Douglas M. Anderson

A Micropeptide Encoded by a Putative Long Noncoding RNA Regulates Muscle Performance

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.

A peptide encoded by a transcript annotated as long noncoding RNA enhances SERCA activity in muscle

Another micropeptide flexes its muscle Genome annotation is a complex but imperfect art. Attesting to its limitations is the growing evidence that certain transcripts annotated as long noncoding RNAs (lncRNAs) in fact code for small peptides with biologically important functions. One such lncRNA-derived micropeptide in mammals is myoregulin, which reduces muscle performance by inhibiting the activity of a key calcium pump. Nelson et al. describe the opposite activity in a second lncRNA-derived micropeptide in mammalian muscle, called DWORF (see the Perspective by Payre and Desplan). This peptide enhances muscle performance by activating the same calcium pump. DWORF may prove to be useful in improving the cardiac muscle function of mammals with heart disease. Science, this issue p. 271; see also p. 226 A long noncoding RNA encodes a small peptide that activates a calcium pump regulating muscle contraction. [Also see Perspective by Payre and Desplan] Muscle contraction depends on release of Ca2+ from the sarcoplasmic reticulum (SR) and reuptake by the Ca2+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 Ca2+ transient amplitude and SR Ca2+ load while reducing the time constant of cytosolic Ca2+ decay during each cycle of contraction-relaxation. Conversely, slow skeletal muscle lacking DWORF exhibits delayed Ca2+ 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.

Skeletal muscle-specific T-tubule protein STAC3 mediates voltage-induced Ca2+ release and contractility

Excitation–contraction (EC) coupling comprises events in muscle that convert electrical signals to Ca2+ transients, which then trigger contraction of the sarcomere. Defects in these processes cause a spectrum of muscle diseases. We report that STAC3, a skeletal muscle-specific protein that localizes to T tubules, is essential for coupling membrane depolarization to Ca2+ release from the sarcoplasmic reticulum (SR). Consequently, homozygous deletion of src homology 3 and cysteine rich domain 3 (Stac3) in mice results in complete paralysis and perinatal lethality with a range of musculoskeletal defects that reflect a blockade of EC coupling. Muscle contractility and Ca2+ release from the SR of cultured myotubes from Stac3 mutant mice could be restored by application of 4-chloro-m-cresol, a ryanodine receptor agonist, indicating that the sarcomeres, SR Ca2+ store, and ryanodine receptors are functional in Stac3 mutant skeletal muscle. These findings reveal a previously uncharacterized, but required, component of the EC coupling machinery of skeletal muscle and introduce a candidate for consideration in myopathic disorders.

Transcription of the non-coding RNA upperhand controls Hand2 expression and heart development

HAND2 is an ancestral regulator of heart development and one of four transcription factors that control the reprogramming of fibroblasts into cardiomyocytes. Deletion of Hand2 in mice results in right ventricle hypoplasia and embryonic lethality. Hand2 expression is tightly regulated by upstream enhancers that reside within a super-enhancer delineated by histone H3 acetyl Lys27 (H3K27ac) modifications. Here we show that transcription of a Hand2-associated long non-coding RNA, which we named upperhand (Uph), is required to maintain the super-enhancer signature and elongation of RNA polymerase II through the Hand2 enhancer locus. Blockade of Uph transcription, but not knockdown of the mature transcript, abolished Hand2 expression, causing right ventricular hypoplasia and embryonic lethality in mice. Given the substantial number of uncharacterized promoter-associated long non-coding RNAs encoded by the mammalian genome, the Uph–Hand2 regulatory partnership offers a mechanism by which divergent non-coding transcription can establish a permissive chromatin environment.

Mohawk is a novel homeobox gene expressed in the developing mouse embryo

Homeodomain‐containing proteins comprise a superfamily of transcription factors that participate in the regulation of almost all aspects of embryonic development. Here, we describe the mouse embryonic expression pattern of Mohawk, a new member of the TALE superclass of atypical homeobox genes that is most‐closely related to the Iroquois class. During mouse development, Mohawk was transcribed in cell lineages derived from the somites. As early as embryonic day 9.0, Mohawk was expressed in an anterior to posterior gradient in the dorsomedial and ventrolateral lips of the dermomyotome of the somites that normally give rise to skeletal muscle. Mohawk transcription in the dorsomedial region required the expression of the transcription factor paraxis. As somites matured, Mohawk transcription was observed in the tendon‐specific syndetome and the sclerotome‐derived condensing mesenchyme that prefigures the proximal ribs and vertebral bodies. In the limbs, Mohawk was expressed in a pattern consistent with the developing tendons that form along the dorsal and ventral aspect of the phalanges. Finally, Mohawk was detectable in the tips of the ureteric buds in the metanephric kidneys and the testis cords of the male gonad. Together, these observations suggest that Mohawk is an important regulator of vertebrate development. Developmental Dynamics 235:792–801, 2006.

A mouse model for adult cardiac-specific gene deletion with CRISPR/Cas9.

Significance The recent development of the clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas)9 system has greatly simplified the process of genomic editing. However, it has remained difficult to induce mutations in postnatal animals due to delivery challenges of the CRISPR/Cas9 components. Here, we report the generation of a transgenic mouse line that expresses Cas9 exclusively in cardiomyocytes. By using Adeno-Associated Virus 9 to deliver single-guide RNA (sgRNA) this method can rapidly induce genomic insertions and deletions in the heart. As proof of concept, administration of sgRNA against the Myh6 gene induced Myh6 editing, resulting in cardiomyopathy and heart failure in the cardiac-specific Cas9 mouse. This transgenic mouse model offers a valuable tool for cardiovascular research, as a straightforward strategy to edit genes of interest in the heart. Clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas)9 genomic editing has revolutionized the generation of mutant animals by simplifying the creation of null alleles in virtually any organism. However, most current approaches with this method require zygote injection, making it difficult to assess the adult, tissue-specific functions of genes that are widely expressed or which cause embryonic lethality when mutated. Here, we describe the generation of cardiac-specific Cas9 transgenic mice, which express high levels of Cas9 in the heart, but display no overt defects. In proof-of-concept experiments, we used Adeno-Associated Virus 9 (AAV9) to deliver single-guide RNA (sgRNA) that targets the Myh6 locus exclusively in cardiomyocytes. Intraperitoneal injection of postnatal cardiac-Cas9 transgenic mice with AAV9 encoding sgRNA against Myh6 resulted in robust editing of the Myh6 locus. These mice displayed severe cardiomyopathy and loss of cardiac function, with elevation of several markers of heart failure, confirming the effectiveness of this method of adult cardiac gene deletion. Mice with cardiac-specific expression of Cas9 provide a tool that will allow rapid and accurate deletion of genes following a single injection of AAV9-sgRNAs, thereby circumventing embryonic lethality. This method will be useful for disease modeling and provides a means of rapidly editing genes of interest in the heart.

Widespread control of calcium signaling by a family of SERCA-inhibiting micropeptides

A family of micropeptides inhibits calcium reuptake by the endoplasmic reticulum in diverse cell types. Inhibiting SERCA in more tissues Calcium triggers critical signaling events in all cell types. The membrane transporter SERCA moves calcium from the cytoplasm into the sarcoplasmic or endoplasmic reticulum to keep the basal cytoplasmic concentration of calcium low. Micropeptides that inhibit SERCA have been characterized in muscle. However, SERCA isoforms are found in all tissues, leading Anderson et al. to search for micropeptides that could perform this inhibitory function in nonmuscle tissues. They discovered two such micropeptides, endoregulin (ELN) and another-regulin (ALN) which inhibit the activity of SERCA isoforms that are abundant in nonmuscle tissues. These results raise the possibility that other membrane transporters related to SERCA could also be regulated by micropeptides. Micropeptides function as master regulators of calcium-dependent signaling in muscle. Sarco/endoplasmic reticulum Ca2+ ATPase (SERCA), the membrane pump that promotes muscle relaxation by taking up Ca2+ 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 Ca2+ dynamics in both muscle and nonmuscle cell types.

The homeobox gene Mohawk represses transcription by recruiting the sin3A/HDAC co-repressor complex.

Mohawk is an atypical homeobox gene expressed in embryonic progenitor cells of skeletal muscle, tendon, and cartilage. We demonstrate that Mohawk functions as a transcriptional repressor capable of blocking the myogenic conversion of 10T1/2 fibroblasts. The repressor activity is located in three small, evolutionarily conserved domains (MRD1–3) in the carboxy‐terminal half of the protein. Point mutation analysis revealed six residues in MRD1 are sufficient for repressor function. The carboxy‐terminal half of Mohawk is able to recruit components of the Sin3A/HDAC co‐repressor complex (Sin3A, Hdac1, and Sap18) and a subset of Polymerase II general transcription factors (Tbp, TFIIA1 and TFIIB). Furthermore, Sap18, a protein that bridges the Sin3A/HDAC complex to DNA‐bound transcription factors, is co‐immunoprecipitated by MRD1. These data predict that Mohawk can repress transcription through recruitment of the Sin3A/HDAC co‐repressor complex, and as a result, repress target genes required for the differentiation of cells to the myogenic lineage. Developmental Dynamics 238:572–580, 2009.

Mouse sperm exhibit chemotaxis to allurin, a truncated member of the cysteine-rich secretory protein family.

Allurin, a 21 kDa protein isolated from egg jelly of the frog Xenopus laevis, has previously been demonstrated to attract frog sperm in two-chamber and microscopic assays. cDNA cloning and sequencing has shown that allurin is a truncated member of the Cysteine-Rich Secretory Protein (CRISP) family, whose members include mammalian sperm-binding proteins that have been postulated to play roles in spermatogenesis, sperm capacitation and sperm-egg binding in mammals. Here, we show that allurin is a chemoattractant for mouse sperm, as determined by a 2.5-fold stimulation of sperm passage across a porous membrane and by analysis of sperm trajectories within an allurin gradient as observed by time-lapse microscopy. Chemotaxis was accompanied by an overall change in trajectory from circular to linear thereby increasing sperm movement along the gradient axis. Allurin did not increase sperm velocity although it did produce a modest increase in flagellar beat frequency. Oregon Green 488-conjugated allurin was observed to bind to the sub-equatorial region of the mouse sperm head and to the midpiece of the flagellum. These findings demonstrate that sperm have retained the ability to bind and respond to truncated Crisp proteins over 300 million years of vertebrate evolution.

Characterization of the DNA-binding Properties of the Mohawk Homeobox Transcription Factor

Background: Mkx is a transcriptional repressor that regulates muscle and tendon differentiation. Results: MKX binds to nnACA recognition sites as a homodimer. Mkx regulates transcription through recognition sites in the Mkx and Sox6 loci. Conclusion: MKX has a novel DNA recognition mode and promotes slow muscle fiber type specification through Sox6. Significance: We provide insight into Mkx regulation of musculoskeletal-specific transcription. The homeobox transcription factor Mohawk (Mkx) is a potent transcriptional repressor expressed in the embryonic precursors of skeletal muscle, cartilage, and bone. MKX has recently been shown to be a critical regulator of musculoskeletal tissue differentiation and gene expression; however, the genetic pathways through which MKX functions and its DNA-binding properties are currently unknown. Using a modified bacterial one-hybrid site selection assay, we determined the core DNA-recognition motif of the mouse monomeric Mkx homeodomain to be A-C-A. Using cell-based assays, we have identified a minimal Mkx-responsive element (MRE) located within the Mkx promoter, which is composed of a highly conserved inverted repeat of the core Mkx recognition motif. Using the minimal MRE sequence, we have further identified conserved MREs within the locus of Sox6, a transcription factor that represses slow fiber gene expression during skeletal muscle differentiation. Real-time PCR and immunostaining of in vitro differentiated muscle satellite cells isolated from Mkx-null mice revealed an increase in the expression of Sox6 and down-regulation of slow fiber structural genes. Together, these data identify the unique DNA-recognition properties of MKX and reveal a novel role for Mkx in promoting slow fiber type specification during skeletal muscle differentiation.