LncRNA-TBP overexpression upregulated the expression level of SQSTM1, whereas expressions of autophagy-related genes (such as MAP1LC3B, and ULK1) and content of LC3BII were downregulated (Fig. 4I, J). Conversely, LncRNA-TBP knockdown activated autophagy (Fig. S4I, J), suggesting that LncRNA-TBP may promote muscle hypertrophy by decreasing basal autophagy flux.
LncRNA-TBP directly interacts with TBP
Recent studies have found that many nuclear lncRNAs perform their functions through interaction with proteins [28]. The nuclear localization of LncRNA-TBP suggested that this lncRNA may modulates the transcriptional regulation of target genes. Thus, we attempted to identify the protein partners of LncRNA-TBP. First, the potential LncRNA-TBP-binding proteins were predicted using the RNA-protein interaction prediction (RPISeq), and TBP was found may interact with LncRNA-TBP (Fig. S5A). To validate this interaction between LncRNA-TBP and TBP, we performed a RNA immunoprecipitation (RIP) analysis in CPMs. As expected, reverse transcription-polymerase chain reaction (RT-PCR) analysis of antibody-enriched RNA revealed that TBP antibody pulled down significantly more LncRNA-TBP than the IgG control (Fig. 5A), suggesting that TBP interacts with LncRNA-TBP. To determine the core protein-binding domain of LncRNA-TBP, we constructed a series of truncated LncRNA-TBP fragments. We found that like full-length LncRNA-TBP, all of the truncated fragments could physically bind TBP (Fig. 5B, C). Collectively, these findings showed that LncRNA-TBP directly interacts with TBP.
LncRNA-TBP regulates transcriptional activity of TBP-target genes by binding to TBP
The general transcription factor (TBP) is a key initiation factor involved in transcription by all three eukaryotic RNA polymerases, is required for every single transcription event in eukaryotes [29–32]. Through our previous ATAC sequencing analysis, a total of 20 target genes (e.g., glycolysis-related genes (GPI), cell proliferation-related genes (CDKN1A and KLF4) and fast muscle-related genes (TNNI2)) were predicted be regulated by TBP (Fig. S5B and Table S3). Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis found that these TBP-target genes were mainly enriched in biological processes such as cellular process, metabolic process, cellular component organization or biogenesis, and biological regulation, as well as participated in biological processes including metabolic pathways, carbon metabolism and so on (Fig. S5C, D). By performing ChIP-qPCR, we validated that TBP can bind and regulate the promoter of KLF4, GPI, TNNI2, and CDKN1A (Fig. 5D).
Because LncRNA-TBP specifically interacts with TBP, we investigated whether there is a mutual regulation relationship between LncRNA-TBP and TBP in CPMs. The results showed that LncRNA-TBP knockdown or overexpression did not significantly influence TBP mRNA and protein expression (Fig. S6A-D). These results suggested that LncRNA-TBP may regulate myogenesis through its interaction with TBP rather than by regulating TBP gene expression. Given that TBP functions in regulate the promoter activity of its target genes, we also performed ChIP-qPCR to elucidate whether LncRNA-TBP affects the capacity of TBP to bind the promoters of its target genes. LncRNA-TBP overexpression significantly increased the enrichment of TBP to the promoter of KLF4, GPI, TNNI2, and CDKN1A, whereas the results were reversed after LncRNA-TBP knockdown (Fig. 5E and Fig. S7A). Next, to determine whether LncRNA-TBP regulates promoter activity of TBP-target genes such as KLF4, GPI, TNNI2, and CDKN1A, luciferase reporter assays were performed. Overexpression of LncRNA-TBP promoted the promoter activity of KLF4 and CDKN1A while inhibiting the promoter activity of GPI and TNNI2 (Fig. 5F-I). Consistently, the knockdown of LncRNA-TBP had opposite effects in CPMs (Fig. S7B-E). We further examined the mRNA and protein expressions of KLF4, GPI, TNNI2, and CDKN1A. As expected, LncRNA-TBP would promote the expression of KLF4 and CDKN1A, while decreasing the expression of GPI and TNNI2 (Fig. 5J, K and Fig. S7F, G), suggesting that LncRNA-TBP can modulate transcriptional activity of TBP-target genes by binding to TBP protein.
TBP is involved in Myogenesis
TBP was upregulated during myoblast differentiation (Fig. S8A), implying that it may play an important role in skeletal muscle development. Moreover, Subcellular location annotation showed that TBP protein exists in the nucleus (Fig. S8B). To explore the potential biological functions of TBP in myogenesis, we examined the effects of TBP in myoblasts proliferation and differentiation in vivo. TBP was successfully overexpressed or knockdown in CPMs (Fig. 6A, K). Overexpression of TBP reduced cell-cycle-promoting genes while increasing the expression of cell-cycle-inhibiting genes. The EdU and CCK-8 assays showed that overexpression of TBP decreased EdU incorporation and repressed myoblast viability, whereas its inhibition promoted myoblast proliferation (Fig. 6B-E, L-O). Moreover, flow cytometric analysis revealed that TBP overexpression reduced the number of S-phase cells (Fig. 6F). Conversely, TBP inhibition resulted in a greater number of S-phase cells (Fig. 6P).
Next, immunofluorescence staining was performed to detected the role of TBP in myogenetic differentiation. TBP overexpression significantly facilitated myoblast differentiation and increased the total areas of myotubes (Fig. 6G, H). Meanwhile, qPCR and western blotting showed that expressions of myoblast differentiation marker genes were upregulated with TBP overexpression (Fig. 6I, J). In contrast, TBP interference repressed myoblast differentiation (Fig. 6Q-T). Taken together, these data indicated that TBP suppresses myoblast proliferation and promotes myoblast differentiation, which is similar to LncRNA-TBP in function.