This study describes comprehensive polling of transcription start and termination sites and analysis of previously unidentified full-length complementary DNAs derived from the mouse genome. We identify the 5' and 3' boundaries of 181,047 transcripts with extensive variation in transcripts arising from alternative promoter usage, splicing, and polyadenylation. There are 16,247 new mouse protein-coding transcripts, including 5154 encoding previously unidentified proteins. Genomic mapping of the transcriptome reveals transcriptional forests, with overlapping transcription on both strands, separated by deserts in which few transcripts are observed. The data provide a comprehensive platform for the comparative analysis of mammalian transcriptional regulation in differentiation and development.

Abstract Muscle contraction depends on release of Ca(2+) from the sarcoplasmic reticulum (SR) and reuptake by the Ca(2+)adenosine triphosphatase SERCA. We discovered a putative muscle-specific long noncoding RNA that encodes a peptide of 34 amino acids and that we named dwarf open reading frame (DWORF). DWORF localizes to the SR membrane, where it enhances SERCA activity by displacing the SERCA inhibitors, phospholamban, sarcolipin, and myoregulin. In mice, overexpression of DWORF in cardiomyocytes increases peak Ca(2+) transient amplitude and SR Ca(2+) load while reducing the time constant of cytosolic Ca(2+) decay during each cycle of contraction-relaxation. Conversely, slow skeletal muscle lacking DWORF exhibits delayed Ca(2+) clearance and relaxation and reduced SERCA activity. DWORF is the only endogenous peptide known to activate the SERCA pump by physical interaction and provides a means for enhancing muscle contractility. Copyright 脗漏 2016, American Association for the Advancement of Science.

Abstract Functional micropeptides can be concealed within RNAs that appear to be noncoding. We discovered a conserved micropeptide, which we named myoregulin (MLN), encoded by a skeletal muscle-specific RNA annotated as a putative long noncoding RNA. MLN shares structural and functional similarity with phospholamban (PLN) and sarcolipin (SLN), which inhibit SERCA, the membrane pump that controls muscle relaxation by regulating Ca(2+) uptake into the sarcoplasmic reticulum (SR). MLN interacts directly with SERCA and impedes Ca(2+) uptake into the SR. In contrast to PLN and SLN, which are expressed in cardiac and slow skeletal muscle in mice, MLN is robustly expressed in all skeletal muscle. Genetic deletion of MLN in mice enhances Ca(2+) handling in skeletal muscle and improves exercise performance. These findings identify MLN as an important regulator of skeletal muscle physiology and highlight the possibility that additional micropeptides are encoded in the many RNAs currently annotated as noncoding. Copyright 脗漏 2015 Elsevier Inc. All rights reserved.

Abstract Micropeptides function as master regulators of calcium-dependent signaling in muscle. Sarco/endoplasmic reticulum Ca0562 ATPase (SERCA), the membrane pump that promotes muscle relaxation by taking up Ca0562 into the sarcoplasmic reticulum, is directly inhibited by three muscle-specific micropeptides: myoregulin (MLN), phospholamban (PLN), and sarcolipin (SLN). The widespread and essential function of SERCA across diverse cell types has raised questions as to how SERCA is regulated in cells that lack MLN, PLN, and SLN. We identified two transmembrane micropeptides, endoregulin (ELN) and another-regulin (ALN), that share key amino acids with their muscle-specific counterparts and function as direct inhibitors of SERCA pump activity. The distribution of transcripts encoding ELN and ALN mirrored that of SERCA isoform-encoding transcripts in nonmuscle cell types. Our findings identify additional members of the SERCA-inhibitory micropeptide family, revealing a conserved mechanism for the control of intracellular Ca0562 dynamics in both muscle and nonmuscle cell types.

Abstract High-throughput sequencing has revealed that the majority of RNAs have no capacity to encode protein. Among these non-coding transcripts, recent work has focused on the roles of long noncoding RNAs (lncRNAs) of >200 nucleotides. Although many of their attributes, such as patterns of expression, remain largely unknown, lncRNAs have key functions in transcriptional, post-transcriptional, and epigenetic gene regulation; Also, new work indicates their functions in scaffolding ribonuclear protein complexes. In plants, genome-wide identification of lncRNAs has been conducted in several species, including Zea mays, and recent research showed that lncRNAs regulate flowering time in the photoperiod pathway, and function in nodulation. In this review, we discuss the basic mechanisms by which lncRNAs regulate key cellular processes, using the large body of knowledge on animal and yeast lncRNAs to illustrate the significance of emerging work on lncRNAs in plants.

Abstract Skeletal muscle myogenesis during development and the injury induced regeneration contribute to the formation and maintenance of muscle tissue. Emerging studies have demonstrated that long non-coding RNAs (lncRNAs) participate in the regulation of gene expression during skeletal myogenesis and their aberrant expression is associated with several muscular diseases. In this review, we summarize recent studies of lncRNAs in the regulation of myogenesis and muscle diseases with mechanistic characterization. These findings have greatly enhanced our understanding of gene regulatory mechanisms governing muscle formation and regeneration, which will eventually lead to novel therapeutics against various muscle diseases.

Here, we present a unifying hypothesis about how messenger RNAs, transcribed pseudogenes, and long noncoding RNAs "talk" to each other using microRNA response elements (MREs) as letters of a new language. We propose that this "competing endogenous RNA" (ceRNA) activity forms a large-scale regulatory network across the transcriptome, greatly expanding the functional genetic information in the human genome and playing important roles in pathological conditions, such as cancer.

Long non-coding RNAs are regulators of various biological functions. Gong and Li et聽al. show that LncMyoD is a LncRNA target of MyoD during myogenesis and is required for myoblast differentiation by affecting IMP2-mediated mRNA translation. LncMyoD is functionally conserved between mouse and human, despite limited sequence homology.

Abstract Myostatin, a member of the transforming growth factor-0205 (TGF-0205) superfamily of secreted proteins, is a potent negative regulator of myogenesis. Free myostatin induces the phosphorylation of the Smad family of transcription factors, which, in turn, regulates gene expression, via the canonical TGF-0205 signaling pathway. There is, however, emerging evidence that myostatin can regulate gene expression independent of Smad signaling. As such, we acquired global gene expression data from the gastrocnemius muscle of C57BL/6 mice following a 6-day treatment with recombinant myostatin compared with vehicle-treated animals. Of the many differentially expressed genes, the myostatin-associated decrease (-11.20-fold; P < 0.05) in the noncoding metastasis-associated lung adenocarcinoma transcript 1 (Malat1) was the most significant and the most intriguing because of numerous reports describing its novel role in regulating cell growth. We therefore sought to further characterize the role of Malat1 expression in skeletal muscle myogenesis. RT-PCR-based quantification of C2C12 and primary human skeletal muscle cells revealed a significant and persistent upregulation (4- to 7-fold; P < 0.05) of Malat1 mRNA during the differentiation of myoblasts into myotubes. Conversely, targeted knockdown of Malat1 using siRNA suppressed myoblast proliferation by arresting cell growth in the G(0)/G(1) phase. These results reveal Malat1 as novel downstream target of myostatin with a considerable ability to regulate myogenesis. The identification of new targets of myostatin will have important repercussions for regenerative biology through inhibition and/or reversal of muscle atrophy and wasting diseases.

Abstract Serum response factor (SRF) controls the transcription of muscle genes by recruiting a variety of partner proteins, including members of the myocardin family of transcriptional coactivators. Mice lacking SRF fail to form mesoderm and die before gastrulation, precluding an analysis of the roles of SRF in muscle tissues. To investigate the functions of SRF in skeletal muscle development, we conditionally deleted the Srf gene in mice by skeletal muscle-specific expression of Cre recombinase. In mice lacking skeletal muscle SRF expression, muscle fibers formed, but failed to undergo hypertrophic growth after birth. Consequently, mutant mice died during the perinatal period from severe skeletal muscle hypoplasia. The myopathic phenotype of these mutant mice resembled that of mice expressing a dominant negative mutant of a myocardin family member in skeletal muscle. These findings reveal an essential role for the partnership of SRF and myocardin-related transcription factors in the control of skeletal muscle growth and maturation in vivo.

Metastasis-associated lung adenocarcinoma transcript 1 (Malat1) is an example of a functional long noncoding RNA involved in many biologic processes. However, the mechanisms for Malat1 in myogenesis are unclear. Serum response factor (SRF) is a pivotal transcription factor for muscle proliferation and differentiation and is reported to be a target gene for muscle-specific microRNA-133 (miR-133). In this study, we initially found that silencing Malat1 in the mouse myoblast C2C12 cell line inhibited myocyte differentiation and decreased Srf at both the RNA and protein levels. Srf silencing decreased Malat1 expression as well. Further study revealed that Malat1 contained an miR-133 functional target site, and the interplay between Malat1 and Srf was miR-133 dependent. We demonstrated that Malat1 modulates Srf through miR-133 as a competing endogenous RNA and established a novel connection among Malat1, miR-133, and Srf in myoblast differentiation.

Abstract Recently, a new regulatory circuitry has been identified in which RNAs can crosstalk with each other by competing for shared microRNAs. Such competing endogenous RNAs (ceRNAs) regulate the distribution of miRNA molecules on their targets and thereby impose an additional level of post-transcriptional regulation. Here we identify a muscle-specific long noncoding RNA, linc-MD1, which governs the time of muscle differentiation by acting as a ceRNA in mouse and human myoblasts. Downregulation or overexpression of linc-MD1 correlate with retardation or anticipation of the muscle differentiation program, respectively. We show that linc-MD1 "sponges" miR-133 and miR-133 [corrected] to regulate the expression of MAML1 and MEF2C, transcription factors that activate muscle-specific gene expression. Finally, we demonstrate that linc-MD1 exerts the same control over differentiation timing in human myoblasts, and that its levels are strongly reduced in Duchenne muscle cells. We conclude that the ceRNA network plays an important role in muscle differentiation. Copyright 漏 2011 Elsevier Inc. All rights reserved.

The muscle-specific long noncoding RNA linc-MD1 was shown to be expressed during early phases of muscle differentiation and to trigger the switch to later stages by acting as a sponge for miR-133 and miR-135. Notably, linc-MD1 is also the host transcript of miR-133b, and their biogenesis is mutually exclusive. Here, we describe that this alternative synthesis is controlled by the HuR protein, which favors linc-MD1 accumulation through its ability to bind linc-MD1 and repress Drosha cleavage. We show that HuR is under the repressive control of miR-133 and that the sponging activity of linc-MD1 consolidates HuR expression in a feedforward positive loop. Finally, we show that HuR also acts in the cytoplasm, reinforcing linc-MD1 sponge activity by cooperating for miRNA recruitment. An increase in miR-133 synthesis, mainly from the two unrelated miR-133a coding genomic loci, is likely to trigger the exit from this circuitry and progression to later differentiation stages.

Abstract Abundantly expressed in fetal tissues and adult muscle, the developmentally regulated H19 long noncoding RNA (lncRNA) has been implicated in human genetic disorders and cancer. However, how H19 acts to regulate gene function has remained enigmatic, despite the recent implication of its encoded miR-675 in limiting placental growth. We noted that vertebrate H19 harbors both canonical and noncanonical binding sites for the let-7 family of microRNAs, which plays important roles in development, cancer, and metabolism. Using H19 knockdown and overexpression, combined with in vivo crosslinking and genome-wide transcriptome analysis, we demonstrate that H19 modulates let-7 availability by acting as a molecular sponge. The physiological significance of this interaction is highlighted in cultures in which H19 depletion causes precocious muscle differentiation, a phenotype recapitulated by let-7 overexpression. Our results reveal an unexpected mode of action of H19 and identify this lncRNA as an important regulator of the major let-7 family of microRNAs. Copyright 脗漏 2013 Elsevier Inc. All rights reserved.

Recent studies indicate important roles for long noncoding RNAs (lncRNAs) as essential regulators of myogenesis and adult skeletal muscle regeneration. However, the specific roles of lncRNAs in myogenic differentiation of adult skeletal muscle stem cells and myogenesis are still largely unknown. Here we identify a lncRNA that is specifically enriched in skeletal muscle (myogenesis-associated lncRNA, in short, lnc-mg). In mice, conditional knockout of lnc-mg in skeletal muscle results in muscle atrophy and the loss of muscular endurance during exercise. Alternatively, skeletal muscle-specific overexpression of lnc-mg promotes muscle hypertrophy. In vitro analysis of primary skeletal muscle cells shows that lnc-mg increases gradually during myogenic differentiation and its overexpression improves cell differentiation. Mechanistically, lnc-mg promotes myogenesis, by functioning as a competing endogenous RNA (ceRNA) for microRNA-125b to control protein abundance of insulin-like growth factor 2. These findings identify lnc-mg as a novel noncoding regulator for muscle cell differentiation and skeletal muscle development.

Abstract Emerging studies document the roles of long non-coding RNAs (LncRNAs) in regulating gene expression at chromatin level but relatively less is known how they regulate DNA methylation. Here we identify an lncRNA, Dum (developmental pluripotency-associated 2 (Dppa2) Upstream binding Muscle lncRNA) in skeletal myoblast cells. The expression of Dum is dynamically regulated during myogenesis in vitro and in vivo. It is also transcriptionally induced by MyoD binding upon myoblast differentiation. Functional analyses show that it promotes myoblast differentiation and damage-induced muscle regeneration. Mechanistically, Dum was found to silence its neighboring gene, Dppa2, in cis through recruiting Dnmt1, Dnmt3a and Dnmt3b. Furthermore, intrachromosomal looping between Dum locus and Dppa2 promoter is necessary for Dum/Dppa2 interaction. Collectively, we have identified a novel lncRNA that interacts with Dnmts to regulate myogenesis.Cell Research advance online publication 17 February 2015; doi:10.1038/cr.2015.21.

Repetitive sequences account for more than 50% of the human genome. Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal-dominant disease associated with reduction in the copy number of the D4Z4 repeat mapping to 4q35. By an unknown mechanism, D4Z4 deletion causes an epigenetic switch leading to de-repression of 4q35 genes. Here we show that the Polycomb group of epigenetic repressors targets D4Z4 in healthy subjects and that D4Z4 deletion is associated with reduced Polycomb silencing in FSHD patients. We identify DBE-T, a chromatin-associated noncoding RNA produced selectively in FSHD patients that coordinates de-repression of 4q35 genes. DBE-T recruits the Trithorax group protein Ash1L to the FSHD locus, driving histone H3 lysine 36 dimethylation, chromatin remodeling, and 4q35 gene transcription. This study provides insights into the biological function of repetitive sequences in regulating gene expression and shows how mutations of such elements can influence the progression of a human genetic disease.

Abstract X chromosome inactivation and genomic imprinting are classic epigenetic processes that cause disease when not appropriately regulated in mammals. Whereas X chromosome inactivation evolved to solve the problem of gene dosage, the purpose of genomic imprinting remains controversial. Nevertheless, the two phenomena are united by allelic control of large gene clusters, such that only one copy of a gene is expressed in every cell. Allelic regulation poses significant challenges because it requires coordinated long-range control in cis and stable propagation over time. Long noncoding RNAs have emerged as a common theme, and their contributions to diseases of imprinting and the X chromosome have become apparent. Here, we review recent advances in basic biology, the connections to disease, and preview potential therapeutic strategies for future development. Copyright 脗漏 2013 Elsevier Inc. All rights reserved.

Polycomb proteins play essential roles in stem cell renewal and human disease. Recent studies of HOX genes and X inactivation have provided evidence for RNA cofactors in Polycomb repressive complex 2 (PRC2). Here we develop a RIP-seq method to capture the PRC2 transcriptome and identify聽a genome-wide pool of >9000 PRC2-interacting RNAs in embryonic stem cells. The transcriptome includes antisense, intergenic, and promoter-associated transcripts, as well as many unannotated RNAs. A large number of transcripts occur within imprinted regions, oncogene and tumor suppressor loci, and stem cell-related bivalent domains. We provide evidence for direct RNA-protein interactions, most likely via the Ezh2 subunit. We also identify Gtl2 RNA as a PRC2 cofactor that directs PRC2 to the reciprocally imprinted Dlk1 coding gene. Thus, Polycomb proteins interact with a genome-wide family of RNAs, some of which may be used as biomarkers and therapeutic targets for human disease.

The Dlk1-Gtl2 imprinting locus is located on mouse distal chromosome 12 and consists of multiple maternally expressed non-coding RNAs and several paternally expressed protein-coding genes. The imprinting of this locus plays a crucial role in embryonic development and postnatal growth. At least one cis-element, the intergenic differentially methylated region (IG-DMR) is required for expression of maternally expressed genes and repression of silenced paternally expressed genes. The mechanism by which the IG-DMR functions is largely unknown. However, it has been suggested that the unmethylated IG-DMR acts as a positive regulator activating expression of non-coding RNAs. Gtl2 is the first non-coding RNA gene downstream of the IG-DMR. Although its in vivo function in the mouse is largely unknown, its human ortholog MEG3 has been linked to tumor suppression in human tumor-derived cell lines. We generated a knockout mouse model, in which the first five exons and adjacent promoter region of the Gtl2 gene were deleted. Maternal deletion of Gtl2 resulted in perinatal death and skeletal muscle defects, indicating that Gtl2 plays an important role in embryonic development. The maternal deletion also completely abolished expression of downstream maternally expressed genes, activated expression of silenced paternally expressed genes and resulted in methylation of the IG-DMR. By contrast, the paternal inherited deletion did not have this effect. These data strongly indicate that activation of Gtl2 and its downstream maternal genes play an essential role in regulating Dlk1-Gtl2 imprinting, possibly by maintaining active status of the IG-DMR.

The callipyge ( CLPG ) phenotype is an inherited skeletal muscle hypertrophy described in sheep. It is characterized by an unusual mode of inheritance (鈥減olar overdominance鈥) in which only heterozygous individuals having received the CLPG mutation from their father ( + MAT /CLPG PAT ) express the phenotype [1] . + MAT /CLPG PAT individuals are born normal and develop the muscular hypertrophy at approximately 1 month of age. The CLPG mutation was identified as an A to G transition in a highly conserved dodecamer motif located between the imprinted DLK1 and GTL2 genes 2 and 3 . This motif is thought to be part of a long-range control element (LRCE) because the CLPG mutation was shown, in postnatal skeletal muscle, to enhance the transcript levels of the DLK1, PEG11, GTL2 , and MEG8 genes in cis without altering their imprinting status [4] . As a result, the + MAT /CLPG PAT individuals have a unique expression profile thought to underlie the callipyge phenotype: an overexpression of the paternally expressed protein encoding DLK1 (Figure 1A) and PEG11 transcripts in the absence of an overexpression of the maternally expressed noncoding GTL2 and MEG8 transcripts [4] . However, the way in which this distinct expression profile causes the callipyge muscular hypertrophy has remained unclear. Herein, we demonstrate that the callipyge phenotype is perfectly correlated with ectopic expression of DLK1 protein in hypertrophied muscle of + MAT /CLPG PAT sheep. We demonstrate the causality of this association by inducing a generalized muscular hypertrophy in transgenic mice that express DLK1 in skeletal muscle. The absence of DLK1 protein in skeletal muscle of CLPG/CLPG animals, despite the presence of DLK1 mRNA, supports a trans inhibition mediated by noncoding RNAs expressed from the maternal allele.

Staufen1-mediated mRNA decay (SMD) degrades mRNAs that harbor a Staufen1-binding site (SBS) in their 3' untranslated regions (UTRs). Human SBSs can form by intermolecular base-pairing between a 3' UTR Alu element and an Alu element within a long noncoding RNA (lncRNA) called a 1/2-sbsRNA. Since Alu elements are confined to primates, it was unclear how SMD occurs in rodents. Here we identify mouse mRNA 39 UTRs and lncRNAs that contain a B1, B2, B4, or identifier (ID) element. We show that SMD occurs in mouse cells via mRNA-lncRNA base-pairing of partially complementary elements and that mouse 1/2-sbsRNA (m 1/2-sbsRNA)triggered SMD regulates C2C12 cell myogenesis. Our findings define new roles for lncRNAs as well as B and ID short interspersed elements (SINEs) in mice that undoubtedly influence many developmental and homeostatic pathways.

Alternative splicing (AS) of pre-mRNA is utilized by higher eukaryotes to achieve increased transcriptome and proteomic complexity. The serine/arginine (SR) splicing factors regulate tissue- or cell-type-specific AS in a concentration- and phosphorylation-dependent manner. However, the mechanisms that modulate the cellular levels of active SR proteins remain to be elucidated. In the present study, we provide evidence for a role for the long nuclear-retained regulatory RNA (nrRNA), MALAT1 in AS regulation. MALAT1 interacts with SR proteins and influences the distribution of these and other splicing factors in nuclear speckle domains. Depletion of MALAT1 or overexpression of an SR protein changes the AS of a similar set of endogenous pre-mRNAs. Furthermore, MALAT1 regulates cellular levels of phosphorylated forms of SR proteins. Taken together, our results suggest that MALAT1 regulates AS by modulating the levels of active SR proteins. Our results further highlight the role for an nrRNA in the regulation of gene expression.Highlights? Nuclear speckle-localized MALAT1 interacts with SR splicing factors ? MALAT1 influences the nuclear speckle distribution of splicing factors ? MALAT1 depletion changes alternative splicing of endogenous pre-mRNAs ? MALAT1 regulates the cellular levels of phosphorylated SR proteins

Abstract The process of cellular differentiation culminating in terminally differentiated mammalian cells is thought to be irreversible. Here, we present evidence that terminally differentiated murine myotubes can be induced to dedifferentiate. Ectopic expression of msx1 in C2C12 myotubes reduced the nuclear muscle proteins MyoD, myogenin, MRF4, and p21 to undetectable levels in 20%-50% of the myotubes. Approximately 9% of the myotubes cleave to produce either smaller multinucleated myotubes or proliferating, mononucleated cells. Finally, clonal populations of the myotube-derived mononucleated cells can be induced to redifferentiate into cells expressing chondrogenic, adipogenic, myogenic, and osteogenic markers. These results suggest that terminally differentiated mammalian myotubes can dedifferentiate when stimulated with the appropriate signals and that msx1 can contribute to the dedifferentiation process.

Msx1 is a key factor for the development of tooth and craniofacial skeleton and has been proposed to play a pivotal role in terminal cell differentiation. In this paper, we demonstrated the presence of an endogenous Msx1 antisense RNA (Msx1-AS RNA) in mice, rats, and humans. In situ analysis revealed that this RNA is expressed only in differentiated dental and bone cells with an inverse correlation with Msx1 protein. These in vivo data and overexpression of Msx1 sense and AS RNA in an odontoblastic cell line (MO6-G3) showed that the balance between the levels of the two Msx1 RNAs is related to the expression of Msx1 protein. To analyze the impact of this balance in the Msx-Dlx homeoprotein pathway, we analyzed the effect of Msx1, Msx2, and Dlx5 overexpression on proteins involved in skeletal differentiation. We showed that the Msx1-AS RNA is involved in crosstalk between the Msx-Dlx pathways because its expression was abolished by Dlx5. Msx1 was shown to down-regulate a master gene of skeletal cells differentiation, Cbfa1. All these data strongly suggest that the ratio between Msx1 sense and antisense RNAs is a very important factor in the control of skeletal terminal differentiation. Finally, the initiation site for Msx1-AS RNA transcription was located by primer extension in both mouse and human in an identical region, including a consensus TATA box, suggesting an evolutionary conservation of the AS RNA-mediated regulation of Msx1 gene expression.

Skeletal muscle aging is associated with increased inflammation and oxidative stress, a decrease in the ability to rebuild muscle after injury and in response to exercise. In this perspective, we discuss the mechanisms regulating Sirt1 activity and expression in skeletal muscles, emphasizing their implications in muscle physiology and the impairment of muscle function with age.

Recent studies demonstrate the functions of long non-coding RNAs (lncRNAs) in mediating gene expression at the transcriptional or translational level. Our previous study identified a Sirt1 antisense (AS) lncRNA transcribed from the Sirt1 AS strand. However, its role and regulatory mechanism is still unknown in myogenesis. Here, functional analyses showed that Sirt1 AS lncRNA overexpression promoted myoblast proliferation, but inhibited differentiation. Mechanistically, Sirt1 AS lncRNA was found to activate its sense gene, Sirt1. The luciferase assay provided evidences that Sirt1 AS lncRNA interacted with Sirt1 3鈥 UTR and rescued Sirt1 transcriptional suppression by competing with miR-34a. In addition, RNA stability assay showed that Sirt1 AS lncRNA prolonged Sirt1 mRNA half-life from 2 to 10鈥塰. Ribonuclease protection assay further indicated that it fully bound to Sirt1 mRNA in the myoblast cytoplasm. Moreover, Sirt1 AS overexpression led to less mouse weight than the control because of less lean mass and greater levels of Sirt1, whereas the fat mass and levels of miR-34a were not altered. Based on the findings, a novel regulatory mechanism was found that Sirt1 AS lncRNA preferably interacted with Sirt1 mRNA forming RNA duplex to promote Sirt1 translation by competing with miR-34a, inhibiting muscle formation.

Abstract An in silico screen for myogenic long noncoding RNAs (lncRNAs) revealed nine lncRNAs that are upregulated more than 10-fold in myotubes versus levels in myoblasts. One of these lncRNAs, MyoD upstream noncoding (MUNC, also known as DRR(eRNA)), is encoded 5 kb upstream of the transcription start site of MyoD, a myogenic transcription factor gene. MUNC is specifically expressed in skeletal muscle and exists as in unspliced and spliced isoforms, and its 5' end overlaps with the cis-acting distal regulatory region (DRR) of MyoD. Small interfering RNA (siRNA) of MUNC reduced myoblast differentiation and specifically reduced the association of MyoD to the DRR enhancer and myogenin promoter but not to another MyoD-dependent enhancer. Stable overexpression of MUNC from a heterologous promoter increased endogenous MyoD, Myogenin, and Myh3 (myosin heavy chain, [MHC] gene) mRNAs but not the cognate proteins, suggesting that MUNC can act in trans to promote gene expression but that this activity does not require an induction of MyoD protein. MUNC also stimulates the transcription of other genes that are not recognized as MyoD-inducible genes. Knockdown of MUNC in vivo impaired murine muscle regeneration, implicating MUNC in primary satellite cell differentiation in the animal. We also discovered a human MUNC that is induced during differentiation of myoblasts and whose knockdown decreases differentiation, suggesting an evolutionarily conserved role of MUNC lncRNA in myogenesis. Although MUNC overlaps with the DRR enhancer, our results suggest that MUNC is not a classic cis-acting enhancer RNA (e-RNA) acting exclusively by stimulating the neighboring MyoD gene but more like a promyogenic lncRNA that acts directly or indirectly on multiple promoters to increase myogenic gene expression. Copyright 漏 2015, American Society for Microbiology. All Rights Reserved.

Transcription factors and DNA regulatory binding motifs are fundamental components of the gene regulatory network. Here, by using genome-wide binding profiling, we show extensive occupancy of transcription factors of myogenesis (MyoD and Myogenin) at extragenic enhancer regions coinciding with RNA synthesis (i.e., eRNA). In particular, multiple regions were transcribed to eRNA within the regulatory region of MYOD1, including previously characterized distal regulatory regions (DRR) and core enhancer (CE). While (CE)RNA enhanced RNA polymerase II (Pol II) occupancy and transcription at MYOD1, DRR RNA acted to activate the downstream myogenic genes. The deployment of transcriptional machinery to appropriate loci is contingent on chromatin accessibility, a rate-limiting step preceding Pol II assembly. By nuclease sensitivity assay, we found that eRNAs regulate genomic access of the transcriptional complex to defined regulatory regions. In conclusion, our data suggest that eRNAs contribute to establishing a cell-type-specific transcriptional circuitry by directing chromatin-remodeling events.

Abstract The steroid receptor RNA activator (SRA) has the unusual property to function as both a non-coding RNA (ncRNA) and a protein SRAP. SRA ncRNA is known to increase the activity of a range of nuclear receptors as well as the master regulator of muscle differentiation MyoD. The contribution of SRA to either a ncRNA or a protein is influenced by alternative splicing of the first intron, the retention of which disrupts the SRAP open reading frame. We reported here that the ratio between non-coding and coding SRA isoforms increased during myogenic differentiation of human satellite cells but not myotonic dystrophy patient satellite cells, in which differentiation capacity is affected. Using constructs that exclusively produce SRA ncRNA or SRAP, we demonstrated that whereas SRA ncRNA was indeed an enhancer of myogenic differentiation and myogenic conversion of non-muscle cells through the co-activation of MyoD activity, SRAP prevented this SRA RNA-dependant co-activation. Interestingly, the SRAP inhibitory effect is mediated through the interaction of SRAP with its RNA counterpart via its RRM-like domain interacting with the functional sub-structure of SRA RNA, STR7. This study thus provides a new model for SRA-mediated regulation of MyoD transcriptional activity in the promotion of normal muscle differentiation, which takes into account the nature of SRA molecules present.

Skeletal muscle differentiation is orchestrated by a network of transcription factors, epigenetic regulators, and non-coding RNAs. The transcription factor Yin Yang 1 (YY1) silences multiple target genes in myoblasts (MBs) by recruiting Ezh2 (Enhancer of Zeste Homologue2). To elucidate genome-wide YY1 binding in MBs, we performed chromatin immunoprecipitation (ChIP)-seq and found 1820 specific binding sites in MBs with a large portion residing in intergenic regions. Detailed analysis demonstrated that YY1 acts as an activator for many loci in addition to its known repressor function. No significant co-occupancy was found between YY1 and Ezh2, suggesting an additional Ezh2-independent function for YY1 in MBs. Further analysis of intergenic binding sites showed that YY1 potentially regulates dozens of large intergenic non-coding RNAs (lincRNAs), whose function in myogenesis is underexplored. We characterized a novel muscle-associated lincRNA (Yam-1) that is positively regulated by YY1. Yam-1 is downregulated upon differentiation and acts as an inhibitor of myogenesis. We demonstrated that Yam-1 functions through in cis regulation of miR-715, which in turn targets Wnt7b. Our findings not only provide the first genome-wide picture of YY1 association in muscle cells, but also uncover the functional role of lincRNA Yam-1.

Abstract Long non-coding RNAs (lncRNAs) are important regulators of diverse biological processes. Here we report on functional identification and characterization of a novel long intergenic non-coding RNA with MyoD-regulated and skeletal muscle-restricted expression that promotes the activation of the myogenic program, and is therefore termed Linc-RAM (Linc-RNA Activator of Myogenesis). Linc-RAM is transcribed from an intergenic region of myogenic cells and its expression is upregulated during myogenesis. Notably, in vivo functional studies show that Linc-RAM knockout mice display impaired muscle regeneration due to the differentiation defect of satellite cells. Mechanistically, Linc-RAM regulates expression of myogenic genes by directly binding MyoD, which in turn promotes the assembly of the MyoD-Baf60c-Brg1 complex on the regulatory elements of target genes. Collectively, our findings reveal the functional role and molecular mechanism of a lineage-specific Linc-RAM as a regulatory lncRNA required for tissues-specific chromatin remodelling and gene expression.

Abstract Although long non-coding RNAs (lncRNAs) are non-protein-coding transcripts by definition, recent studies have shown that a fraction of putative small open reading frames within lncRNAs are translated. However, the biological significance of these hidden polypeptides is still unclear. Here we identify and functionally characterize a novel polypeptide encoded by the lncRNA LINC00961. This polypeptide is conserved between human and mouse, is localized to the late endosome/lysosome and interacts with the lysosomal v-ATPase to negatively regulate mTORC1 activation. This regulation of mTORC1 is specific to activation of mTORC1 by amino acid stimulation, rather than by growth factors. Hence, we termed this polypeptide 'small regulatory polypeptide of amino acid response' (SPAR). We show that the SPAR-encoding lncRNA is highly expressed in a subset of tissues and use CRISPR/Cas9 engineering to develop a SPAR-polypeptide-specific knockout mouse while maintaining expression of the host lncRNA. We find that the SPAR-encoding lncRNA is downregulated in skeletal muscle upon acute injury, and using this in vivo model we establish that SPAR downregulation enables efficient activation of mTORC1 and promotes muscle regeneration. Our data provide a mechanism by which mTORC1 activation may be finely regulated in a tissue-specific manner in response to injury, and a paradigm by which lncRNAs encoding small polypeptides can modulate general biological pathways and processes to facilitate tissue-specific requirements, consistent with their restricted and highly regulated expression profile.

The vast majority of the mammalian genome is transcribed giving rise to many different types of noncoding RNAs. Among them, long noncoding RNAs are the most numerous and functionally versatile class. Indeed, the lncRNA repertoire might be as rich as the proteome. LncRNAs have emerged as key regulators of gene expression at multiple levels. They play important roles in the regulation of development, differentiation and maintenance of cell identity and they also contribute to disease. In this review, we present recent advances in the biology of lncRNAs in muscle development and differentiation. We will also discuss the contribution of lncRNAs to muscle disease with a particular focus on Duchenne and facioscapulohumeral muscular dystrophies.

A significant portion of the mammalian genome encodes numerous transcripts that are not translated into proteins, termed long non-coding RNAs. Initial studies identifying long non-coding RNAs inferred these RNA sequences were a consequence of transcriptional noise or promiscuous RNA polymerase II activity. However, the last decade has seen a revolution in the understanding of regulation and function of long non-coding RNAs. Now it has become apparent that long non-coding RNAs play critical roles in a wide variety of biological processes. In this review, we describe the current understanding of long non-coding RNA-mediated regulation of cellular processes: differentiation, development, and disease.

Transcriptome analysis of mammalian genomes has revealed widespread transcription, much of which does not encode protein. Long non-coding RNAs (lncRNAs) are a subset of the non-coding transcriptome that are emerging as critical regulators of various cellular processes. Differentiation of stem and progenitor cells requires a careful execution of specific genetic programs, and recent studies have revealed that lncRNA expression contributes to specification of cell identity. LncRNAs participate in regulating differentiation at multiple levels of gene expression through various mechanisms of action. In this review, functional roles of lncRNAs in regulating cellular differentiation of blood, muscle, skin, cardiomyocytes, adipocytes, and neurons are discussed.

It is known that more than 70% of mammalian genomes are transcribed, yet the vast majority of transcripts do not code for proteins. Are these noncoding transcripts merely transcriptional noise, or do they serve a biological purpose? Recent developments in genomic analysis technologies, especially sequencing methods, have allowed researchers to create a large atlas of transcriptomes, study subcellular localization, and investigate potential interactions with proteins for a growing number of transcripts. Here, we review the current methodologies available for discovering and investigating functions of long noncoding RNAs (lncRNAs), which require a wide variety of applications to study their potential biological roles. This article is part of a Special Issue entitled: Clues to long noncoding RNA taxonomy1, edited by Dr. Tetsuro Hirose and Dr. Shinichi Nakagawa.

RNA-sequencing (RNA-seq) measures the quantitative change in gene expression over the whole transcriptome, but it lacks spatial context. In contrast, in situ hybridization provides the location of gene expression, but only for a small number of genes. Here we detail a protocol for genome-wide profiling of gene expression in situ in fixed cells and tissues, in which RNA is converted into cross-linked cDNA amplicons and sequenced manually on a confocal microscope. Unlike traditional RNA-seq, our method enriches for context-specific transcripts over housekeeping and/or structural RNA, and it preserves the tissue architecture for RNA localization studies. Our protocol is written for researchers experienced in cell microscopy with minimal computing skills. Library construction and sequencing can be completed within 14 d, with image analysis requiring an additional 2 d.

Abstract RNA serves many functions in biology such as splicing, temperature sensing, and innate immunity. These functions are often determined by the structure of RNA. There is thus a pressing need to understand RNA structure and how it changes during diverse biological processes both in vivo and genome-wide. Here, we present Structure-seq2, which provides nucleotide-resolution RNA structural information in vivo and genome-wide. This optimized version of our original Structure-seq method increases sensitivity by at least 4-fold and improves data quality by minimizing formation of a deleterious by-product, reducing ligation bias, and improving read coverage. We also present a variation of Structure-seq2 in which a biotinylated nucleotide is incorporated during reverse transcription, which greatly facilitates the protocol by eliminating two PAGE purification steps. We benchmark Structure-seq2 on both mRNA and rRNA structure in rice (Oryza sativa). We demonstrate that Structure-seq2 can lead to new biological insights. Our Structure-seq2 datasets uncover hidden breaks in chloroplast rRNA and identify a previously unreported N1-methyladenosine (m1A) in a nuclear-encoded Oryza sativa rRNA. Overall, Structure-seq2 is a rapid, sensitive, and unbiased method to probe RNA in vivo and genome-wide that facilitates new insights into RNA biology. 漏 The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) represent two classes of important non-coding RNAs in eukaryotes. Although these non-coding RNAs have been implicated in organismal development and in various human diseases, surprisingly little is known about their transcriptional regulation. Recent advances in chromatin immunoprecipitation with next-generation DNA sequencing (ChIP-Seq) have provided methods of detecting transcription factor binding sites (TFBSs) with unprecedented sensitivity. In this study, we describe ChIPBase (http://deepbase.sysu.edu.cn/chipbase/), a novel database that we have developed to facilitate the comprehensive annotation and discovery of transcription factor binding maps and transcriptional regulatory relationships of lncRNAs and miRNAs from ChIP-Seq data. The current release of ChIPBase includes high-throughput sequencing data that were generated by 543 ChIP-Seq experiments in diverse tissues and cell lines from six organisms. By analysing millions of TFBSs, we identified tens of thousands of TF-lncRNA and TF-miRNA regulatory relationships. Furthermore, two web-based servers were developed to annotate and discover transcriptional regulatory relationships of lncRNAs and miRNAs from ChIP-Seq data. In addition, we developed two genome browsers, deepView and genomeView, to provide integrated views of multidimensional data. Moreover, our web implementation supports diverse query types and the exploration of TFs, lncRNAs, miRNAs, gene ontologies and pathways.

Abstract Recently, the attention of the research community has been focused on long non-coding RNAs (lncRNAs) and their physiological/pathological implications. As the number of experiments increase in a rapid rate and transcriptional units are better annotated, databases indexing lncRNA properties and function gradually become essential tools to this process. Aim of DIANA-LncBase (www.microrna.gr/LncBase) is to reinforce researchers' attempts and unravel microRNA (miRNA)-lncRNA putative functional interactions. This study provides, for the first time, a comprehensive annotation of miRNA targets on lncRNAs. DIANA-LncBase hosts transcriptome-wide experimentally verified and computationally predicted miRNA recognition elements (MREs) on human and mouse lncRNAs. The analysis performed includes an integration of most of the available lncRNA resources, relevant high-throughput HITS-CLIP and PAR-CLIP experimental data as well as state-of-the-art in silico target predictions. The experimentally supported entries available in DIANA-LncBase correspond to >5000 interactions, while the computationally predicted interactions exceed 10 million. DIANA-LncBase hosts detailed information for each miRNA-lncRNA pair, such as external links, graphic plots of transcripts' genomic location, representation of the binding sites, lncRNA tissue expression as well as MREs conservation and prediction scores.

Abstract BACKGROUND: The GENCODE project has collected over 10,000 human long non-coding RNA (lncRNA) genes. However, the vast majority of them remain to be functionally characterized. Computational investigation of potential functions of human lncRNA genes is helpful to guide further experimental studies on lncRNAs. RESULTS: In this study, based on expression correlation between lncRNAs and protein-coding genes across 19 human normal tissues, we used the hypergeometric test to functionally annotate a single lncRNA or a set of lncRNAs with significantly enriched functional terms among the protein-coding genes that are significantly co-expressed with the lncRNA(s). The functional terms include all nodes in the Gene Ontology (GO) and 4,380 human biological pathways collected from 12 pathway databases. We successfully mapped 9,625 human lncRNA genes to GO terms and biological pathways, and then developed the first ontology-driven user-friendly web interface named lncRNA2Function, which enables researchers to browse the lncRNAs associated with a specific functional term, the functional terms associated with a specific lncRNA, or to assign functional terms to a set of human lncRNA genes, such as a cluster of co-expressed lncRNAs. The lncRNA2Function is freely available at http://mlg.hit.edu.cn/lncrna2function. CONCLUSIONS: The LncRNA2Function is an important resource for further investigating the functions of a single human lncRNA, or functionally annotating a set of human lncRNAs of interest.

Long non-coding RNAs (lncRNAs) play key roles in various cellular contexts and diseases by diverse mechanisms. With the rapid growth of identified lncRNAs and disease-associated single nucleotide polymorphisms (SNPs), there is a great demand to study SNPs in lncRNAs. Aiming to provide a useful resource about lncRNA SNPs, we systematically identified SNPs in lncRNAs and analyzed their potential impacts on lncRNA structure and function. In total, we identified 495,729 and 777,095 SNPs in more than 30,000 lncRNA transcripts in human and mouse, respectively. A large number of SNPs were predicted with the potential to impact on the miRNA-lncRNA interaction. The experimental evidence and conservation of miRNA-lncRNA interaction, as well as miRNA expressions from TCGA were also integrated to prioritize the miRNA-lncRNA interactions and SNPs on the binding sites. Furthermore, by mapping SNPs to GWAS results, we found that 142 human lncRNA SNPs are GWAS tagSNPs and 197,827 lncRNA SNPs are in the GWAS linkage disequilibrium regions. All these data for human and mouse lncRNAs were imported into lncRNASNP database (http://bioinfo.life.hust.edu.cn/lncRNASNP/), which includes two sub-databases lncRNASNP-human and lncRNASNP-mouse. The lncRNASNP database has a user-friendly interface for searching and browsing through the SNP, lncRNA and miRNA sections.

Despite the fact that a large quantity of noncoding RNAs (ncRNAs) have been identified, their functions remain unclear. To enable researchers to have a better understanding of ncRNAs鈥 functions, we updated the NPInter database to version 3.0, which contains experimentally verified interactions between ncRNAs (excluding tRNAs and rRNAs), especially long noncoding RNAs (lncRNAs) and other biomolecules (proteins, mRNAs, miRNAs and genomic DNAs). In NPInter v3.0, interactions pertaining to ncRNAs are not only manually curated from scientific literature but also curated from high-throughput technologies. In addition, we also curated lncRNA鈥搈iRNA interactions fromin silicopredictions supported by AGO CLIP-seq data. When compared with NPInter v2.0, the interactions are more informative (with additional information on tissues or cell lines, binding sites, conservation, co-expression values and other features) and more organized (with divisions on data sets by data sources, tissues or cell lines, experiments and other criteria). NPInter v3.0 expands the data set to 491,416 interactions in 188 tissues (or cell lines) from 68 kinds of experimental technologies. NPInter v3.0 also improves the user interface and adds new web services, including a local UCSC Genome Browser to visualize binding sites. Additionally, NPInter v3.0 defined a high-confidence set of interactions and predicted the functions of lncRNAs in human and mouse based on the interactions curated in the database. NPInter v3.0 is available athttp://www.bioinfo.org/NPInter/. Database URL:http://www.bioinfo.org/NPInter/

Motivation Recent studies have revealed that large numbers of non-coding RNAs are transcribed in humans, but only a few of them have been identified with their functions. Identification of the...

Although microRNAs (miRNAs), other non-coding RNAs (ncRNAs) (e.g. lncRNAs, pseudogenes and circRNAs) and competing endogenous RNAs (ceRNAs) have been implicated in cell-fate determination and in various human diseases, surprisingly little is known about the regulatory interaction networks among the multiple classes of RNAs. In this study, we developed starBase v2.0 (http://starbase.sysu.edu.cn/) to systematically identify the RNA-RNA and protein-RNA interaction networks from 108 CLIP-Seq (PAR-CLIP, HITS-CLIP, iCLIP, CLASH) data sets generated by 37 independent studies. By analyzing millions of RNA-binding protein binding sites, we identified 鈭9000 miRNA-circRNA, 16 000 miRNA-pseudogene and 285,000 protein-RNA regulatory relationships. Moreover, starBase v2.0 has been updated to provide the most comprehensive CLIP-Seq experimentally supported miRNA-mRNA and miRNA-lncRNA interaction networks to date. We identified 鈭10,000 ceRNA pairs from CLIP-supported miRNA target sites. By combining 13 functional genomic annotations, we developed miRFunction and ceRNAFunction web servers to predict the function of miRNAs and other ncRNAs from the miRNA-mediated regulatory networks. Finally, we developed interactive web implementations to provide visualization, analysis and downloading of the aforementioned large-scale data sets. This study will greatly expand our understanding of ncRNA functions and their coordinated regulatory networks.

As a result of the development of high-throughput sequencing and efficient microarray analysis, global gene expression analysis has become an easy and readily available form of data collection. In many research and disease models however, steady state levels of target gene mRNA does not always directly correlate with steady state protein levels. Post-transcriptional gene regulation is a likely explanation of the divergence between the two. Driven by the binding of RNA Binding Proteins (RBP), post-transcriptional regulation affects mRNA localization, stability and translation by forming a Ribonucleoprotein (RNP) complex with target mRNAs. Identifying these unknown de novo mRNA targets from cellular extracts in the RNP complex is pivotal to understanding mechanisms and functions of the RBP and their resulting effect on protein output. This protocol outlines a method termed RNP immunoprecipitation-microarray (RIP-Chip), which allows for the identification of specific mRNAs associated in the ribonucleoprotein complex, under changing experimental conditions, along with options to further optimize an experiment for the individual researcher. With this important experimental tool, researchers can explore the intricate mechanisms associated with post-transcriptional gene regulation as well as other ribonucleoprotein interactions.

Abstract Long noncoding RNAs (lncRNAs) are key regulators of chromatin state, yet the nature and sites of RNA-chromatin interaction are mostly unknown. Here we introduce Chromatin Isolation by RNA Purification (ChIRP), where tiling oligonucleotides retrieve specific lncRNAs with bound protein and DNA sequences, which are enumerated by deep sequencing. ChIRP-seq of three lncRNAs reveal that RNA occupancy sites in the genome are focal, sequence-specific, and numerous. Drosophila roX2 RNA occupies male X-linked gene bodies with increasing tendency toward the 3' end, peaking at CES sites. Human telomerase RNA TERC occupies telomeres and Wnt pathway genes. HOTAIR lncRNA preferentially occupies a GA-rich DNA motif to nucleate broad domains of Polycomb occupancy and histone H3 lysine 27 trimethylation. HOTAIR occupancy occurs independently of EZH2, suggesting the order of RNA guidance of Polycomb occupancy. ChIRP-seq is generally applicable to illuminate the intersection of RNA and chromatin with newfound precision genome wide. Copyright 脗漏 2011 Elsevier Inc. All rights reserved.

Capture Hybridization Analysis of RNA Targets (CHART) has recently been developed to map the genome-wide binding profile of chromatin-associated RNAs. This protocol uses a small number of 22–28 nucleo

Abstract Post-transcriptional gene regulation is robustly regulated by RNA-binding proteins (RBPs). Here we describe the collection of RNAs regulated by AUF1 (AU-binding factor 1), an RBP linked to cancer, inflammation and aging. Photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) analysis reveals that AUF1 primarily recognizes U-/GU-rich sequences in mRNAs and noncoding RNAs and influences target transcript fate in three main directions. First, AUF1 lowers the steady-state levels of numerous target RNAs, including long noncoding RNA NEAT1, in turn affecting the organization of nuclear paraspeckles. Second, AUF1 does not change the abundance of many target RNAs, but ribosome profiling reveals that AUF1 promotes the translation of numerous mRNAs in this group. Third, AUF1 unexpectedly enhances the steady-state levels of several target mRNAs encoding DNA-maintenance proteins. Through its actions on target RNAs, AUF1 preserves genomic integrity, in agreement with the AUF1-elicited prevention of premature cellular senescence.

HULC is a long noncoding RNA overexpressed in hepatocellular carcinoma (HCC), but its functional contributions in this setting have not been determined. In this study, we explored the hypothesis that HULC contributes to malignant development by supporting abnormal lipid metabolism in hepatoma cells. HULC modulated the deregulation of lipid metabolism in HCC by activating the acyl-CoA synthetase subunit ACSL1. Immunohistochemical analysis of tissue microarrays revealed that approximately 77% (180/233) of HCC tissues were positive for ACSL1. Moreover, HULC mRNA levels correlated positively with ACSL1 levels in 60 HCC cases according to real-time PCR analysis. Mechanistic investigations showed that HULC upregulated the transcriptional factor PPARA, which activated the ACSL1 promoter in hepatoma cells. HULC also suppressed miR-9 targeting of PPARA mRNA by eliciting methylation of CpG islands in the miR-9 promoter. We documented the ability of HULC to promote lipogenesis, thereby stimulating accumulation of intracellular triglycerides and cholesterol in vitro and in vivo. Strikingly, ACSL1 overexpression that generates cholesterol was sufficient to enhance the proliferation of hepatoma cells. Further, cholesterol addition was sufficient to upregulate HULC expression through a positive feedback loop involving the retinoid receptor RXRA, which activated the HULC promoter. Overall, we concluded that HULC functions as an oncogene in hepatoma cells, acting mechanistically by deregulating lipid metabolism through a signaling pathway involving miR-9, PPARA, and ACSL1 that is reinforced by a feed-forward pathway involving cholesterol and RXRA to drive HULC signaling.

Abstract Uncoupling protein 1 (UCP1) is localized on the inner mitochondrial membrane and generates heat by uncoupling ATP synthesis from proton transit across the inner membrane. UCP1 is a key element of nonshivering thermogenesis and is most likely important in the regulation of body adiposity. Pigs (Artiodactyl family Suidae ) lack a functional UCP1 gene, resulting in poor thermoregulation and susceptibility to cold, which is an economic and pig welfare issue owing to neonatal mortality. Pigs also have a tendency toward fat accumulation, which may be linked to their lack of UCP1, and thus influences the efficiency of pig production. Here, we report application of a CRISPR/Cas9-mediated, homologous recombination (HR)-independent approach to efficiently insert mouse adiponectin-UCP1 into the porcine endogenous UCP1 locus. The resultant UCP1 knock-in (KI) pigs showed an improved ability to maintain body temperature during acute cold exposure, but they did not have alterations in physical activity levels or total daily energy expenditure (DEE). Furthermore, ectopic UCP1 expression in white adipose tissue (WAT) dramatically decreased fat deposition by 4.89% ( P < 0.01), consequently increasing carcass lean percentage (CLP; P < 0.05). Mechanism studies indicated that the loss of fat upon UCP1 activation in WAT was linked to elevated lipolysis. UCP1 KI pigs are a potentially valuable resource for agricultural production through their combination of cold adaptation, which improves pig welfare and reduces economic losses, with reduced fat deposition and increased lean meat production. Published under the PNAS license.

Long non-coding RNA (long ncRNA) is a novel class of ncRNA that may be involved in critical cellular processes. A considerable number of mammalian long ncRNAs have now been isolated but only a small number of these nucleic acids have been functionally well characterized. In this study, to determine the structure, regulation and function of long ncRNA in pigs, TncRNA was isolated from this mammal and its potential function during pig foetus development was identified. We anticipated that this would provide new insights into functional genomic studies in the pig. Using LongSAGE libraries generated from Chinese indigenous Tongcheng and Landrace pigs at three prenatal stages, a novel porcine long ncRNA was identified, TncRNA , which was found to be differentially expressed during myogenesis. The full-length cDNA for this gene is 3409 bp, and it harbours a typical polyadenylation signal sequence located 18 bp upstream from the 3' poly (A) tail. Genomic sequence analysis showed that pig TncRNA is alternatively spliced and several transcripts were detected. Using the INRA芒聙聯University of Minnesota porcine radiation hybrid panel, TncRNA was assigned to SSC2 and found to be closely linked to the microsatellite marker SW256 . Porcine TncRNA was found to be expressed in all tissues examined but in variable amounts. Comparisons between the expression profiles of TncRNA at different development stages in Tongcheng and Landrace pigs revealed up-regulation of this molecule in prenatal skeletal muscle, and differential expression in 90-day-old foetal skeletal muscle between these two pig breeds. This is the first report to describe a long ncRNA in pig. Moreover, the distinct expression pattern and structure of porcine TncRNA suggest that it performs complex and critical functions during foetal development.

61We report here the first analysis of lncRNA landscape during muscle development.61lncMDis a muscle-specific lncRNA which is tightly regulated during myogenesis.61lncMDpromotes muscle differentiation by acting as a ceRNA for miR-125b.61IGF2is an important target of miR-125b in bovine myogenesis.61lncMDincreasesIGF2expression in a miR-125b-dependent manner.

Long non-coding RNAs (lncRNAs) play important roles in epigenetic regulation of skeletal muscle development. In our previous RNA-seq study (accession numberGSE58755), we found that lncRNA-Six1 is an lncRNA that is differentially expressed between White Recessive Rock (WRR) and Xinghua (XH) chicken. In this study, we have further demonstrated that lncRNA-Six1 is located 432 bp upstream of the gene encoding the protein Six homeobox 1 (Six1). A dual-luciferase reporter assay identified that lncRNA-Six1 overlaps theSix1proximal promoter. In lncRNA-Six1, a micropeptide of about 7.26 kDa was found to play an important role in the lncRNA-Six1in cisactivity. Overexpression of lncRNA-Six1 promoted the mRNA and protein expression level of theSix1gene, while knockdown of lncRNA-Six1 inhibitedSix1expression. Moreover, tissue expression profiles showed that both the lncRNA-Six1 and theSix1mRNA were highly expressed in chicken breast tissue. LncRNA-Six1 overexpression promoted cell proliferation and induced cell division. Conversely, its loss of function inhibited cell proliferation and reduced cell viability. Similar effects were observed after overexpression or knockdown of theSix1gene. In addition, overexpression or knockdown ofSix1promoted or inhibited, respectively, the expression levels of muscle-growth-related genes, such asMYOG, MYHC, MYOD, IGF1R, andINSR. Taken together, these data demonstrate that lncRNA-Six1 carries outcis-acting regulation of the protein-encodingSix1gene, and encodes a micropeptide to activateSix1gene, thus promoting cell proliferation and being involved in muscle growth.

Abstract Long noncoding RNAs (lncRNAs) are key regulatory factors for gene expression in a variety of biological processes; however, the role of lncRNAs in muscle formation and development is poorly understood, particularly in cattle. Here, we identified a highly expressed lncRNA in muscle, lncYYW, by high-throughput sequencing in bovine longissimus, scapular, intercostal, and gluteus muscles. The expression of lncYYW increased gradually during myoblast differentiation. Overexpression of lncYYW increased the number of cells in the DNA synthesis (S) stage of the cell cycle and upregulated the expression of two well-established myogenic markers, myogenin and myosin heavy chain. A microarray analysis showed that lncYYW positively regulates the expression of growth hormone 1 and its downstream genes, AKT1 and PIK3CD, in bovine myoblasts. This discovery provides a good foundation for further study of the mechanism of action of lncYYW during bovine myoblast development. Taken together, our results reveal a novel lncRNA associated with bovine myoblast proliferation and differentiation. This lncRNA will play a crucial and critical role in future studies of bovine muscle development.

Long intergenic non-coding RNAs (lincRNAs) play important roles in many cellular processes. Here, we present the first systematic identification and characterization of lincRNAs in fetal porcine skeletal muscle. We obtained a total of 55.02 million 90-bp paired-end reads and assembled 54,550 transcripts using cufflinks. We developed a pipeline to identify 570 multi-exon lincRNAs by integrating a set of previous approaches. These putative porcine lincRNAs share many characteristics with mammalian lincRNAs, such as a relatively short length, small number of exons and low level of sequence conservation. We found that the porcine lincRNAs were preferentially located near genes mediating transcriptional regulation rather than those with developmental functions. We further experimentally analyzed the features of a conserved mouse lincRNA gene and found that isoforms 1 and 4 of this lincRNA were enriched in the cell nucleus and were associated with polycomb repressive complex 2 (PRC2). Our results provide a catalog of fetal porcine lincRNAs for further experimental investigation of the functions of these genes in the skeletal muscle developmental process.

Abstract Long non-coding RNAs (lncRNAs) participated in growth and development of skeletal muscle; however, little is known about their response to testosterone deficiency in porcine skeletal muscle. We compared lean mass related carcass traits and lncRNAs expression files in Longissimus dorsi (LD) muscle between intact and castrated Huainan male pigs. The results showed that castration significantly reduced eye muscle area and lean meat percentage (P02<020.05), but increased the fat mass weight (P02<020.05). Meanwhile, 8946 lncRNAs, including 6743 intergenic lncRNAs (lincRNAs), 498 anti-sense lncRNAs, and 1705 intronic lncRNAs, were identified in porcine LD, among which, 385 lncRNAs were considered as the differentially expressed candidates between intact groups and castrated groups (q-value02<020.05). Functional analysis indicated that these differently expressed lncRNAs and their target genes were involved in the estrogen receptor signaling pathway and skeletal and muscular system development and function. We first detect porcine muscular lncRNA response to castration, and the results suggested that lncRNAs and their target genes participated in the regulation of testosterone deficiency-related skeletal muscle growth. 08 2017 Japanese Society of Animal Science.

Vertebrate genomes encode thousands of non-coding RNAs including short non-coding RNAs (such as microRNAs) and long non-coding RNAs (lncRNAs). Chicken ( Gallus gallus ) is an important model organism for developmental biology, and the recently assembled genome sequences for chicken will facilitate the understanding of the functional roles of non-coding RNA genes during development. The present study concerns the first systematic identification of lncRNAs using RNA-Seq to sample the transcriptome during chicken muscle development. A computational approach was used to identify 281 new intergenic lncRNAs in the chicken genome. Novel lncRNAs in general are less conserved than protein-coding genes and slightly more conserved than random non-coding sequences. The present study has provided an initial chicken lncRNA catalog and greatly increased the number of chicken ncRNAs in the non-protein coding RNA database. Furthermore, the computational pipeline presented in the current work will be useful for characterizing lncRNAs obtained from deep sequencing data.

Embryonic growth and development of skeletal muscle is a major determinant of muscle mass, and has a significant effect on meat production in chicken. To assess the protein expression profiles during embryonic skeletal muscle development, we performed a proteomics analysis using isobaric tags for relative and absolute quantification (iTRAQ) in leg muscle tissues of female Xinghua chicken at embryonic age (E) 11, E16, and 1-day post hatch (D1). We identified 3,240 proteins in chicken embryonic muscle and 491 of them were differentially expressed (fold change 鈮 1.5 or 鈮 0.666 andp< 0.05). There were 19 up- and 32 down-regulated proteins in E11 vs. E16 group, 238 up- and 227 down-regulated proteins in E11 vs. D1 group, and 13 up- and 5 down-regulated proteins in E16 vs. D1 group. Protein interaction network analyses indicated that these differentially expressed proteins were mainly involved in the pathway of protein synthesis, muscle contraction, and oxidative phosphorylation. Integrative analysis of proteome and our previous transcriptome data found 189 differentially expressed proteins that correlated with their mRNA level. The interactions between these proteins were also involved in muscle contraction and oxidative phosphorylation pathways. The lncRNA-protein interaction network found four proteins DMD, MYL3, TNNI2, and TNNT3 that are all involved in muscle contraction and may be lncRNA regulated. These results provide several candidate genes for further investigation into the molecular mechanisms of chicken embryonic muscle development, and enable us to better understanding their regulation networks and biochemical pathways.

Long non-coding RNAs (lncRNAs) play important roles in transcriptional and post-transcriptional regulation. However, little is currently known about the mechanisms by which they regulate skeletal muscle development in the chicken. In this study, we used RNA sequencing to profile the leg muscle transcriptome (lncRNA and mRNA) at three stages of skeletal muscle development in the chicken: embryonic day 11 (E11), embryonic day 16 (E16), and 1 day after hatching (D1). In total, 129, 132, and 45 differentially expressed lncRNAs, and 1798, 3072, and 1211 differentially expressed mRNAs were identified in comparisons of E11 vs. E16, E11 vs. D1, and E16 vs. D1, respectively. Moreover, we identified thecis- andtrans-regulatory target genes of differentially expressed lncRNAs, and constructed lncRNA-gene interaction networks. In total, 126 and 200cis-targets, and two and threetrans-targets were involved in lncRNA-gene interaction networks that were constructed based on the E11 vs. E16, and E11 vs. D1 comparisons, respectively. The comparison of the E16 vs. D1 lncRNA-gene network comprised 25cis-targets. We determined that lncRNA target genes are potentially involved in cellular development, and cellular growth and proliferation using Ingenuity Pathway Analysis. The gene networks identified for the E11 vs. D1 comparison were involved in embryonic development, organismal development and tissue development. The present study provides an RNA sequencing based evaluation of lncRNA function during skeletal muscle development in the chicken. Comprehensive analysis facilitated the identification of lncRNAs and target genes that might contribute to the regulation of different stages of skeletal muscle development.

Long non-coding RNAs (lncRNAs) have been studied extensively over the past few years. Large numbers of lncRNAs have been identified in mouse, rat, and human, and some of them have been shown to play important roles in muscle development and myogenesis. However, there are few reports on the characterization of lncRNAs covering all the development stages of skeletal muscle in livestock. RNA libraries constructed from developinglongissimus dorsimuscle of fetal (45, 60, and 10502days of gestation) and postnatal (302days after birth) goat (Capra hircus) were sequenced. A total of 1,034,049,894 clean reads were generated. Among them, 3981 lncRNA transcripts corresponding to 2739 lncRNA genes were identified, including 3515 intergenic lncRNAs and 466 anti-sense lncRNAs. Notably, in pairwise comparisons between the libraries of skeletal muscle at the different development stages, a total of 577 transcripts were differentially expressed (P<650.05) which were validated by qPCR using randomly selected six lncRNA genes. The identified goat lncRNAs shared some characteristics, such as fewer exons and shorter length, with the lncRNAs in other mammals. We also found 1153 lncRNAs genes were neighbored 1455 protein-coding genes (<1002kb upstream and downstream) and functionally enriched in transcriptional regulation and development-related processes, indicating they may be incis-regulatory relationships. Additionally, Pearson’s correlation coefficients of co-expression levels suggested 1737 lncRNAs and 19,422 mRNAs were possibly intrans-regulatory relationships (r>650.95 orr<65610.95). These co-expressed mRNAs were enriched in development-related biological processes such as muscle system processes, regulation of cell growth, muscle cell development, regulation of transcription, and embryonic morphogenesis. This study provides a catalog of goat muscle-related lncRNAs, and will contribute to a fuller understanding of the molecular mechanism underpinning muscle development in mammals. The online version of this article (doi:10.1186/s12864-016-3009-3) contains supplementary material, which is available to authorized users.

Abstract As an important type of noncoding RNA molecules, long non-coding RNAs (lncRNAs) act as versatile players in various biological processes. However, little is known about lncRNA regulators during sheep muscle growth. To explore functional lncRNAs during sheep muscle growth, we systematically investigated lncRNAs using strand-specific Ribo-Zero RNA sequencing at three key developmental stages in Hu sheep. A total of 6924 lncRNAs were obtained, and the differentially expressed lncRNAs and genes were screened from (control vs. experiment) fetus vs. lamb, lamb vs. adult, and fetus vs. adult comparisons, respectively. The quantitative real-time polymerase chain reaction (qRT-PCR) analysis results correlated well with the sequencing data. Moreover, functional annotation analysis based on the Gene Ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) databases showed that the target genes of the differentially expressed lncRNAs were significantly enriched in organ morphogenesis, skeletal system development as well as response to stimulus and some other terms related to muscle. Furthermore, a co-expression network of the differentially expressed target genes and lncRNAs was constructed and well-known muscle growth regulators such as retrotransposon-like 1 and Junctophilin-2 were included. Finally, we investigated the expression profiles of seven lncRNAs and their target genes, and found that they played vital roles in muscle growth. This study extends the sheep muscle lncRNA database and provides novel candidate regulators for future genetic and molecular studies on sheep muscle growth, which is helpful for optimizing the production of mutton.

Background The advent of large-scale gene expression technologies has helped to reveal in eukaryotic cells, the existence of thousands of non-coding transcripts, whose function and significance remain mostly poorly understood. Among these non-coding transcripts, long non-coding RNAs (lncRNAs) are the least well-studied but are emerging as key regulators of diverse cellular processes. In the present study, we performed a survey in bovine Longissimus thoraci of lincRNAs (long intergenic non-coding RNAs not overlapping protein-coding transcripts). To our knowledge, this represents the first such study in bovine muscle. Results To identify lincRNAs, we used paired-end RNA sequencing (RNA-Seq) to explore the transcriptomes of Longissimus thoraci from nine Limousin bull calves. Approximately 14鈥45 million paired-end reads were obtained per library. A total of 30,548 different transcripts were identified. Using a computational pipeline, we defined a stringent set of 584 different lincRNAs with 418 lincRNAs found in all nine muscle samples. Bovine lincRNAs share characteristics seen in their mammalian counterparts: relatively short transcript and gene lengths, low exon number and significantly lower expression, compared to protein-encoding genes. As for the first time, our study identified lincRNAs from nine different samples from the same tissue, it is possible to analyse the inter-individual variability of the gene expression level of the identified lincRNAs. Interestingly, there was a significant difference when we compared the expression variation of the 418 lincRNAs with the 10,775 known selected protein-encoding genes found in all muscle samples. In addition, we found 2,083 pairs of lincRNA/protein-encoding genes showing a highly significant correlated expression. Fourteen lincRNAs were selected and 13 were validated by RT-PCR . Some of the lincRNAs expressed in muscle are located within quantitative trait loci for meat quality traits. Conclusions Our study provides a glimpse into the lincRNA content of bovine muscle and will facilitate future experimental studies to unravel the function of these molecules. It may prove useful to elucidate their effect on mechanisms underlying the genetic variability of meat quality traits. This catalog will complement the list of lincRNAs already discovered in cattle and therefore will help to better annotate the bovine genome.