Non-Coding RNA, Volume 7, Issue 4 (December 2021) – 22 articles

The ability to differentiate between benign, suspicious, and malignant pulmonary nodules is imperative for definitive intervention in patients with early stage lung cancers. Here, we report that plasma protein functional effector sncRNAs (pfeRNAs) serve as non-invasive biomarkers for determining both the existence and the nature of pulmonary nodules in a three-stage study that included the healthy group, patients with benign pulmonary nodules, patients with suspicious nodules, and patients with malignant nodules. Following the standards required for a clinical laboratory improvement amendments (CLIA)-compliant laboratory-developed test (LDT), we identified a pfeRNA classifier containing 8 pfeRNAs in 108 biospecimens from 60 patients by sncRNA deep sequencing, deduced prediction rules using a separate training cohort of 198 plasma specimens, and then applied the prediction rules to another 230 plasma specimens in an independent validation cohort. The pfeRNA classifier could (1) differentiate patients with or without pulmonary nodules with an average sensitivity and specificity of 96.2% and 97.35% and (2) differentiate malignant versus benign pulmonary nodules with an average sensitivity and specificity of 77.1% and 74.25%. Our biomarkers are cost-effective, non-invasive, sensitive, and specific, and the qPCR-based method provides the possibility for automatic testing of robotic applications. Full article

Complex RNA–RNA interactions are increasingly known to play key roles in numerous biological processes from gene expression control to ribonucleoprotein granule formation. By contrast, the nature of these interactions and characteristics of their interfaces, especially those that involve partially or wholly structured RNAs, remain elusive. Herein, we discuss different modalities of RNA–RNA interactions with an emphasis on those that depend on secondary, tertiary, or quaternary structure. We dissect recently structurally elucidated RNA–RNA complexes including RNA triplexes, riboswitches, ribozymes, and reverse transcription complexes. These analyses highlight a reciprocal relationship that intimately links RNA structure formation with RNA–RNA interactions. The interactions not only shape and sculpt RNA structures but also are enabled and modulated by the structures they create. Understanding this two-way relationship between RNA structure and interactions provides mechanistic insights into the expanding repertoire of noncoding RNA functions, and may inform the design of novel therapeutics that target RNA structures or interactions. Full article

Over the last decade, tens of thousands of new long non-coding RNAs (lncRNAs) have been identified in the human genome. Nevertheless, except for a handful of genes, the genetic characteristics and functions of most of these lncRNAs remain elusive; this is partially due to their relatively low expression, high tissue specificity, and low conservation across species. A major limitation for determining the function of lncRNAs was the lack of methodologies suitable for studying these genes. The recent development of CRISPR/Cas9 technology has opened unprecedented opportunities to uncover the genetic and functional characteristics of the non-coding genome via targeted and high-throughput approaches. Specific CRISPR/Cas9-based approaches were developed to target lncRNA loci. Some of these approaches involve modifying the sequence, but others were developed to study lncRNAs by inducing transcriptional and epigenetic changes. The discovery of other programable Cas proteins broaden our possibilities to target RNA molecules with greater precision and accuracy. These approaches allow for the knock-down and characterization of lncRNAs. Here, we review how various CRISPR-based strategies have been used to characterize lncRNAs with important functions in different biological contexts and how these approaches can be further utilized to improve our understanding of the non-coding genome. Full article

Milk is an integral and therefore complex structural element of mammalian nutrition. Therefore, it is simple to conclude that lactation, the process of producing milk, is as complex as the mammary gland, the organ responsible for this biochemical activity. Nutrition, genetics, epigenetics, disease pathogens, climatic conditions, and other environmental variables all impact breast productivity. In the last decade, the number of studies devoted to epigenetics has increased dramatically. Reports are increasingly describing the direct participation of microRNAs (miRNAs), small noncoding RNAs that regulate gene expression post-transcriptionally, in the regulation of mammary gland development and function. This paper presents a summary of the current state of knowledge about the roles of miRNAs in mammary gland development, health, and functions, particularly during lactation. The significance of miRNAs in signaling pathways, cellular proliferation, and the lipid metabolism in agricultural ruminants, which are crucial in light of their role in the nutrition of humans as consumers of dairy products, is discussed. Full article

Long non-coding RNAs (lncRNAs) are widely recognized as important regulators of gene expression. Their molecular functions range from miRNA sponging to chromatin-associated mechanisms, leading to effects in disease progression and establishing them as diagnostic and therapeutic targets. Still, only a few representatives of this diverse class of RNAs are well studied, while the vast majority is poorly described beyond the existence of their transcripts. In this review we survey common in silico approaches for lncRNA annotation. We focus on the well-established sets of features used for classification and discuss their specific advantages and weaknesses. While the available tools perform very well for the task of distinguishing coding sequence from other RNAs, we find that current methods are not well suited to distinguish lncRNAs or parts thereof from other non-protein-coding input sequences. We conclude that the distinction of lncRNAs from intronic sequences and untranslated regions of coding mRNAs remains a pressing research gap. Full article

The role of trypsin genes in pharmacological sensitivity has been described in numerous arthropod species, including the sea louse Caligus rogercresseyi. This ectoparasite species is mainly controlled by xenobiotic drugs in Atlantic salmon farming. However, the post-transcriptional regulation of trypsin genes and the molecular components involved in drug response remain unclear. In particular, the miRNA bantam family has previously been associated with drug response in arthropods and is also found in C. rogercresseyi, showing a high diversity of isomiRs. This study aimed to uncover molecular interactions among trypsin genes and bantam miRNAs in the sea louse C. rogercresseyi in response to delousing drugs. Herein, putative mRNA/miRNA sequences were identified and localized in the C. rogercresseyi genome through genome mapping and blast analyses. Expression analyses were obtained from the mRNA transcriptome and small-RNA libraries from groups with differential sensitivity to three drugs used as anti-sea lice agents: azamethiphos, deltamethrin, and cypermethrin. The validation was conducted by qPCR analyses and luciferase assay of selected bantam and trypsin genes identified from in silico transcript prediction. A total of 60 trypsin genes were identified in the C. rogercresseyi genome, and 39 bantam miRNAs were differentially expressed in response to drug exposure. Notably, expression analyses and correlation among values obtained from trypsin and bantam revealed an opposite trend and potential binding sites with significant ΔG values. The luciferase assay showed a reduction of around 50% in the expression levels of the trypsin 2-like gene, which could imply that this gene is a potential target for bantam. The role of trypsin genes and bantam miRNAs in the pharmacological sensitivity of sea lice and the use of miRNAs as potential markers in these parasites are discussed in this study. Full article

Neuroendocrine prostate cancer (NEPC) represents a variant of prostate cancer that occurs in response to treatment resistance or, to a much lesser extent, de novo. Unravelling the molecular mechanisms behind transdifferentiation of cancer cells to neuroendocrine-like cancer cells is essential for development of new treatment opportunities. This review focuses on summarizing the role of small molecules, predominantly microRNAs, in this phenomenon. A published literature search was performed to identify microRNAs, which are reported and experimentally validated to modulate neuroendocrine markers and/or regulators and to affect the complex neuroendocrine phenotype. Next, available patients’ expression datasets were surveyed to identify deregulated microRNAs, and their effect on NEPC and prostate cancer progression is summarized. Finally, possibilities of miRNA detection and quantification in body fluids of prostate cancer patients and their possible use as liquid biopsy in prostate cancer monitoring are discussed. All the addressed clinical and experimental contexts point to an association of NEPC with upregulation of miR-375 and downregulation of miR-34a and miR-19b-3p. Together, this review provides an overview of different roles of non-coding RNAs in the emergence of neuroendocrine prostate cancer. Full article

Altered expression of protein coding gene (PCG) and long non-coding RNA (lncRNA) have been identified in SARS-CoV-2 infected cells and tissues from COVID-19 patients. The functional role and mechanism (s) of transcriptional regulation of deregulated genes in COVID-19 remain largely unknown. In the present communication, reanalyzing publicly available gene expression data, we observed that 66 lncRNA and 5491 PCG were deregulated in more than one experimental condition. Combining our earlier published results and using different publicly available resources, it was observed that 72 deregulated lncRNA interacted with 3228 genes/proteins. Many targets of deregulated lncRNA could also interact with SARS-CoV-2 coded proteins, modulated by IFN treatment and identified in CRISPR screening to modulate SARS-CoV-2 infection. The majority of the deregulated lncRNA and PCG were targets of at least one of the transcription factors (TFs), interferon responsive factors (IRFs), signal transducer, and activator of transcription (STATs), NFκB, MYC, and RELA/p65. Deregulated 1069 PCG was joint targets of lncRNA and TF. These joint targets are significantly enriched with pathways relevant for SARS-CoV-2 infection indicating that joint regulation of PCG could be one of the mechanisms for deregulation. Over all this manuscript showed possible involvement of lncRNA and mechanisms of deregulation of PCG in the pathogenesis of COVID-19. Full article

Protein biosynthesis is essential for any organism, yet how this process is regulated is not fully understood at the molecular level. During evolution, ribosomal RNA expanded in specific regions, referred to as rRNA expansion segments (ES). First functional roles of these expansions have only recently been discovered. Here we address the role of ES7L_a located in the large ribosomal subunit for factor recruitment to the yeast ribosome and the potential consequences for translation. Truncation of ES7L_a has only minor effects on ribosome biogenesis, translation efficiency and cell doubling. Using yeast rRNA deletion strains coupled with ribosome-specific mass spectrometry we analyzed the interactome of ribosomes lacking ES7L_a. Three aminoacyl-tRNA synthetases showed reduced ribosome association. Synthetase activities however remained unaltered suggesting that the pool of aminoacylated tRNAs is unaffected by the ES deletion. These results demonstrated that aminoacylation activities of tRNA synthetases per se do not rely on ribosome association. These findings suggest a role of ribosome-associated aminoacyl-tRNA synthetase beyond their core enzymatic functions. Full article

Currently there are nine known examples of transmissible cancers in nature. They have been observed in domestic dog, Tasmanian devil, and six bivalve species. These tumours can overcome host immune defences and spread to other members of the same species. Non-coding RNAs (ncRNAs) are known to play roles in tumorigenesis and immune system evasion. Despite their potential importance in transmissible cancers, there have been no studies on ncRNA function in this context to date. Here, we present possible applications of the CRISPR/Cas system to study the RNA biology of transmissible cancers. Specifically, we explore how ncRNAs may play a role in the immortality and immune evasion ability of these tumours. Full article

As more sequencing data accumulate and novel puzzling genetic regulations are discovered, the need for accurate automated modeling of RNA structure increases. RNA structure modeling from chemical probing experiments has made tremendous progress, however accurately predicting large RNA structures is still challenging for several reasons: RNA are inherently flexible and often adopt many energetically similar structures, which are not reliably distinguished by the available, incomplete thermodynamic model. Moreover, computationally, the problem is aggravated by the relevance of pseudoknots and non-canonical base pairs, which are hardly predicted efficiently. To identify nucleotides involved in pseudoknots and non-canonical interactions, we scrutinized the SHAPE reactivity of each nucleotide of the 188 nt long lariat-capping ribozyme under multiple conditions. Reactivities analyzed in the light of the X-ray structure were shown to report accurately the nucleotide status. Those that seemed paradoxical were rationalized by the nucleotide behavior along molecular dynamic simulations. We show that valuable information on intricate interactions can be deduced from probing with different reagents, and in the presence or absence of Mg²⁺. Furthermore, probing at increasing temperature was remarkably efficient at pointing to non-canonical interactions and pseudoknot pairings. The possibilities of following such strategies to inform structure modeling software are discussed. Full article

Non-coding RNAs (ncRNAs) comprise a set of abundant and functionally diverse RNA molecules. Since the discovery of the first ncRNA in the 1960s, ncRNAs have been shown to be involved in nearly all steps of the central dogma of molecular biology. In recent years, the pace of discovery of novel ncRNAs and their cellular roles has been greatly accelerated by high-throughput sequencing. Advances in sequencing technology, library preparation protocols as well as computational biology helped to greatly expand our knowledge of which ncRNAs exist throughout the kingdoms of life. Moreover, RNA sequencing revealed crucial roles of many ncRNAs in human health and disease. In this review, we discuss the most recent methodological advancements in the rapidly evolving field of high-throughput sequencing and how it has greatly expanded our understanding of ncRNA biology across a large number of different organisms. Full article

Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (Mtb), with 10.4 million new cases per year reported in the human population. Recent studies on the Mtb transcriptome have revealed the abundance of noncoding RNAs expressed at various phases of mycobacteria growth, in culture, in infected mammalian cells, and in patients. Among these noncoding RNAs are both small RNAs (sRNAs) between 50 and 350 nts in length and smaller RNAs (sncRNA) < 50 nts. In this review, we provide an up-to-date synopsis of the identification, designation, and function of these Mtb-encoded sRNAs and sncRNAs. The methodological advances including RNA sequencing strategies, small RNA antagonists, and locked nucleic acid sequence-specific RNA probes advancing the studies on these small RNA are described. Initial insights into the regulation of the small RNA expression and putative processing enzymes required for their synthesis and function are discussed. There are many open questions remaining about the biological and pathogenic roles of these small non-coding RNAs, and potential research directions needed to define the role of these mycobacterial noncoding RNAs are summarized. Full article

Over recent years, the long known class of small nucleolar RNAs (snoRNAs) have gained interest among the scientific community, especially in the clinical context. The main molecular role of this interesting family of non-coding RNAs is to serve as scaffolding RNAs to mediate site-specific RNA modification of ribosomal RNAs (rRNAs) and small nuclear RNAs (snRNAs). With the development of new sequencing techniques and sophisticated analysis pipelines, new members of the snoRNA family were identified and global expression patterns in disease backgrounds could be determined. We will herein shed light on the current research progress in snoRNA biology and their clinical role by influencing disease outcome in hematological diseases. Astonishingly, in recent studies snoRNAs emerged as potent biomarkers in a variety of these clinical setups, which is also highlighted by the frequent deregulation of snoRNA levels in the hema-oncological context. However, research is only starting to reveal how snoRNAs might influence cellular functions and the connected disease hallmarks in hematological malignancies. Full article

The cohesin complex is a multi-subunit protein complex initially discovered for its role in sister chromatid cohesion. However, cohesin also has several other functions and plays important roles in transcriptional regulation, DNA double strand break repair, and chromosome architecture thereby influencing gene expression and development in organisms from yeast to man. While most of these functions rely on protein–protein interactions, post-translational protein, as well as DNA modifications, non-coding RNAs are emerging as additional players that facilitate and modulate the function or expression of cohesin and its individual components. This review provides a condensed overview about the architecture as well as the function of the cohesin complex and highlights its multifaceted interplay with both short and long non-coding RNAs. Full article

Long non-coding RNAs (lncRNA) comprise a diverse group of non-protein-coding RNAs >200 bp in length that are involved in various normal cellular processes and disease states, and can affect coding gene expression through mechanisms in cis or in trans. Since the discovery of the first functional lncRNAs transcribed by RNA Polymerase II, H19 and Xist, many others have been identified and noted for their unusual transcriptional pattern, whereby expression from one chromosome homolog is strongly favored over the other, also known as mono-allelic or differential allelic expression. lncRNAs with differential allelic expression have been observed to play critical roles in developmental gene regulation, chromosome structure, and disease. Here, we will focus on known examples of differential allelic expression of lncRNAs and highlight recent research describing functional lncRNAs expressed from both imprinted and random mono-allelic expression domains. Full article

Despite advances in treatments and therapies, cardiovascular diseases (CVDs) remain one of the leading causes of death worldwide. The discovery that most of the human genome, although transcribed, does not encode proteins was crucial for focusing on the potential of long non-coding RNAs (lncRNAs) as essential regulators of cell function at the epigenetic, transcriptional, and post-transcriptional levels. This class of non-coding RNAs is related to the pathophysiology of the cardiovascular system. The different expression profiles of lncRNAs, in different contexts of CVDs, change a great potential in their use as a biomarker and targets of therapeutic intervention. Furthermore, regular physical exercise plays a protective role against CVDs; on the other hand, little is known about its underlying molecular mechanisms. In this review, we look at the accumulated knowledge on lncRNAs and their functions in the cardiovascular system, focusing on the cardiovascular pathology of arterial hypertension, coronary heart disease, acute myocardial infarction, and heart failure. We discuss the potential of these molecules as biomarkers for clinical use, their limitations, and how the manipulation of the expression profile of these transcripts through physical exercise can begin to be suggested as a strategy for the treatment of CVDs. Full article

Many RNA-RNA interactions depend on molecular chaperones to form and remain stable in living cells. A prime example is the RNA chaperone Hfq, which is a critical effector involved in regulatory interactions between small RNAs (sRNAs) and cognate target mRNAs in Enterobacteriaceae. While there is a great deal of in vitro biochemical evidence supporting the model that Hfq enhances rates or affinities of sRNA:mRNA interactions, there is little corroborating in vivo evidence. Here we used in vivo tools including reporter genes, co-purification assays, and super-resolution microscopy to analyze the role of Hfq in RyhB-mediated regulation, and we found that Hfq is often unnecessary for efficient RyhB:mRNA complex formation in vivo. Remarkably, our data suggest that a primary function of Hfq is to promote RyhB-induced cleavage of mRNA targets by RNase E. Moreover, our work indicates that Hfq plays a more limited role in dictating regulatory outcomes following sRNAs RybB and DsrA complex formation with specific target mRNAs. Our investigation helps evaluate the roles played by Hfq in some RNA-mediated regulation. Full article

Cutaneous melanoma (CM) is the most lethal tumor among skin cancers, and its incidence is constantly increasing. A deeper understanding of the molecular processes guiding melanoma pathogenesis could improve diagnosis, treatment and prognosis. MicroRNAs play a key role in melanoma biology. Recently, next generation sequencing (NGS) experiments, designed to assess small-RNA expression, revealed the existence of microRNA variants with different length and sequence. These microRNA isoforms are known as isomiRs and provide an additional layer to the complex non-coding RNA world. Here, we collected data from NGS experiments to provide a comprehensive characterization of miRNA and isomiR dysregulation in benign nevi (BN) and early-stage melanomas. We observed that melanoma and BN express different and specific isomiRs and have a different isomiR abundance distribution. Moreover, isomiRs from the same microRNA can have opposite expression trends between groups. Using The Cancer Genome Atlas (TCGA) dataset of skin cancers, we analyzed isomiR expression in primary melanoma and melanoma metastasis and tested their association with NF1, BRAF and NRAS mutations. IsomiRs differentially expressed were identified and catalogued with reference to the canonical form. The reported non-random dysregulation of specific isomiRs contributes to the understanding of the complex melanoma pathogenesis and serves as the basis for further functional studies. Full article

The vascular endothelium comprises the interface between the circulation and the vessel wall and, as such, is under the dynamic regulation of vascular signalling, nutrients, and hypoxia. Understanding the molecular drivers behind endothelial cell (EC) and vascular smooth muscle cell (VSMC) function and dysfunction remains a pivotal task for further clinical progress in tackling vascular disease. A newly emerging era in vascular biology with landmark deep sequencing approaches has provided us with the means to profile diverse layers of transcriptional regulation at a single cell, chromatin, and epigenetic level. This review describes the roles of major vascular long non-coding RNA (lncRNAs) in the epigenetic regulation of EC and VSMC function and discusses the recent progress in their discovery, detection, and functional characterisation. We summarise new findings regarding lncRNA-mediated epigenetic mechanisms—often regulated by hypoxia—within the vascular endothelium and smooth muscle to control vascular homeostasis in health and disease. Furthermore, we outline novel molecular techniques being used in the field to delineate the lncRNA subcellular localisation and interaction with proteins to unravel their biological roles in the epigenetic regulation of vascular genes. Full article

Complete 3′UTRs unambiguously assigned to specific mRNA isoforms from the Atlantic salmon full-length (FL) transcriptome were collected into a 3′UTRome. miRNA response elements (MREs) and other cis-regulatory motifs were subsequently predicted and assigned to 3′UTRs of all FL-transcripts. The MicroSalmon GitHub repository provides all results. RNAHybrid and sRNAtoolbox tools predicted the MREs. UTRscan and the Teiresias algorithm predicted other 3′UTR cis-acting motifs, both known vertebrate motifs and putative novel motifs. MicroSalmon provides search programs to retrieve all FL-transcripts targeted by a miRNA (median number 1487), all miRNAs targeting an FL-transcript (median number 27), and other cis-acting motifs. As thousands of FL-transcripts may be targets of each miRNA, additional experimental strategies are necessary to reduce the likely true and relevant targets to a number that may be functionally validated. Low-complexity motifs known to affect mRNA decay in vertebrates were over-represented. Many of these were enriched in the terminal end, while purine- or pyrimidine-rich motifs with unknown functions were enriched immediately downstream of the stop codon. Furthermore, several novel complex motifs were over-represented, indicating conservation and putative function. In conclusion, MicroSalmon is an extensive and useful, searchable resource for study of Atlantic salmon transcript regulation by miRNAs and cis-acting 3′UTR motifs. Full article

The COVID-19 pandemic caused by the novel coronavirus SARS-CoV-2 (2019-nCoV) has devastated global healthcare and economies. Despite the stabilization of infectivity rates in some developed nations, several countries are still under the grip of the pathogenic viral mutants that are causing a significant increase in infections and hospitalization. Given this urgency, targeting of key host factors regulating SARS-CoV-2 life cycle is postulated as a novel strategy to counter the virus and its associated pathological outcomes. In this regard, Poly (ADP)-ribose polymerase-1 (PARP-1) is being increasingly recognized as a possible target. PARP-1 is well studied in human diseases such as cancer, central nervous system (CNS) disorders and pathology of RNA viruses. Emerging evidence indicates that regulation of PARP-1 by non-coding RNAs such as microRNAs is integral to cell survival, redox balance, DNA damage response, energy homeostasis, and several other cellular processes. In this short perspective, we summarize the recent findings on the microRNA/PARP-1 axis and its therapeutic potential for COVID-19 pathologies. Full article