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. Key features • The protocol refines previous methods of cardiac extracts preparation to use for CLIP assays. • The protocol allows the quantitative RNA-seq analysis of transcripts interacting with selected proteins. • Depending on the embryonal stage, a high number of hearts can be required as starting material. • The steps are adaptable to other tissues and biochemical assays.

Exploring the landscape of tools and resources for the analysis of long non-coding RNAs.

In recent years, research on long non-coding RNAs (lncRNAs) has gained considerable attention due to the increasing number of newly identified transcripts. Several characteristics make their functional evaluation challenging, which called for the urgent need to combine molecular biology with other disciplines, including bioinformatics. Indeed, the recent development of computational pipelines and resources has greatly facilitated both the discovery and the mechanisms of action of lncRNAs. In this review, we present a curated collection of the most recent computational resources, which have been categorized into distinct groups: databases and annotation, identification and classification, interaction prediction, and structure prediction. As the repertoire of lncRNAs and their analysis tools continues to expand over the years, standardizing the computational pipelines and improving the existing annotation of lncRNAs will be crucial to facilitate functional genomics studies.

The long noncoding RNA Charme supervises cardiomyocyte maturation by controlling cell differentiation programs in the developing heart.

Long noncoding RNAs (lncRNAs) are emerging as critical regulators of heart physiology and disease, although the studies unveiling their modes of action are still limited to few examples. We recently identified pCharme, a chromatin-associated lncRNA whose functional knockout in mice results in defective myogenesis and morphological remodeling of the cardiac muscle. Here, we combined Cap-Analysis of Gene Expression (CAGE), single-cell (sc)RNA sequencing, and whole-mount in situ hybridization analyses to study pCharme cardiac expression. Since the early steps of cardiomyogenesis, we found the lncRNA being specifically restricted to cardiomyocytes, where it assists the formation of specific nuclear condensates containing MATR3, as well as important RNAs for cardiac development. In line with the functional significance of these activities, pCharme ablation in mice results in a delayed maturation of cardiomyocytes, which ultimately leads to morphological alterations of the ventricular myocardium. Since congenital anomalies in myocardium are clinically relevant in humans and predispose patients to major complications, the identification of novel genes controlling cardiac morphology becomes crucial. Our study offers unique insights into a novel lncRNA-mediated regulatory mechanism promoting cardiomyocyte maturation and bears relevance to Charme locus for future theranostic applications.

Advances in endogenous RNA pull-down: A straightforward dextran sulfate-based method enhancing RNA recovery.

Detecting RNA/RNA interactions in the context of a given cellular system is crucial to gain insights into the molecular mechanisms that stand beneath each specific RNA molecule. When it comes to non-protein coding RNA (ncRNAs), and especially to long noncoding RNAs (lncRNAs), the reliability of the RNA purification is dramatically dependent on their abundance. Exogenous methods, in which lncRNAs are in vitro transcribed and incubated with protein extracts or overexpressed by cell transfection, have been extensively used to overcome the problem of abundance. However, although useful to study the contribution of single RNA sub-modules to RNA/protein interactions, these exogenous practices might fail in revealing biologically meaningful contacts occurring in vivo and risk to generate non-physiological artifacts. Therefore, endogenous methods must be preferred, especially for the initial identification of partners specifically interacting with elected RNAs. Here, we apply an endogenous RNA pull-down to lncMN2-203, a neuron-specific lncRNA contributing to the robustness of motor neurons specification, through the interaction with miRNA-466i-5p. We show that both the yield of lncMN2-203 recovery and the specificity of its interaction with the miRNA dramatically increase in the presence of Dextran Sulfate Sodium (DSS) salt. This new set-up may represent a powerful means for improving the study of RNA-RNA interactions of biological significance, especially for those lncRNAs whose role as microRNA (miRNA) sponges or regulators of mRNA stability was demonstrated.

A multifunctional locus controls motor neuron differentiation through short and long noncoding RNAs.

The transition from dividing progenitors to postmitotic motor neurons (MNs) is orchestrated by a series of events, which are mainly studied at the transcriptional level by analyzing the activity of specific programming transcription factors. Here, we identify a post-transcriptional role of a MN-specific transcriptional unit (MN2) harboring a lncRNA (lncMN2-203) and two miRNAs (miR-325-3p and miR-384-5p) in this transition. Through the use of in vitro mESC differentiation and single-cell sequencing of CRISPR/Cas9 mutants, we demonstrate that lncMN2-203 affects MN differentiation by sponging miR-466i-5p and upregulating its targets, including several factors involved in neuronal differentiation and function. In parallel, miR-325-3p and miR-384-5p, co-transcribed with lncMN2-203, act by repressing proliferation-related factors. These findings indicate the functional relevance of the MN2 locus and exemplify additional layers of specificity regulation in MN differentiation.

Targeting the Expression of Long Noncoding RNAs in Murine Satellite Cells from Single Myofibers.

LncRNAs have been recently implicated in the epigenetic control of muscle differentiation and their functional characterization has traditionally relied upon in vitro models of myogenic differentiation. However, the use of experimental paradigms to specifically target lncRNAs expression in muscle stem cells (MuSCs), also known as satellite cells, represents an important requisite to interrogate their function in more physiological contexts. Since isolation and culture of single myofibers preserves satellite cells within their physiological niche underneath the surrounding basal lamina, this procedure represents the optimal approach to follow satellite cell dynamics ex-vivo, such as activation from quiescence, expansion of committed progenitors, differentiation, and self-renewal. Here, we detail an optimized protocol to isolate viable single myofibers from the extensor digitorum longus (EDL) skeletal muscle of adult mice and to manipulate the expression of lncRNAs by antisense LNA GapmeRs-mediated knock-down (KD). Furthermore, we describe a method of EdU incorporation that, coupled to lncRNA KD and subsequent immunofluorescence analysis of proliferating, differentiating, and satellite cell-specific markers, permits the inference of lncRNAs function on muscle stem cells dynamics. Graphic abstract: Graphical representation of the single myofiber isolation method. Experimental workflow showing the main steps of the protocol procedure: EDL muscle harvesting from the mouse hindlimb; EDL digestion into single myofibers; transfection with antisense oligos and culture for 96h; immunofluorescence protocol and image outcome.

Muscle Regeneration and RNA: New Perspectives for Ancient Molecules.

The ability of the ribonucleic acid (RNA) to self-replicate, combined with a unique cocktail of chemical properties, suggested the existence of an RNA world at the origin of life. Nowadays, this hypothesis is supported by innovative high-throughput and biochemical approaches, which definitively revealed the essential contribution of RNA-mediated mechanisms to the regulation of fundamental processes of life. With the recent development of SARS-CoV-2 mRNA-based vaccines, the potential of RNA as a therapeutic tool has received public attention. Due to its intrinsic single-stranded nature and the ease with which it is synthesized in vitro, RNA indeed represents the most suitable tool for the development of drugs encompassing every type of human pathology. The maximum effectiveness and biochemical versatility is achieved in the guise of non-coding RNAs (ncRNAs), which are emerging as multifaceted regulators of tissue specification and homeostasis. Here, we report examples of coding and ncRNAs involved in muscle regeneration and discuss their potential as therapeutic tools. Small ncRNAs, such as miRNA and siRNA, have been successfully applied in the treatment of several diseases. The use of longer molecules, such as lncRNA and circRNA, is less advanced. However, based on the peculiar properties discussed below, they represent an innovative pool of RNA biomarkers and possible targets of clinical value.

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 cell (MuSC) activity. This ability requires the proper balance between MuSC expansion and differentiation, which is critical for muscle homeostasis and contributes, if deregulated, to muscle diseases. Here, we functionally characterize a novel chromatin-associated long noncoding RNA (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 MuSC 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.

Visualization of Nuclear and Cytoplasmic Long Noncoding RNAs at Single-Cell Level by RNA-FISH.

The RNA fluorescence in situ hybridization (RNA-FISH) methodology offers an attractive strategy to deepen our knowledge on the long noncoding RNA biology. In this chapter, we provide a comprehensive overview of the current RNA-FISH protocols available for imaging nuclear and cytoplasmic lncRNAs within cells or tissues. We describe a multicolor approach optimized for the simultaneous visualization of these transcripts with their specific molecular interactors, such as proteins or DNA sequences. Common challenges faced by this methodology such as cell-type specific permeabilization, target accessibility, image acquisition, and post-acquisition analyses are also discussed.

Intronic Determinants Coordinate Charme lncRNA Nuclear Activity through the Interaction with MATR3 and PTBP1.

Chromatin architect of muscle expression (Charme) is a muscle-restricted long noncoding RNA (lncRNA) that plays an important role in myogenesis. Earlier evidence indicates that the nuclear Charme isoform, named pCharme, acts on the chromatin by assisting the formation of chromatin domains where myogenic transcription occurs. By combining RNA antisense purification (RAP) with mass spectrometry and loss-of-function analyses, we have now identified the proteins that assist these chromatin activities. These proteins-which include a sub-set of splicing regulators, principally PTBP1 and the multifunctional RNA/DNA binding protein MATR3-bind to sequences located within the alternatively spliced intron-1 to form nuclear aggregates. Consistent with the functional importance of pCharme interactome in vivo, a targeted deletion of the intron-1 by a CRISPR-Cas9 approach in mouse causes the release of pCharme from the chromatin and results in cardiac defects similar to what was observed upon knockout of the full-length transcript.

HOTAIRM1 regulates neuronal differentiation by modulating NEUROGENIN 2 and the downstream neurogenic cascade.

Neuronal differentiation is a timely and spatially regulated process, relying on precisely orchestrated gene expression control. The sequential activation/repression of genes driving cell fate specification is achieved by complex regulatory networks, where transcription factors and noncoding RNAs work in a coordinated manner. Herein, we identify the long noncoding RNA HOTAIRM1 (HOXA Transcript Antisense RNA, Myeloid-Specific 1) as a new player in neuronal differentiation. We demonstrate that the neuronal-enriched HOTAIRM1 isoform epigenetically controls the expression of the proneural transcription factor NEUROGENIN 2 that is key to neuronal fate commitment and critical for brain development. We also show that HOTAIRM1 activity impacts on NEUROGENIN 2 downstream regulatory cascade, thus contributing to the achievement of proper neuronal differentiation timing. Finally, we identify the RNA-binding proteins HNRNPK and FUS as regulators of HOTAIRM1 biogenesis and metabolism. Our findings uncover a new regulatory layer underlying NEUROGENIN 2 transitory expression in neuronal differentiation and reveal a previously unidentified function for the neuronal-induced long noncoding RNA HOTAIRM1.

The Noncoding Side of Cardiac Differentiation and Regeneration.

Large scale projects such as FANTOM and ENCODE led to a revolution in our comprehension of the mammalian transcriptomes by revealing that ~53% of the produced RNAs do not encode for proteins. These transcripts, defined as noncoding RNAs (ncRNAs), constitute a heterogeneous group of molecules which can be categorized in two main classes, namely small and long, according to their length. In animals, the first-class includes Piwi-interacting RNAs (piRNAs), small interfering RNAs (siRNAs) and microRNAs (miRNAs). Among them, the best-characterized subgroup is represented by miRNAs, which are known to regulate gene expression largely at the post-transcriptional level. In contrast, long noncoding RNAs (lncRNAs) represent a more heterogeneous group of > 200 nucleotides long transcripts, that act through a variety of mechanisms at both transcriptional and posttranscriptional level. Here, we discuss how miRNAs and lncRNAs are emerging as pivotal regulators of cardiac muscle development and how the alteration of ncRNA expression was seen to disturb the physiology of all the different cell types forming the cardiac tissue. Particular emphasis is given to those species that are expressed and are known to regulate the capacity of cardiac progenitor cells (CPCs), currently used in regenerative medicine protocols, to proliferate and differentiate. Understanding how the ncRNAmediated circuitries regulate heart homeostasis is one of the research areas expected to have a high impact, improving the therapeutic efficacy of stem/progenitor-cells treatments for translation into clinical applications.

Non-coding RNAs Shaping Muscle.

In 1957, Francis Crick speculated that RNA, beyond its protein-coding capacity, could have its own function. Decade after decade, this theory was dramatically boosted by the discovery of new classes of non-coding RNAs (ncRNAs), including long ncRNAs (lncRNAs) and circular RNAs (circRNAs), which play a fundamental role in the fine spatio-temporal control of multiple layers of gene expression. Recently, many of these molecules have been identified in a plethora of different tissues, and they have emerged to be more cell-type specific than protein-coding genes. These findings shed light on how ncRNAs are involved in the precise tuning of gene regulatory mechanisms governing tissues homeostasis. In this review, we discuss the recent findings on the mechanisms used by lncRNAs and circRNAs to sustain skeletal and cardiac muscle formation, paying particular attention to the technological developments that, over the last few years, have aided their genome-wide identification and study. Together with lncRNAs and circRNAs, the emerging contribution of Piwi-interacting RNAs and transfer RNA-derived fragments to myogenesis will be also discussed, with a glimpse on the impact of their dysregulation in muscle disorders, such as myopathies, muscle atrophy, and rhabdomyosarcoma degeneration.

Deficiency in the nuclear long noncoding RNA Charme 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.

The Long Non-coding RNA lnc-31 Interacts with Rock1 mRNA and Mediates Its YB-1-Dependent Translation.

Cytoplasmic long non-coding RNAs have been shown to act at many different levels to control post-transcriptional gene expression, although their role in translational control is poorly understood. Here, we show that lnc-31, a non-coding RNA required for myoblast proliferation, promotes ROCK1 protein synthesis by stabilizing its translational activator, YB-1. We find that lnc-31 binds to the Rock1 mRNA as well as to the YB-1 protein and that translational activation requires physical interaction between the two RNA species. These results suggest a localized effect of YB-1 stabilization on the Rock1 mRNA. ROCK1 upregulation by lnc-31, in proliferative conditions, correlates well with the differentiation-repressing activity of ROCK1. We also show that, upon induction of differentiation, the downregulation of lnc-31, in conjunction with miR-152 targeting of Rock1, establishes a regulatory loop that reinforces ROCK1 repression and promotes myogenesis.

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.

Protein complex scaffolding predicted as a prevalent function of long non-coding RNAs.

The human transcriptome contains thousands of long non-coding RNAs (lncRNAs). Characterizing their function is a current challenge. An emerging concept is that lncRNAs serve as protein scaffolds, forming ribonucleoproteins and bringing proteins in proximity. However, only few scaffolding lncRNAs have been characterized and the prevalence of this function is unknown. Here, we propose the first computational approach aimed at predicting scaffolding lncRNAs at large scale. We predicted the largest human lncRNA-protein interaction network to date using the catRAPID omics algorithm. In combination with tissue expression and statistical approaches, we identified 847 lncRNAs (∼5% of the long non-coding transcriptome) predicted to scaffold half of the known protein complexes and network modules. Lastly, we show that the association of certain lncRNAs to disease may involve their scaffolding ability. Overall, our results suggest for the first time that RNA-mediated scaffolding of protein complexes and modules may be a common mechanism in human cells.

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 diseases.