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Lamins and transmembrane proteins within the nuclear envelope regulate nuclear structure and chromatin organization. Nuclear envelope transmembrane protein 39 (Net39) is a muscle nuclear envelope protein whose functions in vivo have not been explored. We show that mice lacking Net39 succumb to severe myopathy and juvenile lethality, with concomitant disruption in nuclear integrity, chromatin accessibility, gene expression, and metabolism. These abnormalities resemble those of Emery–Dreifuss muscular dystrophy (EDMD), caused by mutations in A-type lamins (LMNA) and other genes, like Emerin (EMD). We observe that Net39 is downregulated in EDMD patients, implicating Net39 in the pathogenesis of this disorder. Our findings highlight the role of Net39 at the nuclear envelope in maintaining muscle chromatin organization, gene expression and function, and its potential contribution to the molecular etiology of EDMD., The nuclear envelope tethers chromatin to the nuclear periphery to control genome architecture. Here, the authors show that Net39 preserves the integrity and gene expression of muscle nuclei in mice, and it may contribute to the pathogenesis of Emery–Dreifuss muscular dystrophy.

Micromanaging muscle cell fusion Adult skeletal muscles are characterized by long, multinucleated cells called myofibers. Myofibers form when muscle precursor cells, or myoblasts, differentiate and fuse together during embryogenesis. The fusion process is not fully understood. Studying cell culture and mouse models, Bi et al. identified an 84–amino acid peptide that promotes myoblast fusion. This small peptide, called Myomixer, physically interacts with and stimulates the activity of a fusogenic membrane protein called Myomaker. Notably, the Myomaker-Myomixer pair can also promote the fusion of nonmuscle cells, such as fibroblasts. Science , this issue p. 323

The formation of new myofibers in vertebrates occurs by myoblast fusion and requires fusogenic activity of the muscle-specific membrane protein myomaker. Here, using in silico (BLAST) genome analyses, we show that the myomaker gene from trout includes 14 minisatellites, indicating that it has an unusual structure compared with those of other animal species. We found that the trout myomaker gene encodes a 434 –amino acid (aa) protein, in accordance with its apparent molecular mass (40 kDa) observed by immunoblotting. The first half of the trout myomaker protein (1–220 aa) is similar to the 221-aa mouse myomaker protein, whereas the second half (222–234 aa) does not correspond to any known motifs and arises from two protein extensions. The first extension (70 aa) apparently appeared with the radiation of the bony fish clade Euteleostei, whereas the second extension (up to 236 aa) is restricted to the superorder Protacanthopterygii (containing salmonids and pike) and corresponds to the insertion of minisatellites having a length of 30 nucleotides. According to gene expression analyses, trout myomaker expression is consistently associated with the formation of new myofibers during embryonic development, postlarval growth, and muscle regeneration. Using cell-mixing experiments, we observed that trout myomaker has retained the ability to drive the fusion of mouse fibroblasts with C2C12 myoblasts. Our work reveals that trout myomaker has fusogenic function despite containing two protein extensions., SCOPUS: ar.j, DecretOANoAutActif, info:eu-repo/semantics/published

Maintenance of muscle function requires assembly of contractile proteins into highly organized sarcomeres. Mutations in Kelch-like protein 41 (KLHL41) cause nemaline myopathy, a fatal muscle disorder associated with sarcomere disarray. We generated KLHL41 mutant mice, which display lethal disruption of sarcomeres and aberrant expression of muscle structural and contractile proteins, mimicking the hallmarks of the human disease. We show that KLHL41 is poly-ubiquitinated and acts, at least in part, by preventing aggregation and degradation of Nebulin, an essential component of the sarcomere. Furthermore, inhibition of KLHL41 poly-ubiquitination prevents its stabilization of nebulin, suggesting a unique role for ubiquitination in protein stabilization. These findings provide new insights into the molecular etiology of nemaline myopathy and reveal a mechanism whereby KLHL41 stabilizes sarcomeres and maintains muscle function by acting as a molecular chaperone. Similar mechanisms for protein stabilization likely contribute to the actions of other Kelch proteins., eLife digest Together with the tendon and joints, muscles move our bones by contracting and relaxing. Muscles are formed of bundles of lengthy cells, which are made up of small units called sarcomeres. To contract, the proteins in the sarcomere need to be able to slide past each other. In healthy muscle cells, the proteins in the sarcomeres are evenly distributed in an organized pattern to make sure that the muscle can contract. However, when some of the proteins in the sarcomere become faulty, it can lead to diseases that affect the muscles and movement. For example, in a genetic muscle disease called nemaline myopathy, the proteins in the sarcomere are no longer organized, which leads to a build-up of proteins in the muscle fiber. These protein masses form rod-like structures that are lodged in between sarcomeres, which makes it more difficult for the muscles to contract. This can cause muscle weakness, difficulties eating or breathing, and eventually death. A common cause of nemaline myopathy is mutations in the gene that encodes the nebulin protein, which serves as a scaffold for the sarcomere assembly. Other proteins, including proteins named KLHL40 and KLHL41, have also been linked to the disease. These two proteins are both ‘Kelch’ proteins, most of which help to degrade specific proteins. However, a recent study has shown that KLHL40 actually stabilizes nebulin. As the two Kelch proteins KLHL40 and 41 are very similar in structure, scientists wanted to find out if KLHL41 plays a similar role to KLHL40. Now, Ramirez-Martinez et al. have created genetically modified mice that lacked KLHL41. These mice showed symptoms similar to people with nemaline myopathy, in which the sarcomeres were disorganized and could not form properly. Further experiments with cells grown in the laboratory showed that KLHL41 stabilized nebulin by using a specific chemical process that usually helps to degrade proteins. These results suggest that Kelch proteins have additional roles beyond degrading proteins and that some proteins linked to nemaline myopathy may actively prevent others from accumulating. A next step will be to find drugs that can compensate for the lack of KLHL41. A better understanding of the causes of this fatal disease will contribute towards developing better treatments.

Multinucleate cellular syncytial formation is a hallmark of skeletal muscle differentiation. Myomaker, encoded by Mymk (Tmem8c), is a well-conserved plasma membrane protein required for myoblast fusion to form multinucleated myotubes in mouse, chick, and zebrafish. Here, we report that autosomal recessive mutations in MYMK (OMIM 615345) cause Carey-Fineman-Ziter syndrome in humans (CFZS; OMIM 254940) by reducing but not eliminating MYMK function. We characterize MYMK-CFZS as a congenital myopathy with marked facial weakness and additional clinical and pathologic features that distinguish it from other congenital neuromuscular syndromes. We show that a heterologous cell fusion assay in vitro and allelic complementation experiments in mymk knockdown and mymkinsT/insT zebrafish in vivo can differentiate between MYMK wild type, hypomorphic and null alleles. Collectively, these data establish that MYMK activity is necessary for normal muscle development and maintenance in humans, and expand the spectrum of congenital myopathies to include cell-cell fusion deficits., During embryogenesis, the cytoplasmic protein Myomarker (MYMK) mediates muscle fibre formation by fusion of myoblasts. Here, the authors identify autosomal recessive mutations in MYMK that cause Carey-Fineman-Ziter syndrome in humans, and model the disease variants in zebrafish.

MicroRNAs (miRNAs) perform critical functions in normal physiology and disease by associating with Argonaute proteins and downregulating partially complementary messenger RNAs (mRNAs). Here we use clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) genome-wide loss-of-function screening coupled with a fluorescent reporter of miRNA activity in human cells to identify new regulators of the miRNA pathway. By using iterative rounds of screening, we reveal a novel mechanism whereby target engagement by Argonaute 2 (AGO2) triggers its hierarchical, multi-site phosphorylation by CSNK1A1 on a set of highly conserved residues (S824-S834), followed by rapid dephosphorylation by the ANKRD52-PPP6C phosphatase complex. Although genetic and biochemical studies demonstrate that AGO2 phosphorylation on these residues inhibits target mRNA binding, inactivation of this phosphorylation cycle globally impairs miRNA-mediated silencing. Analysis of the transcriptome-wide binding profile of non-phosphorylatable AGO2 reveals a pronounced expansion of the target repertoire bound at steady-state, effectively reducing the active pool of AGO2 on a per-target basis. These findings support a model in which an AGO2 phosphorylation cycle stimulated by target engagement regulates miRNA:target interactions to maintain the global efficiency of miRNA-mediated silencing.