Functional Classification and Experimental Dissection of Long Noncoding RNAs.

Over the last decade, it has been increasingly demonstrated that the genomes of many species are pervasively transcribed, resulting in the production of numerous long noncoding RNAs (lncRNAs). At the same time, it is now appreciated that many types of DNA regulatory elements, such as enhancers and promoters, regularly initiate bi-directional transcription. Thus, discerning functional noncoding transcripts from a vast transcriptome is a paramount priority, and challenge, for the lncRNA field. In this review, we aim to provide a conceptual and experimental framework for classifying and elucidating lncRNA function. We categorize lncRNA loci into those that regulate gene expression in cis versus those that perform functions in trans and propose an experimental approach to dissect lncRNA activity based on these classifications. These strategies to further understand lncRNAs promise to reveal new and unanticipated biology with great potential to advance our understanding of normal physiology and disease.

Noncoding RNA NORAD Regulates Genomic Stability by Sequestering PUMILIO Proteins.

Long noncoding RNAs (lncRNAs) have emerged as regulators of diverse biological processes. Here, we describe the initial functional analysis of a poorly characterized human lncRNA (LINC00657) that is induced after DNA damage, which we termed "noncoding RNA activated by DNA damage", or NORAD. NORAD is highly conserved and abundant, with expression levels of approximately 500-1,000 copies per cell. Remarkably, inactivation of NORAD triggers dramatic aneuploidy in previously karyotypically stable cell lines. NORAD maintains genomic stability by sequestering PUMILIO proteins, which repress the stability and translation of mRNAs to which they bind. In the absence of NORAD, PUMILIO proteins drive chromosomal instability by hyperactively repressing mitotic, DNA repair, and DNA replication factors. These findings introduce a mechanism that regulates the activity of a deeply conserved and highly dosage-sensitive family of RNA binding proteins and reveal unanticipated roles for a lncRNA and PUMILIO proteins in the maintenance of genomic stability.

A weekly 4-methylpyrazole treatment attenuates the development of non-obese metabolic dysfunction-associated steatotic liver disease (MASLD) in male mice: Role of JNK.

BACKGROUND: 4-methylpyrazole (4MP, fomepizole) is a competitive inhibitor of alcohol dehydrogenase (ADH) preventing the metabolism of ethylene glycol and methanol, respectively, into their toxic metabolites. 4MP seems also to possess a potential in the treatment of intoxication from other substance, for example, acetaminophen, and to modulate JNK-dependent signalling. Here, we determined if a treatment with 4MP once weekly affects the development of diet-induced non-obese metabolic dysfunction-associated steatotic liver disease (MASLD) in C57BL/6 mice. METHODS: Male C57BL/6 mice (6-8 weeks old, n = 7-8/group) were pair-fed either a liquid control diet (C) or a liquid sucrose-, fat- and cholesterol-rich diet (SFC) for 8 weeks while being concomitantly treated with 4MP (50 mg/kg bw i.p.) or vehicle once a week. Liver damage, inflammatory markers and glucose tolerance were assessed. Moreover, in endotoxin-challenged J774A.1 cells pretreated with 4MP, pro-inflammatory markers were assessed. RESULTS: The concomitant treatment of SFC-fed mice with 4MP attenuated the increase in JNK phosphorylation and pro-inflammatory markers like IFNγ, IL-6 and 3-nitrotyrosine protein adducts in liver tissue found in vehicle-treated SFC-fed mice, while not affecting impairments of glucose tolerance or the increase in portal endotoxin levels. Moreover, a pretreatment of endotoxin-stimulated J774A.1 cells with 4MP significantly attenuated the increases in JNK phosphorylation and pro-inflammatory mediators like IL-6 and Mcp1. CONCLUSIONS: Taken together, our results suggest that a treatment with 4MP once weekly attenuates the activation of JNK and dampens the development of non-obese MASLD in mice.

Long Noncoding RNAs in Diet-Induced Metabolic Diseases.

The prevalence of metabolic diseases, including type 2 diabetes and metabolic dysfunction-associated steatotic liver disease (MASLD), is steadily increasing. Although many risk factors, such as obesity, insulin resistance, or hyperlipidemia, as well as several metabolic gene programs that contribute to the development of metabolic diseases are known, the underlying molecular mechanisms of these processes are still not fully understood. In recent years, it has become evident that not only protein-coding genes, but also noncoding genes, including a class of noncoding transcripts referred to as long noncoding RNAs (lncRNAs), play key roles in diet-induced metabolic disorders. Here, we provide an overview of selected lncRNA genes whose direct involvement in the development of diet-induced metabolic dysfunctions has been experimentally demonstrated in suitable in vivo mouse models. We further summarize and discuss the associated molecular modes of action for each lncRNA in the respective metabolic disease context. This overview provides examples of lncRNAs with well-established functions in diet-induced metabolic diseases, highlighting the need for appropriate in vivo models and rigorous molecular analyses to assign clear biological functions to lncRNAs.

Long noncoding RNAs as versatile molecular regulators of cellular stress response and homeostasis.

Normal cell and body functions need to be maintained and protected against endogenous and exogenous stress conditions. Different cellular stress response pathways have evolved that are utilized by mammalian cells to recognize, process and overcome numerous stress stimuli in order to maintain homeostasis and to prevent pathophysiological processes. Although these stress response pathways appear to be quite different on a molecular level, they all have in common that they integrate various stress inputs, translate them into an appropriate stress response and eventually resolve the stress by either restoring homeostasis or inducing cell death. It has become increasingly appreciated that non-protein-coding RNA species, such as long noncoding RNAs (lncRNAs), can play critical roles in the mammalian stress response. However, the precise molecular functions and underlying modes of action for many of the stress-related lncRNAs remain poorly understood. In this review, we aim to provide a framework for the categorization of mammalian lncRNAs in stress response and homeostasis based on their experimentally validated modes of action. We describe the molecular functions and physiological roles of selected lncRNAs and develop a concept of how lncRNAs can contribute as versatile players in mammalian stress response and homeostasis. These concepts may be used as a starting point for the identification of novel lncRNAs and lncRNA functions not only in the context of stress, but also in normal physiology and disease.

An Argonaute phosphorylation cycle promotes microRNA-mediated silencing.

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.

Molecular functions and biological roles of long non-coding RNAs in human physiology and disease.

The human genome has been demonstrated to be pervasively transcribed, with a large fraction of the transcriptome representing non-protein-coding RNA species. A relatively novel class of non-coding RNAs, referred to as long non-coding RNAs (lncRNAs), has attracted increasing attention over recent years. Long non-coding RNAs comprise a very heterogenous class of transcripts that exert various functions in mammalian cells. Although the number of identified and annotated lncRNAs has been increasing steadily, our understanding of the biological roles for the vast majority of these transcripts remains limited. In this review, a broad concept of the currently known lncRNA modes of action is provided. Examples of mammalian lncRNAs with well-established biological roles are highlighted and their molecular functions and mechanisms are discussed in detail.

Salinomycin treatment reduces metastatic tumor burden by hampering cancer cell migration.

BACKGROUND: Tumor spreading is the major threat for cancer patients. The recently published anti-cancer drug salinomycin raised hope for an improved treatment by targeting therapy-refractory cancer stem cells. However, an unambiguous role of salinomycin against cancer cell migration and metastasis formation remains elusive. FINDINGS: We report that salinomycin effectively inhibits cancer cell migration in a variety of cancer types as determined by Boyden chamber assays. Additionally, cells were treated with doxorubicin at a concentration causing a comparable low cytotoxicity, emphasizing the anti-migratory potential of salinomycin. Moreover, single-cell tracking by time-lapse microscopy demonstrated a remarkable effect of salinomycin on breast cancer cell motility. Ultimately, salinomycin treatment significantly reduced the metastatic tumor burden in a syngenic mouse tumor model. CONCLUSIONS: Our findings clearly show that salinomycin can strongly inhibit cancer cell migration independent of the induction of cell death. We furthermore demonstrate for the first time that salinomycin treatment reduces metastasis formation in vivo, strengthening its role as promising anti-cancer therapeutic.