The ever-evolving concept of the gene: The use of RNA/Protein experimental techniques to understand genome functions

The completion of the human genome sequence together with advances in sequencing technologies have shifted the paradigm of the genome, as composed of discrete and hereditable coding entities, and have shown the abundance of functional noncoding DNA. This part of the genome, previously dismissed as "junk" DNA, increases proportionally with organismal complexity and contributes to gene regulation beyond the boundaries of known protein-coding genes. Different classes of functionally relevant nonprotein-coding RNAs are transcribed from noncoding DNA sequences. Among them are the long noncoding RNAs (lncRNAs), which are thought to participate in the basal regulation of protein-coding genes at both transcriptional and post-transcriptional levels. Although knowledge of this field is still limited, the ability of lncRNAs to localize in different cellular compartments, to fold into specific secondary structures and to interact with different molecules (RNA or proteins) endows them with multiple regulatory mechanisms. It is becoming evident that lncRNAs may play a crucial role in most biological processes such as the control of development, differentiation and cell growth. This review places the evolution of the concept of the gene in its historical context, from Darwin's hypothetical mechanism of heredity to the post-genomic era. We discuss how the original idea of protein-coding genes as unique determinants of phenotypic traits has been reconsidered in light of the existence of noncoding RNAs. We summarize the technological developments which have been made in the genome-wide identification and study of lncRNAs and emphasize the methodologies that have aided our understanding of the complexity of lncRNA-protein interactions in recent years

Divergent lncRNAs take the lead on pluripotent cell differentiation

[No abstract available

Long Non-Coding RNAs: New Players in Hematopoiesis and Leukemia

Long non-coding RNAs (lncRNAs) are important regulators of gene expression that influence almost every step in the life cycle of genes, from transcription to mRNA splicing, RNA decay, and translation. Besides their participation to normal physiology, lncRNA expression and function have been already associated to cancer development and progression. Here, we review the functional role and mechanisms of action of lncRNAs in normal hematopoiesis and how their misregulation may be implicated in the development of blood cell cancer, such as leukemia

Preparation of cardiac extracts from embryonal hearts to capture RNA–protein interactions by CLIP

The interaction of RNA with specific RNA-binding proteins (RBP) leads to the establishment of complex regulatory networks through which gene expression is controlled. Careful consideration should be given to the exact environment where a given RNA/RBP interplay occurs, as the functional responses might depend on the type of organism as well as the specific cellular or subcellular contexts. This requisite becomes particularly crucial for the study of long non-coding RNAs (lncRNA), as a consequence of their peculiar tissue-specificity and timely regulated expression. The functional characterization of lncRNAs has traditionally relied on the use of established cell lines that, although useful, are unable to fully recapitulate the complexity of a tissue or organ. Here, we detail an optimized protocol, with comments and tips, to identify the RNA interactome of given RBPs by performing cross-linking immunoprecipitation (CLIP) from mouse embryonal hearts. We tested the efficiency of this protocol on the murine pCharme, a muscle-specific lncRNA interacting with Matrin3 (MATR3) and forming RNA-enriched condensates of biological significance in the nucleus

Non-coding RNAs in muscle differentiation and musculoskeletal disease

RNA is likely to be the most rediscovered macromolecule in biology. Periodically, new non-canonical functions have been ascribed to RNA, such as the ability to act as a catalytic molecule or to work independently from its coding capacity. Recent annotations show that more than half of the transcriptome encodes for RNA molecules lacking coding activity. Here we illustrate how these transcripts affect skeletal muscle differentiation and related disorders. We discuss the most recent scientific discoveries that have led to the identification of the molecular circuitries that are controlled by RNA during the differentiation process and that, when deregulated, lead to pathogenic events. These findings will provide insights that can aid in the development of new therapeutic interventions for muscle disease

Novel long noncoding RNAs (lncRNAs) in Myogenesis: A miR-31 overlapping lncRNA transcript controls myoblast differentiation

Transcriptome analysis allowed the identification of new long noncoding RNAs differentially expressed during murine myoblast differentiation. These transcripts were classified on the basis of their expression under proliferating versus differentiated conditions, muscle-restricted activation, and subcellular localization. Several species displayed preferential expression in dystrophic (mdx) versus wild-type muscles, indicating their possible link with regenerative processes. One of the identified transcripts, lnc-31, even if originating from the same nuclear precursor of miR-31, is produced by a pathway mutually exclusive. We show that lnc-31 and its human homologue hsa-lnc-31 are expressed in proliferating myoblasts, where they counteract differentiation. In line with this, both species are more abundant in mdx muscles and in human Duchenne muscular dystrophy (DMD) myoblasts, than in their normal counterparts. Altogether, these data suggest a crucial role for lnc-31 in controlling the differentiation commitment of precursor myoblasts and indicate that its function is maintained in evolution despite the poor sequence conservation with the human counterpart

Epigenetic regulation of Wnt7b expression by the cis-acting long noncoding RNA Lnc-Rewind in muscle stem cells

Skeletal muscle possesses an outstanding capacity to regenerate upon injury due to the adult muscle stem cells (MuSCs) activity. This ability requires the proper balance between MuSCs expansion and differentiation which is critical for muscle homeostasis and contributes, if deregulated, to muscle diseases. Here, we functionally characterize a novel chromatin-associated lncRNA, Lnc-Rewind, which is expressed in murine MuSCs and conserved in human. We find that, in mouse, Lnc-Rewind acts as an epigenetic regulator of MuSCs proliferation and expansion by influencing the expression of skeletal muscle genes and several components of the WNT (Wingless-INT) signalling pathway. Among them, we identified the nearby Wnt7b gene as a direct Lnc-Rewind target. We show that Lnc-Rewind interacts with the G9a histone lysine methyltransferase and mediates the in cis repression of Wnt7b by H3K9me2 deposition. Overall, these findings provide novel insights into the epigenetic regulation of adult muscle stem cells fate by lncRNAs

Deficiency in the nuclear long noncoding RNA causes myogenic defects and heart remodeling in mice

Myogenesis is a highly regulated process that involves the conversion of progenitor cells into multinucleated myofibers. Besides proteins and miRNAs, long noncoding RNAs (lncRNAs) have been shown to participate in myogenic regulatory circuitries. Here, we characterize a murine chromatin-associated muscle-specific lncRNA, Charme, which contributes to the robustness of the myogenic program in vitro and in vivo. In myocytes, Charme depletion triggers the disassembly of a specific chromosomal domain and the downregulation of myogenic genes contained therein. Notably, several Charme-sensitive genes are associated with human cardiomyopathies and Charme depletion in mice results in a peculiar cardiac remodeling phenotype with changes in size, structure, and shape of the heart. Moreover, the existence of an orthologous transcript in human, regulating the same subset of target genes, suggests an important and evolutionarily conserved function for Charme. Altogether, these data describe a new example of a chromatin-associated lncRNA regulating the robustness of skeletal and cardiac myogenesis

A single-oral bolus of 100,000 IU of cholecalciferol at hospital admission did not improve outcomes in the COVID-19 disease: the COVID-VIT-D—a randomised multicentre international clinical trial

BACKGROUND: Vitamin D status has been implicated in COVID-19 disease. The objective of the COVID-VIT-D trial was to investigate if an oral bolus of cholecalciferol (100,000 IU) administered at hospital admission influences the outcomes of moderate-severe COVID-19 disease. In the same cohort, the association between baseline serum calcidiol levels with the same outcomes was also analysed. METHODS: The COVID-VIT-D is a multicentre, international, randomised, open label, clinical trial conducted throughout 1 year. Patients older than 18 years with moderate-severe COVID-19 disease requiring hospitalisation were included. At admission, patients were randomised 1:1 to receive a single oral bolus of cholecalciferol (n=274) or nothing (n=269). Patients were followed from admission to discharge or death. Length of hospitalisation, admission to intensive care unit (ICU) and mortality were assessed. RESULTS: In the randomised trial, comorbidities, biomarkers, symptoms and drugs used did not differ between groups. Median serum calcidiol in the cholecalciferol and control groups were 17.0 vs. 16.1 ng/mL at admission and 29.0 vs. 16.4 ng/mL at discharge, respectively. The median length of hospitalisation (10.0 [95%CI 9.0-10.5] vs. 9.5 [95%CI 9.0-10.5] days), admission to ICU (17.2% [95%CI 13.0-22.3] vs. 16.4% [95%CI 12.3-21.4]) and death rate (8.0% [95%CI 5.2-12.1] vs. 5.6% [95%CI 3.3-9.2]) did not differ between the cholecalciferol and control group. In the cohort analyses, the highest serum calcidiol category at admission (>25ng/mL) was associated with lower percentage of pulmonary involvement and better outcomes. CONCLUSIONS: The randomised clinical trial showed the administration of an oral bolus of 100,000 IU of cholecalciferol at hospital admission did not improve the outcomes of the COVID-19 disease. A cohort analysis showed that serum calcidiol at hospital admission was associated with outcomes. TRIAL REGISTRATION: COVID-VIT-D trial was authorised by the Spanish Agency for Medicines and Health products (AEMPS) and registered in European Union Drug Regulating Authorities Clinical Trials (EudraCT 2020-002274-28) and in ClinicalTrials.gov ( NCT04552951 )

Long Noncoding RNA Regulation of Pluripotency

Pluripotent stem cells (PSCs) represent a unique kind of stem cell, as they are able to indefinitely self-renew and hold the potential to differentiate into any derivative of the three germ layers. As such, human Embryonic Stem Cells (hESCs) and human induced Pluripotent Stem Cells (hiPSCs) provide a unique opportunity for studying the earliest steps of human embryogenesis and, at the same time, are of great therapeutic interest. The molecular mechanisms underlying pluripotency represent a major field of research. Recent evidence suggests that a complex network of transcription factors, chromatin regulators, and noncoding RNAs exist in pluripotent cells to regulate the balance between self-renewal and multilineage differentiation. Regulatory noncoding RNAs come in two flavors: short and long. The first class includes microRNAs (miRNAs), which are involved in the posttranscriptional regulation of cell cycle and differentiation in PSCs. Instead, long noncoding RNAs (lncRNAs) represent a heterogeneous group of long transcripts that regulate gene expression at transcriptional and posttranscriptional levels. In this review, we focus on the role played by lncRNAs in the maintenance of pluripotency, emphasizing the interplay between lncRNAs and other pivotal regulators in PSCs