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Epigenetic processes involving long non-coding RNAs regulate endothelial gene expression. However, the underlying regulatory mechanisms causing endothelial dysfunction remain to be elucidated. Enhancer of zeste homolog 2 (EZH2) is an important rheostat of histone H3K27 trimethylation (H3K27me3) that represses endothelial targets, but EZH2 RNA binding capacity and EZH2:RNA functional interactions have not been explored in post-ischemic angiogenesis. We used formaldehyde/UV-assisted crosslinking ligation and sequencing of hybrids and identified a new role for maternally expressed gene 3 (MEG3). MEG3 formed the predominant RNA:RNA hybrid structures in endothelial cells. Moreover, MEG3:EZH2 assists recruitment onto chromatin. By EZH2-chromatin immunoprecipitation, following MEG3 depletion, we demonstrated that MEG3 controls recruitment of EZH2/H3K27me3 onto integrin subunit alpha4 ( ITGA4 ) promoter. Both MEG3 knockdown or EZH2 inhibition (A-395) promoted ITGA4 expression and improved endothelial cell migration and adhesion to fibronectin in vitro . The A-395 inhibitor re-directed MEG3 -assisted chromatin remodeling, offering a direct therapeutic benefit by increasing endothelial function and resilience. This approach subsequently increased the expression of ITGA4 in arterioles following ischemic injury in mice, thus promoting arteriogenesis. Our findings show a context-specific role for MEG3 in guiding EZH2 to repress ITGA4 . Novel therapeutic strategies could antagonize MEG3:EZH2 interaction for pre-clinical studies., Competing Interests: The authors declare no competing interests., (© 2024.)

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The early mammalian embryo is characterized by the presence of three germ layers-the outer ectoderm, middle mesoderm and inner endoderm. The mesoderm is organized into paraxial, intermediate and lateral plate mesoderm. The musculature, vasculature and heart of the adult body are the major derivatives of mesoderm. Tracing back the developmental process to generate these specialized tissues has sparked much interest in the field of regenerative medicine focusing on generating specialized tissues to treat patients with degenerative diseases. Several Long Non-Coding RNAs (lncRNAs) have been identified as regulators of development, proliferation and differentiation of various tissues of mesodermal origin. A better understanding of lncRNAs that can regulate the development of these tissues will open potential avenues for their therapeutic utility and enhance our knowledge about disease progression and development. In this review, we aim to summarize the functions and mechanisms of lncRNAs regulating the early mesoderm differentiation, development and homeostasis of skeletal muscle and cardiovascular system with an emphasis on their therapeutic potential., (Copyright © 2019 Sweta, Dudnakova, Sudheer, Baker and Bhushan.)

Background: Recent analyses implicate changes in the expression of the box C/D class of small nucleolar RNAs (snoRNAs) in several human diseases. Methods: Here we report the identification of potential novel RNA targets for box C/D snoRNAs in budding yeast, using the approach of UV crosslinking and sequencing of hybrids (CLASH) with the snoRNP proteins Nop1, Nop56 and Nop58. We also developed a bioinformatics approach to filter snoRNA-target interactions for bona fide methylation guide interactions. Results: We recovered 241,420 hybrids, out of which 190,597 were classed as reproducible, high energy hybrids. As expected, the majority of snoRNA interactions were with the ribosomal RNAs (rRNAs). Following filtering, 117,047 reproducible hybrids included 51 of the 55 reported rRNA methylation sites. The majority of interactions at methylation sites were predicted to guide methylation. However, competing, potentially regulatory, binding was also identified. In marked contrast, following CLASH performed with the RNA helicase Mtr4 only 7% of snoRNA-rRNA interactions recovered were predicted to guide methylation. We propose that Mtr4 functions in dissociating inappropriate snoRNA-target interactions. Numerous snoRNA-snoRNA interactions were recovered, indicating potential cross regulation. The snoRNAs snR4 and snR45 were recently implicated in site-directed rRNA acetylation, and hybrids were identified adjacent to the acetylation sites. We also identified 1,368 reproducible snoRNA-mRNA interactions, representing 448 sites of interaction involving 39 snoRNAs and 382 mRNAs. Depletion of the snoRNAs U3, U14 or snR4 each altered the levels of numerous mRNAs. Targets identified by CLASH were over-represented among these species, but causality has yet to be established. Conclusions: Systematic mapping of snoRNA-target binding provides a catalogue of high-confidence binding sites and indicates numerous potential regulatory interactions., Competing Interests: No competing interests were disclosed.

The RNA-binding protein HuD promotes neurogenesis and favors recovery from peripheral axon injury. HuD interacts with many mRNAs, altering both stability and translation efficiency. We generated a nucleotide resolution map of the HuD RNA interactome in motor neuron-like cells, identifying HuD target sites in 1,304 mRNAs, almost exclusively in the 3' UTR. HuD binds many mRNAs encoding mTORC1-responsive ribosomal proteins and translation factors. Altered HuD expression correlates with the translation efficiency of these mRNAs and overall protein synthesis, in a mTORC1-independent fashion. The predominant HuD target is the abundant, small non-coding RNA Y3, amounting to 70% of the HuD interaction signal. Y3 functions as a molecular sponge for HuD, dynamically limiting its recruitment to polysomes and its activity as a translation and neuron differentiation enhancer. These findings uncover an alternative route to the mTORC1 pathway for translational control in motor neurons that is tunable by a small non-coding RNA., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Tumor suppressor protein, Wt1 is a transcription factor that binds to DNA sequence similar to the Early Growth Response gene, EGR1 consensus binding sequence. Biophysical and biochemical validations have shown that the zinc fingers of Wt1 are capable of binding to both DNA and RNA albeit with different binding affinities which potentially is also isoform specific. SELEX based identification of the RNA binding motifs led to the identification of motifs which could not be translated into the in vivo context. With the advent of recent technologies that allow cross-linking of RNA and protein and high throughput sequencing techniques, it is now possible to analyze the in vivo RNA binding interactome of Wt1. This chapter outlines the initial studies that were aimed at addressing the Wt1 RNA interactome and also provides a detailed overview of some of the recent techniques used.

Wilms' tumours, paediatric kidney cancers, are the archetypal example of tumours caused through the disruption of normal development. The genetically best-defined subgroup of Wilms' tumours is the group caused by biallelic loss of the WT1 tumour suppressor gene. Here, we describe a developmental series of mouse models with conditional loss of Wt1 in different stages of nephron development before and after the mesenchymal-to-epithelial transition (MET). We demonstrate that Wt1 is essential for normal development at all kidney developmental stages under study. Comparison of genome-wide expression data from the mutant mouse models with human tumour material of mutant or wild-type WT1 datasets identified the stage of origin of human WT1-mutant tumours, and emphasizes fundamental differences between the two human tumour groups due to different developmental stages of origin., (© 2015. Published by The Company of Biologists Ltd.)

During the last step in 40S ribosome subunit biogenesis, the PIN-domain endonuclease Nob1 cleaves the 20S pre-rRNA at site D, to form the mature 18S rRNAs. Here we report that cleavage occurs in particles that have largely been stripped of previously characterized pre-40S components, but retain the endonuclease Nob1, its binding partner Pno1 (Dim2) and the atypical ATPase Rio1. Within the Rio1-associated pre-40S particles, in vitro pre-rRNA cleavage was strongly stimulated by ATP and required nucleotide binding by Rio1. In vivo binding sites for Rio1, Pno1 and Nob1 were mapped by UV cross-linking in actively growing cells. Nob1 and Pno1 bind overlapping regions within the internal transcribed spacer 1, and both bind directly over cleavage site D. Binding sites for Rio1 were within the core of the 18S rRNA, overlapping tRNA interaction sites and distinct from the related kinase Rio2. Site D cleavage occurs within pre-40S-60S complexes and Rio1-associated particles efficiently assemble into these complexes, whereas Pno1 appeared to be depleted relative to Nob1. We speculate that Rio1-mediated dissociation of Pno1 from cleavage site D is the trigger for final 18S rRNA maturation., (© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.)

5-Methylcytosine (5mC) is an epigenetic modification involved in regulation of gene activity during differentiation. Tet dioxygenases oxidize 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). Both 5fC and 5caC can be excised from DNA by thymine-DNA glycosylase (TDG) followed by regeneration of unmodified cytosine via the base excision repair pathway. Despite evidence that this mechanism is operative in embryonic stem cells, the role of TDG-dependent demethylation in differentiation and development is currently unclear. Here, we demonstrate that widespread oxidation of 5hmC to 5caC occurs in postimplantation mouse embryos. We show that 5fC and 5caC are transiently accumulated during lineage specification of neural stem cells (NSCs) in culture and in vivo. Moreover, 5caC is enriched at the cell-type-specific promoters during differentiation of NSCs, and TDG knockdown leads to increased 5fC/5caC levels in differentiating NSCs. Our data suggest that active demethylation contributes to epigenetic reprogramming determining lineage specification in embryonic brain., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

In vivo UV crosslinking identified numerous preribosomal RNA (pre-rRNA) binding sites for the large, highly conserved ribosome synthesis factor Rrp5. Intramolecular complementation has shown that the C-terminal domain (CTD) of Rrp5 is required for pre-rRNA cleavage at sites A0-A2 on the pathway of 18S rRNA synthesis, whereas the N-terminal domain (NTD) is required for A3 cleavage on the pathway of 5.8S/25S rRNA synthesis. The CTD was crosslinked to sequences flanking A2 and to the snoRNAs U3, U14, snR30, and snR10, which are required for cleavage at A0-A2. The NTD was crosslinked to sequences flanking A3 and to the RNA component of ribonuclease MRP, which cleaves site A3. Rrp5 could also be directly crosslinked to several large structural proteins and nucleoside triphosphatases. A key role in coordinating preribosomal assembly and processing was confirmed by chromatin spreads. Following depletion of Rrp5, cotranscriptional cleavage was lost and preribosome compaction greatly reduced., (Copyright © 2013 Elsevier Inc. All rights reserved.)

MicroRNAs (miRNAs) play key roles in gene regulation, but reliable bioinformatic or experimental identification of their targets remains difficult. To provide an unbiased view of human miRNA targets, we developed a technique for ligation and sequencing of miRNA-target RNA duplexes associated with human AGO1. Here, we report data sets of more than 18,000 high-confidence miRNA-mRNA interactions. The binding of most miRNAs includes the 5' seed region, but around 60% of seed interactions are noncanonical, containing bulged or mismatched nucleotides. Moreover, seed interactions are generally accompanied by specific, nonseed base pairing. 18% of miRNA-mRNA interactions involve the miRNA 3' end, with little evidence for 5' contacts, and some of these were functionally validated. Analyses of miRNA:mRNA base pairing showed that miRNA species systematically differ in their target RNA interactions, and strongly overrepresented motifs were found in the interaction sites of several miRNAs. We speculate that these affect the response of RISC to miRNA-target binding., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Methylation of cytosine is a DNA modification associated with gene repression. Recently, a novel cytosine modification, 5-hydroxymethylcytosine (5-hmC) has been discovered. Here we examine 5-hmC distribution during mammalian development and in cellular systems, and show that the developmental dynamics of 5-hmC are different from those of 5-methylcytosine (5-mC); in particular 5-hmC is enriched in embryonic contexts compared to adult tissues. A detectable 5-hmC signal appears in pre-implantation development starting at the zygote stage, where the paternal genome is subjected to a genome-wide hydroxylation of 5-mC, which precisely coincides with the loss of the 5-mC signal in the paternal pronucleus. Levels of 5-hmC are high in cells of the inner cell mass in blastocysts, and the modification colocalises with nestin-expressing cell populations in mouse post-implantation embryos. Compared to other adult mammalian organs, 5-hmC is strongly enriched in bone marrow and brain, wherein high 5-hmC content is a feature of both neuronal progenitors and post-mitotic neurons. We show that high levels of 5-hmC are not only present in mouse and human embryonic stem cells (ESCs) and lost during differentiation, as has been reported previously, but also reappear during the generation of induced pluripotent stem cells; thus 5-hmC enrichment correlates with a pluripotent cell state. Our findings suggest that apart from the cells of neuronal lineages, high levels of genomic 5-hmC are an epigenetic feature of embryonic cell populations and cellular pluri- and multi-lineage potency. To our knowledge, 5-hmC represents the first epigenetic modification of DNA discovered whose enrichment is so cell-type specific., (© 2011 IBCB, SIBS, CAS All rights reserved)

Transgenic 3T3 fibroblasts have been generated that express either the wild-type KRP or its truncated mutant lacking the C-terminal domain, which primarily contributes to myosin binding of KRP. It was found that KRP-expressing cells display a significantly increased content of myosin filaments and a reduced level of rMLC phosphorylation, whereas the mock transfected cells or cells expressing the C-terminally truncated KRP do not. Our results suggest that (1) KRP promotes the polymerization of myosin II and reduces the rMLC phosphorylation level in cells, (2) KRP acts through direct binding to myosin II, and (3) transgenic 3T3 fibroblasts stably expressing KRP represent a useful and versatile model to study the role of myosin II filament dynamics in cell motility.

Dilated cardiomyopathy (DCM) is characterized by enlargement and dilation of all heart compartments associated with serious decrease of its contractile function. DCM hallmark is the combination of dystrophic and hypertrophic alterations of cardiomyocytes. Since the power output of cardiac cells is directly related to remodeling of their contractile machinery we investigated expression of selected contractile and cytoskeletal proteins in the left ventricle of DCM patients using immunoblotting. The content of the recognized protein markers of cardiomyocyte hypertrophy such as tubulin, desmin and slow skeletal myosin heavy chain isoform, MHCbeta, was significantly elevated in DCM compared to normal myocardium. In addition, marked increase in the content of several smooth muscle proteins (smooth muscle alpha-actin, Myosin Light Chain Kinase, Kinase Related protein SM22) that are normally expressed in embryonic myocardium, was observed in DCM hearts. Thus, cardiomyocyte hypertrophy in DCM is associated with activation of embryonic protein expression program and smooth muscle proteins could serve as markers of this process. Understanding their involvement in sarcomere assembly and pathways of their expression activation during cardiac hypertrophy may bring new insights in treatment of various forms of cardiomyopathy.

The biochemical and morphological study of the cytoskeleton and extracellular matrix of rat heart was carried out after single injection of adriamycin (2.2 or 0.44 mg/kg). Hearts were taken for the study after 2 hours and 3 weeks after injection. The light and electronic microscopy, immunohistochemical determination of type I, III and IV collagens and fibronectin using specific antibodies were implied for morphological study; electrophoresis and immunoblotting were implied for the determination of the content of some proteins of cardiomyocytes (KRP or telokin, desmin, tubulin and vinculin), and extracellular matrix (fibronectin) and vascular smooth muscle cells (MLCK, myosin light chain kinase). Adriamycin injection in the dose 2.2 mg/kg which is close to therapeutic and known to alter intracellular membranes approximately in the half of cardiomyocytes, did not influence the relative volume and structure of collagen network but distinctly reduced the density of fibronectin-distribution. The content of tubulin, fibronectin, MLCK and KRP was significantly decreased by 18-24%, while contents of desmin and vinculin were changed insignificantly. After 3 weeks, an increased density and extension of collagen network indicating the development of diffuse fibrosis were observed. Contents of tubulin and KRP were increased above control level by 50 and 20%, respectively. Similar hyperrestitution of tubulin, fibronectin and KRP content by 15-25% was determined after smaller dose of adriamycin (0.44 mg/kg). Only content of MLCK out of proteins studied remained at lower level in both groups by 25-34%. Isolated chick embryo cardiomyocytes subjected to adriamycin responded by increased level of KRP expression by 20% in 4 days while the level of tubulin expression remained unchanged. Results showed that damage of cardiomyocytes and extracellular matrix after single injection of adriamycin in the dose close to therapeutic was followed by increased expression of some proteins of cytoskeleton and extracellular matrix. KRP seems to play active role in this reparative response while the steadily reduced level of MLCK expression may disturb the control of coronary vessels.

Aim: To elucidate alterations in myocardial ultrastructure and protein expression caused by isoproterenol., Methods: Biochemical, immunohistochemical and electron microscopic studies of rat myocardium were carried out 2 hours and 3 weeks after single injections of isoproterenol (50 and 10 mg/kg). Relative content of myospecific proteins (KRP - kinase-related protein, desmin), cytoskeletal proteins (tubulin, vinculin, and myosin light chain kinase - MLCK) and extracellular matrix protein, fibronectin, was determined by immunoblotting., Results: In 2 hours after injection of 50 mg/kg of isoproterenol destruction of some cardiomyocytes, contracture of myofibrils, and mild edema of intercellular space occurred; the content of KRP decreased by l6%, and that of tubulin, vinculin and fibronectin - by 27-29%. Reduced level of these proteins and also of MLCK persisted until 3 weeks after injection and was associated with altered cardiomyocyte ultrastructure. Glycogen granules were sparse, mitochondria contained arrow-like inclusions characteristic for calcium overload, huge mitochondria connected by specialized intermitochondrial contacts were present. Enlarged intercellular space contained areas of fibrosis with increased amount of type I and II collagens and fibronectin. Lower dose of isoproterenol (10 mg/kg) did not cause noticeable damaging action in the acute period, but in 3 weeks thickening of extracellular matrix occurred accompanied by increases of KRP and tubulin contents (by 26-32% compared with control level). Similar rise in expression of these proteins, and also of MLCK was observed after addition of isoproterenol to culture of chicken cardiomyocytes., Conclusion: These results indicate that even single injection of isoproterenol causes long lasting structural alterations in cardiac muscle accompanied by increased expression of extracellular matrix proteins as well as sarcoplasmic proteins apparently involved in the hypertrophic response of the cardiomyocytes.