Dystrophin myonuclear domain restoration governs treatment efficacy in dystrophic muscle.

Dystrophin is essential for muscle health: its sarcolemmal absence causes the fatal, X-linked condition, Duchenne muscular dystrophy (DMD). However, its normal, spatial organization remains poorly understood, which hinders the interpretation of efficacy of its therapeutic restoration. Using female reporter mice heterozygous for fluorescently tagged dystrophin (DmdEGFP), we here reveal that dystrophin distribution is unexpectedly compartmentalized, being restricted to myonuclear-defined sarcolemmal territories extending ~80 µm, which we called "basal sarcolemmal dystrophin units (BSDUs)." These territories were further specialized at myotendinous junctions, where both Dmd transcripts and dystrophin protein were enriched. Genome-level correction in X-linked muscular dystrophy mice via CRISPR/Cas9 gene editing restored a mosaic of separated dystrophin domains, whereas transcript-level Dmd correction, following treatment with tricyclo-DNA antisense oligonucleotides, restored dystrophin initially at junctions before extending along the entire fiber-with levels ~2% sufficient to moderate the dystrophic process. We conclude that widespread restoration of fiber dystrophin is likely critical for therapeutic success in DMD, perhaps most importantly, at muscle-tendon junctions.

RBM10 regulates centriole duplication in HepG2 cells by ectopically assembling PLK4-STIL complexes in the nucleus.

RNA-binding motif protein 10 (RBM10) primarily regulates alternative splicing of certain genes. Loss-of-function mutations in RBM10 have been frequently reported in patients with various cancers. However, how RBM10 levels affect cell proliferation and tumorigenesis remains unknown. To elucidate the role of RBM10 in cell proliferation, we established HepG2-RBM10 knockout cell lines and derivative doxycycline-inducible RBM10-expressing cells. RBM10 over-expression caused growth arrest in the M phase with a monopolar spindle because of impaired centriole duplication. Two RBM10 splicing mutants, one with F345A/F347A and the other with only the C-terminal half (401-930), were sufficient to cause growth arrest, whereas an RBM10 mutant with cytoplasmic localization forced by an NES did not show growth arrest. RBM10 over-expression induced the formation of many large nuclear domains containing RBM10, PLK4, STIL and SAS6, which are the regulatory proteins involved in centriole duplication. Consistently, the centrioles in the RBM10-over-expressing HepG2 cells lost PLK4 and STIL, accounting for the unsuccessful centriole duplication. In contrast, RBM10 depletion resulted in elevated levels of cytoplasmic PLK4 with a concomitant increase in the number of centrioles in HepG2 cells but not in A549 cells. Thus, nuclear RBM10 regulates normal chromosomal division in a cell-type-specific manner, independent of alternative RNA splicing.

Aquatic Training after Joint Immobilization in Rats Promotes Adaptations in Myotendinous Junctions.

The myotendinous junction (MTJ) is the muscle-tendon interface and constitutes an integrated mechanical unit to force transmission. Joint immobilization promotes muscle atrophy via disuse, while physical exercise can be used as an adaptative stimulus. In this study, we aimed to investigate the components of the MTJ and their adaptations and the associated elements triggered with aquatic training after joint immobilization. Forty-four male Wistar rats were divided into sedentary (SD), aquatic training (AT), immobilization (IM), and immobilization/aquatic training (IMAT) groups. The samples were processed to measure fiber area, nuclear fractal dimension, MTJ nuclear density, identification of telocytes, sarcomeres, and MTJ perimeter length. In the AT group, the maintenance of ultrastructure and elements in the MTJ region were observed; the IM group presented muscle atrophy effects with reduced MTJ perimeter; the IMAT group demonstrated that aquatic training after joint immobilization promotes benefits in the muscle fiber area and fractal dimension, in the MTJ region shows longer sarcomeres and MTJ perimeter. We identified the presence of telocytes in the MTJ region in all experimental groups. We concluded that aquatic training is an effective rehabilitation method after joint immobilization due to reduced muscle atrophy and regeneration effects on MTJ in rats.

Quantitative analysis of multilayer organization of proteins and RNA in nuclear speckles at super resolution.

Nuclear speckles are self-assembled organelles composed of RNAs and proteins. They are proposed to act as structural domains that control distinct steps in gene expression, including transcription, splicing and mRNA export. Earlier studies identified differential localization of a few components within the speckles. It was speculated that the spatial organization of speckle components might contribute directly to the order of operations that coordinate distinct processes. Here, by performing multi-color structured illumination microscopy, we characterized the multilayer organization of speckles at a higher resolution. We found that SON and SC35 (also known as SRSF2) localize to the central region of the speckle, whereas MALAT1 and small nuclear (sn)RNAs are enriched at the speckle periphery. Coarse-grained simulations indicate that the non-random organization arises due to the interplay between favorable sequence-encoded intermolecular interactions of speckle-resident proteins and RNAs. Finally, we observe positive correlation between the total amount of RNA present within a speckle and the speckle size. These results imply that speckle size may be regulated to accommodate RNA accumulation and processing. Accumulation of RNA from various actively transcribed speckle-associated genes could contribute to the observed speckle size variations within a single cell.

Expression of Human Cytomegalovirus IE1 Leads to Accumulation of Mono-SUMOylated PML That Is Protected from Degradation by Herpes Simplex Virus 1 ICP0.

To countermeasure the host cellular intrinsic defense, cytomegalovirus (CMV) and herpes simplex viruses (HSV) have evolved the ability to disperse nuclear domain 10 (ND10, aka PML body). However, mechanisms underlying their action on ND10 differ. HSV infection produces ICP0, which degrades the ND10-forming protein PML. Human CMV (HCMV) infection expresses IE1 that deSUMOylates PML to result in dispersion of ND10. It has been demonstrated that HSV ICP0 degraded only the SUMOylated PML, so we hypothesized that HCMV IE1 can protect PML from degradation by ICP0. HCMV IE1-expressing cell lines (U-251 MG-IE1 and HELF-IE1) were used for infection of HSV-1 or transfection of ICP0-expressing plasmid. Multilabeling by immunocytochemistry assay and protein examination by Western blot assay were performed to determine the resultant fate of PML caused by ICP0 in the presence or absence of HCMV IE1. Here, we report that deSUMOylation of human PML (hPML) by HCMV IE1 was incomplete, as mono-SUMOylated PML remained in the IE1-expressing cells, which is consistent with the report by E. M. Schilling, M. Scherer, N. Reuter, J. Schweininger, et al. (J Virol 91:e02049-16, 2017, https://doi.org/10.1128/JVI.02049-16). As expected, we found that IE1 protected PML from degradation by ICP0 or HSV-1 infection. An in vitro study found that IE1 with mutation of L174P failed to deSUMOylate PML and did not protect PML from degradation by ICP0; hence, we conclude that the deSUMOylation of PML is important for IE1 to protect PML from degradation by ICP0. In addition, we revealed that murine CMV failed to deSUMOylate and to protect the HSV-mediated degradation of hPML, and that HCMV failed to deSUMOylate and protect the HSV-mediated degradation of mouse PML. However, IE1-expressing cells did not enhance wild-type HSV-1 replication but significantly increased ICP0-defective HSV-1 replication at a low multiplicity of infection. Therefore, our results uncovered a host-virus functional interaction at the posttranslational level.IMPORTANCE Our finding that HCMV IE1 protected hPML from degradation by HSV ICP0 is important, because the PML body (aka ND10) is believed to be the first line of host intrinsic defense against herpesviral infection. How the infected viruses overcome the nuclear defensive structure (PML body) has not been fully understood. Herpesviral proteins, ICP0 of HSV and IE1 of CMV, have been identified to interact with PML. Here, we report that HCMV IE1 incompletely deSUMOylated PML, resulting in the mono-SUMOylated PML, which is consistent with the report of Schilling et al. (J Virol 91:e02049-16, 2017, https://doi.org/10.1128/JVI.02049-16). The mono-SUMOylated PML was subjected to degradation by HSV ICP0. However, it was protected by IE1 from degradation by ICP0 or HSV-1 infection. In contrast, IE1 with L174P mutation lost the function of deSUMOylating PML and failed to protect the degradation of the mono-SUMOylated PML. Whether the mono-SUMOylated PML has any defensive function against viral infection will be further investigated.

The Human Cytomegalovirus IE1 Protein Antagonizes PML Nuclear Body-Mediated Intrinsic Immunity via the Inhibition of PML De Novo SUMOylation.

PML nuclear bodies (NBs) are accumulations of cellular proteins embedded in a scaffold-like structure built by SUMO-modified PML/TRIM19. PML and other NB proteins act as cellular restriction factors against human cytomegalovirus (HCMV); however, this intrinsic defense is counteracted by the immediate early protein 1 (IE1) of HCMV. IE1 directly interacts with the PML coiled-coil domain via its globular core region and disrupts NB foci by inducing a loss of PML SUMOylation. Here, we demonstrate that IE1 acts via abrogating the de novo SUMOylation of PML. In order to overcome reversible SUMOylation dynamics, we made use of a cell-based assay that combines inducible IE1 expression with a SUMO mutant resistant to SUMO proteases. Interestingly, we observed that IE1 expression did not affect preSUMOylated PML; however, it clearly prevented de novo SUMO conjugation. Consistent results were obtained by in vitro SUMOylation assays, demonstrating that IE1 alone is sufficient for this effect. Furthermore, IE1 acts in a selective manner, since K160 was identified as the main target lysine. This is strengthened by the fact that IE1 also prevents As2O3-mediated hyperSUMOylation of K160, thereby blocking PML degradation. Since IE1 did not interfere with coiled-coil-mediated PML dimerization, we propose that IE1 affects PML autoSUMOylation either by directly abrogating PML E3 ligase function or by preventing access to SUMO sites. Thus, our data suggest a novel mechanism for how a viral protein counteracts a cellular restriction factor by selectively preventing the de novo SUMOylation at specific lysine residues without affecting global protein SUMOylation. IMPORTANCE: The human cytomegalovirus IE1 protein acts as an important antagonist of a cellular restriction mechanism that is mediated by subnuclear structures termed PML nuclear bodies. This function of IE1 is required for efficient viral replication and thus constitutes a potential target for antiviral strategies. In this paper, we further elucidate the molecular mechanism for how IE1 antagonizes PML NBs. We show that tight binding of IE1 to PML interferes with the de novo SUMOylation of a distinct lysine residue that is also the target of stress-mediated hyperSUMOylation of PML. This is of importance since it represents a novel mechanism used by a viral antagonist of intrinsic immunity. Furthermore, it highlights the possibility of developing small molecules that specifically abrogate this PML-antagonistic activity of IE1 and thus inhibit viral replication.

The ND10 Component Promyelocytic Leukemia Protein Acts as an E3 Ligase for SUMOylation of the Major Immediate Early Protein IE1 of Human Cytomegalovirus.

Previous studies identified the nuclear domain 10 (ND10) components promyelocytic leukemia protein (PML), hDaxx, and Sp100 as factors of an intrinsic immune response against human cytomegalovirus (HCMV). This antiviral function of ND10, however, is antagonized by viral effector proteins like IE1p72, which induces dispersal of ND10. Furthermore, we have shown that both major immediate early proteins of HCMV, IE1p72 and IE2p86, transiently colocalize with ND10 subnuclear structures and undergo modification by the covalent attachment of SUMO. Since recent reports indicate that PML acts as a SUMO E3 ligase, we asked whether the SUMOylation of IE1p72 and IE2p86 is regulated by PML. To address this, PML-depleted fibroblasts, as well as cells overexpressing individual PML isoforms, were infected with HCMV. Western blot experiments revealed a clear correlation between the degree of IE1p72 SUMO conjugation and the abundance of PML. On the other hand, the SUMOylation of IE2p86 was not affected by PML. By performing in vitro SUMOylation assays, we were able to provide direct evidence that IE1p72 is a substrate for PML-mediated SUMOylation. Interestingly, disruption of the RING finger domain of PML, which is proposed to confer SUMO E3 ligase activity, abolished PML-induced SUMOylation of IE1p72. In contrast, IE1p72 was still efficiently SUMO modified by a SUMOylation-defective PML mutant, indicating that intact ND10 bodies are not necessary for this effect. Thus, this is the first report that the E3 ligase PML is capable of stimulating the SUMOylation of a viral protein which is supposed to serve as a cellular mechanism to compromise specific functions of IE1p72.IMPORTANCE The major immediate early proteins of human cytomegalovirus, termed IE1p72 and IE2p86, have previously been shown to undergo posttranslational modification by covalent coupling to SUMO moieties at specific lysine residues. However, the enzymatic activities that are responsible for this modification have not been identified. Here, we demonstrate that the PML protein, which mediates an intrinsic immune response against HCMV, specifically serves as an E3 ligase for SUMO modification of IE1p72. Since SUMO modification of IE1p72 has previously been shown to interfere with STAT factor binding, thus compromising the interferon-antagonistic function of this viral effector protein, our finding highlights an additional mechanism through which PML is able to restrict viral infections.

Identification of Genomic Loci Responsible for the Formation of Nuclear Domains Using Lampbrush Chromosomes.

In the cell nuclei, various types of nuclear domains assemble as a result of transcriptional activity at specific chromosomal loci. Giant transcriptionally active lampbrush chromosomes, which form in oocyte nuclei of amphibians and birds enable the mapping of genomic sequences with high resolution and the visualization of individual transcription units. This makes avian and amphibian oocyte nuclei an advantageous model for studying locus-specific nuclear domains. We developed two strategies for identification and comprehensive analysis of the genomic loci involved in nuclear domain formation on lampbrush chromosomes. The first approach was based on the sequential FISH-mapping of BAC clones containing genomic DNA fragments with a known chromosomal position close to the locus of a nuclear domain. The second approach involved mechanical microdissection of the chromosomal region adjacent to the nuclear domain followed by the generation of FISH-probes and DNA sequencing. Furthermore, deciphering the DNA sequences from the dissected material by high throughput sequencing technologies and their mapping to the reference genome helps to identify the genomic region responsible for the formation of the nuclear domain. For those nuclear domains structured by nascent transcripts, identification of genomic loci of their formation is a crucial step in the identification of scaffold RNAs.

In Situ Hi-C for Plants: An Improved Method to Detect Long-Range Chromatin Interactions.

Recently, long noncoding RNAs (lncRNAs) are shown to be implicating nuclear domain organization and gene regulation by mediating long-range chromatin interactions. Chromosome conformation capture (3C) is a method used to study such long-range interaction between two different loci in the 3D nuclear space. Through successive improvement in resolution and throughput, 3C, chromosome conformation capture on chip (4C), and chromosome conformation capture carbon copy (5C) to Hi-C methods were developed to study interactions between loci from one versus one scale to an unprecedented genome-wide resolution. In situ Hi-C is a variant of Hi-C in which proximity ligation is performed at the intact nuclei to improve the signal-to-noise ratio and throughput of the experiment to provide useful genome-wide contact frequency matrix/maps. The contact frequency maps obtained could be used for physical ordering of scaffolds in complex genome assembly projects, in deducing the nuclear domain organization in high resolution and in identifying specific long-range interactions between genomic regions of interest. In this chapter, we describe in detail a protocol for in situ Hi-C used on crops like barley, wheat, rye, oat, and evening primrose.

Herpesvirus tegument and immediate early proteins are pioneers in the battle between viral infection and nuclear domain 10-related host defense.

The sophisticated anti-viral functions of nuclear domain 10 (ND10) are revealed by identifying the role of each component and the countermeasures applied by viruses. Several ND10 proteins suppress herpesviruses at initial and early phases of infection. Herpesviruses need to antagonize these anti-viral proteins to start a productive infection. In this review the recently identified similarities and differences among the strategies adopted by the three subfamilies of herpesviruses are discussed, highlighting that one of the significant purposes of incorporating tegument proteins into the viral particles might be to counteract ND10 proteins immediately after the viral genome enters the host nucleus. Once the infection progresses, a sufficient amount of immediate early proteins is expressed to disperse and hydrolyze ND10 proteins, accelerating the development of infection.

The essential role of guinea pig cytomegalovirus (GPCMV) IE1 and IE2 homologs in viral replication and IE1-mediated ND10 targeting.

Guinea pig cytomegalovirus (GPCMV) immediate early proteins, IE1 and IE2, demonstrated structural and functional homologies with human cytomegalovirus (HCMV). GPCMV IE1 and IE2 co-localized in the nucleus with each other, the viral polymerase and guinea pig ND10 components (gpPML, gpDaxx, gpSp100, gpATRX). IE1 showed direct interaction with ND10 components by immunoprecipitation unlike IE2. Additionally, IE1 protein disrupted ND10 bodies. IE1 mutagenesis mapped the nuclear localization signal to the C-terminus and identified the core domain for gpPML interaction. Individual knockout of GPCMV GP122 or GP123 (IE2 and IE1 unique exons respectively) was lethal to the virus. However, an IE1 mutant (codons 234-474 deleted), was viable with attenuated viral growth kinetics and increased susceptibility to type I interferon (IFN-I). In HCMV, the IE proteins are important T cell target antigens. Consequently, characterization of the homologs in GPCMV provides a basis for their evaluation in candidate vaccines against congenital infection.

Nuclear domain 10 of the viral aspect.

Nuclear domain 10 (ND10) are spherical bodies distributed throughout the nucleoplasm and measuring around 0.2-1.0 µm. First observed under an electron microscope, they were originally described as dense bodies found in the nucleus. They are known by a number of other names, including Promyelocytic Leukemia bodies (PML bodies), Kremer bodies, and PML oncogenic domains. ND10 are frequently associated with Cajal bodies and cleavage bodies. It has been suggested that they play a role in regulating gene transcription. ND10 were originally characterized using human autoantisera, which recognizes Speckled Protein of 100 kDa, from patients with primary biliary cirrhosis. At the immunohistochemical level, ND10 appear as nuclear punctate structures, with 10 indicating the approximate number of dots per nucleus observed. ND10 do not colocalize with kinetochores, centromeres, sites of mRNA processing, or chromosomes. Resistance of ND10 antigens to nuclease digestion and salt extraction suggest that ND10 are associated with the nuclear matrix. They are often identified by immunofluorescent assay using specific antibodies against PML, Death domain-associated protein, nuclear dot protein (NDP55), and so on. The role of ND10 has long been the subject of investigation, with the specific connection of ND10 and viral infection having been a particular focus for almost 20 years. This review summarizes the relationship of ND10 and viral infection. Some future study directions are also discussed.

Protein oxidation in aging and the removal of oxidized proteins.

Reactive oxygen species (ROS) are generated constantly within cells at low concentrations even under physiological conditions. During aging the levels of ROS can increase due to a limited capacity of antioxidant systems and repair mechanisms. Proteins are among the main targets for oxidants due to their high rate constants for several reactions with ROS and their abundance in biological systems. Protein damage has an important influence on cellular viability since most protein damage is non-repairable, and has deleterious consequences on protein structure and function. In addition, damaged and modified proteins can form cross-links and provide a basis for many senescence-associated alterations and may contribute to a range of human pathologies. Two proteolytic systems are responsible to ensure the maintenance of cellular functions: the proteasomal (UPS) and the lysosomal system. Those degrading systems provide a last line of antioxidative protection, removing irreversible damaged proteins and recycling amino acids for the continuous protein synthesis. But during aging, both systems are affected and their proteolytic activity declines significantly. Here we highlight the recent advantages in the understanding of protein oxidation and the fate of these damaged proteins during aging. This article is part of a Special Issue entitled: Posttranslational Protein modifications in biology and Medicine.

The human cytomegalovirus major immediate-early proteins as antagonists of intrinsic and innate antiviral host responses.

The major immediate-early (IE) gene of human cytomegalovirus (CMV) is believed to have a decisive role in acute infection and its activity is an important indicator of viral reactivation from latency. Although a variety of gene products are expressed from this region, the 72-kDa IE1 and the 86-kDa IE2 nuclear phosphoproteins are the most abundant and important. Both proteins have long been recognized as promiscuous transcriptional regulators. More recently, a critical role of the IE1 and IE2 proteins in counteracting non-adaptive host cell defense mechanisms has been revealed. In this review we will briefly summarize the available literature on IE1- and IE2-dependent mechanisms contributing to CMV evasion from intrinsic and innate immune responses.

Interplay between Herpesvirus Infection and Host Defense by PML Nuclear Bodies.

In recent studies we and others have identified the cellular proteins PML, hDaxx, and Sp100, which form a subnuclear structure known as nuclear domain 10 (ND10) or PML nuclear bodies (PML-NBs), as host restriction factors that counteract herpesviral infections by inhibiting viral replication at different stages. The antiviral function of ND10, however, is antagonized by viral regulatory proteins (e.g., ICP0 of herpes simplex virus; IE1 of human cytomegalovirus) which induce either a modification or disruption of ND10. This review will summarize the current knowledge on how viral replication is inhibited by ND10 proteins. Furthermore, herpesviral strategies to defeat this host defense mechanism are discussed.