Research Note: Association of single nucleotide polymorphism of AKT3 with egg production traits in White Muscovy ducks (Cairina moschata).

Prior studies on transcriptomes of hypothalamus and ovary revealed that AKT3 is one of the candidate genes that might affect egg production in White Muscovy ducks. The role of AKT3 in the uterus during reproductive processes cannot be overemphasized. However, functional role of this gene in the tissues and on egg production traits of Muscovy ducks remains unknown. To identify the relationship between AKT3 and egg production traits in ducks, relative expression profile was first examined prior to identifying the variants within AKT3 that may underscore egg production traits [age at first egg (AFE), number of eggs at 300 d (N300D), and number of eggs at 59 wk (N59W)] in 549 ducks. The mRNA expression of AKT3 gene in high producing (HP) ducks was significantly higher than low producing (LP) ducks in the ovary, oviduct, and hypothalamus (P \u3c 0.05 or 0.001). Three variants in AKT3 (C-3631A, C-3766T, and C-3953T) and high linkage block between C-3766T and C-3953T which are significantly (P \u3c 0.05) associated with N300D and N59W were discovered. This study elucidates novel knowledge on the molecular mechanism of AKT3 that might be regulating egg production traits in Muscovy ducks

Characterization of miR-206 Promoter and Its Association with Birthweight in Chicken

miRNAs have been widely investigated in terms of cell proliferation and differentiation. However, little is known about their effects on bird growth. Here we characterized the promoter of miR-206 in chicken and found that the preferable promoter was located in 1200 bp upstream of pri-miR-206. In this region, many key transcription factors, including MyoD, c-Myb, CEBPα/β, AP-4, RAP1, Brn2, GATA-1/2/3, E47, Sn, upstream stimulatory factor (USF) and CdxA, were predicted to bind and interact with miR-206 promoter. Overexpression of MyoD sharply increased miR-206 expression in both fibroblast and myoblast cells, and also the regulation in the myoblast cells was much stronger, indicating that miR-206 was regulated by MyoD combined with other muscle specific transcriptional factors. Aiming to further investigate the relationship between miR-206 mutation and transcriptional expression, total of 23 SNPs were identified in the two distinct bird lines by sequencing. Interestingly, the motif bound by MyoD was individually destroyed by G-to-C mutation located at 419 bp upstream of miR-206 precursor. Co-transfecting MyoD and miR-206 promoter in DF-1 cells, the luciferase activity of promoter containing homozygous GG types was significantly higher than CC ones (p < 0.05). Thus, this mutation caused low expression of miR-206. Consistently, eight variants including G-419C mutation exhibited a great effect on birthweight through maker-trait association analysis in F2 population (p < 0.05). Additionally, the regulation of miR-206 on embryo muscle mass mainly by increasing MyoG and muscle creatine kinase (MCK) expression (p < 0.05) with little change in MyoD, TMEM8C and myosin heavy chain (MHC). In conclusion, our findings provide a novel mutation destroying the promoter activity of miR-206 in birds and shed new light to understand the regulation mechanism of miR-206 on the embryonic muscle growth

MiR-34b-5p Mediates the Proliferation and Differentiation of Myoblasts by Targeting IGFBP2

As key post-transcriptional regulators, microRNAs (miRNAs) play an indispensable role in skeletal muscle development. Our previous study suggested that miR-34b-5p and IGFBP2 could have a potential role in skeletal muscle growth. Our goal in this study is to explore the function and regulatory mechanism of miR-34b-5p and IGFBP2 in myogenesis. In this study, the dual-luciferase reporter assay and Western blot analysis showed that IGFBP2 is a direct target of miR-34b-5p. Flow cytometric analysis and EdU assay showed that miR-34b-5p could repress the cell cycle progression of myoblasts, and miR-34b-5p could promote the formation of myotubes by promoting the expression of MyHC. On the contrary, the overexpression of IGFBP2 significantly facilitated the proliferation of myoblasts and hampered the formation of myotubes. Together, our results indicate that miR-34b-5p could mediate the proliferation and differentiation of myoblasts by targeting IGFBP2

lncRNA-Six1 Is a Target of miR-1611 That Functions as a ceRNA to Regulate Six1 Protein Expression and Fiber Type Switching in Chicken Myogenesis

Emerging studies indicate important roles for non-coding RNAs (ncRNAs) as essential regulators in myogenesis, but relatively less is known about their function. In our previous study, we found that lncRNA-Six1 can regulate Six1 in cis to participate in myogenesis. Here, we studied a microRNA (miRNA) that is specifically expressed in chickens (miR-1611). Interestingly, miR-1611 was found to contain potential binding sites for both lncRNA-Six1 and Six1, and it can interact with lncRNA-Six1 to regulate Six1 expression. Overexpression of miR-1611 represses the proliferation and differentiation of myoblasts. Moreover, miR-1611 is highly expressed in slow-twitch fibers, and it drives the transformation of fast-twitch muscle fibers to slow-twitch muscle fibers. Together, these data demonstrate that miR-1611 can mediate the regulation of Six1 by lncRNA-Six1, thereby affecting proliferation and differentiation of myoblasts and transformation of muscle fiber types

LncIRS1 controls muscle atrophy via sponging miR‐15 family to activate IGF1‐PI3K/AKT pathway

Abstract Background Recent studies indicate important roles for long noncoding RNAs (lncRNAs) in the regulation of gene expression by acting as competing endogenous RNAs (ceRNAs). However, the specific role of lncRNAs in skeletal muscle atrophy is still unclear. Our study aimed to identify the function of lncRNAs that control skeletal muscle myogenesis and atrophy. Methods RNA sequencing was performed to identify the skeletal muscle transcriptome (lncRNA and messenger RNA) between hypertrophic broilers and leaner broilers. To study the ‘sponge’ function of lncRNA, we constructed a lncRNA‐microRNA (miRNA)‐gene interaction network by integrated our previous submitted skeletal muscle miRNA sequencing data. The primary myoblast cells and animal model were used to assess the biological function of the lncIRS1 in vitro or in vivo. Results We constructed a myogenesis‐associated lncRNA‐miRNA‐gene network and identified a novel ceRNA lncRNA named lncIRS1 that is specifically enriched in skeletal muscle. LncIRS1 could regulate myoblast proliferation and differentiation in vitro, and muscle mass and mean muscle fibre in vivo. LncIRS1 increases gradually during myogenic differentiation. Mechanistically, lncIRS1 acts as a ceRNA for miR‐15a, miR‐15b‐5p, and miR‐15c‐5p to regulate IRS1 expression, which is the downstream of the IGF1 receptor. Overexpression of lncIRS1 not only increased the protein abundance of IRS1 but also promoted phosphorylation level of AKT (p‐AKT) a central component of insulin‐like growth factor‐1 pathway. Furthermore, lncIRS1 regulates the expression of atrophy‐related genes and can rescue muscle atrophy. Conclusions The newly identified lncIRS1 acts as a sponge for miR‐15 family to regulate IRS1 expression, resulting in promoting skeletal muscle myogenesis and controlling atrophy

Long noncoding RNA ZFP36L2-AS functions as a metabolic modulator to regulate muscle development.

Skeletal muscle is the largest metabolic organ in the body, and its metabolic flexibility is essential for maintaining systemic energy homeostasis. Metabolic inflexibility in muscles is a dominant cause of various metabolic disorders, impeding muscle development. In our previous study, we found lncRNA ZFP36L2-AS (for ZFP36L2-antisense transcript ) is specifically enriched in skeletal muscle. Here, we report that ZFP36L2-AS is upregulated during myogenic differentiation, and highly expressed in breast and leg muscle. In vitro, ZFP36L2-AS inhibits myoblast proliferation but promotes myoblast differentiation. In vivo, ZFP36L2-AS facilitates intramuscular fat deposition, as well as activates fast-twitch muscle phenotype and induces muscle atrophy. Mechanistically, ZFP36L2-AS interacts with acetyl-CoA carboxylase alpha (ACACA) and pyruvate carboxylase (PC) to induce ACACA dephosphorylation and damaged PC protein stability, thus modulating muscle metabolism. Meanwhile, ZFP36L2-AS can activate ACACA to reduce acetyl-CoA content, which enhances the inhibition of PC activity. Our findings present a novel model about the regulation of lncRNA on muscle metabolism

Long noncoding RNA SMUL suppresses SMURF2 production-mediated muscle atrophy via nonsense-mediated mRNA decay.

As the world population grows, muscle atrophy leading to muscle wasting could become a bigger risk. Long noncoding RNAs (lncRNAs) are known to play important roles in muscle growth and muscle atrophy. Meanwhile, it has recently come to light that many putative small open reading frames (sORFs) are hidden in lncRNAs; however, their translational capabilities and functions remain unclear. In this study, we uncovered 104 myogenic-associated lncRNAs translated, in at least a small peptide, by integrated transcriptome and proteomic analyses. Furthermore, an upstream ORF (uORF) regulatory network was constructed, and a novel muscle atrophy-associated lncRNA name