Non-Coding RNA, Volume 7, Issue 3 (September 2021) – 22 articles

Recently, we discovered a new family of unusually short RNAs mapping to 5.8S ribosomal RNA (rRNA) and which we named dodecaRNAs (doRNAs), according to the number of core nucleotides (12 nt) their members contain. To confirm these small RNA-sequencing (RNA-Seq) data, validate the existence of the two overly abundant doRNAs—the minimal core 12-nt doRNA sequence and its + 1-nt variant bearing a 5′ Cytosine, C-doRNA—and streamline their analysis, we developed a new specific and sensitive splinted 5′ ligation reverse transcription (RT)-quantitative polymerase chain reaction (qPCR) method. This method is based on a splint-assisted ligation of an adapter to the 5′ end of doRNAs, followed by RT-qPCR amplification and quantitation. Our optimized protocol, which may discriminate between doRNA, C-doRNA, mutated and precursor sequences, can accurately detect as low as 240 copies and is quantitatively linear over a range of 7 logs. This method provides a unique tool to expand and facilitate studies exploring the molecular and cellular biology of RNA species shorter than microRNAs. Full article

MicroRNAs (miRNAs) are small non-coding RNAs that are about 22 nucleotides in length. They regulate gene expression post-transcriptionally by guiding the effector protein Argonaute to its target mRNA in a sequence-dependent manner, causing the translational repression and destabilization of the target mRNAs. Both Drosha and Dicer, members of the RNase III family proteins, are essential components in the canonical miRNA biogenesis pathway. miRNA is transcribed into primary-miRNA (pri-miRNA) from genomic DNA. Drosha then cleaves the flanking regions of pri-miRNA into precursor-miRNA (pre-miRNA), while Dicer cleaves the loop region of the pre-miRNA to form a miRNA duplex. Although the role of Drosha and Dicer in miRNA maturation is well known, the modulation processes that are important for regulating the downstream gene network are not fully understood. In this review, we summarized and discussed current reports on miRNA biogenesis caused by Drosha and Dicer. We also discussed the modulation mechanisms regulated by double-stranded RNA binding proteins (dsRBPs) and the function and substrate specificity of dsRBPs, including the TAR RNA binding protein (TRBP) and the adenosine deaminase acting on RNA (ADAR). Full article

Small non-coding RNAs (sncRNAs) represent an important class of regulatory RNAs involved in the regulation of transcription, RNA splicing or translation. Among these sncRNAs, small nucleolar RNAs (snoRNAs) mostly originate from intron splicing in humans and are central to posttranscriptional regulation of gene expression. However, the characterization of the complete repertoire of sncRNAs in a given cellular context and the functional annotation of the human transcriptome are far from complete. Here, we report the large-scale identification of sncRNAs in the size range of 50 to 200 nucleotides without a priori on their biogenesis, structure and genomic origin in the context of normal human muscle cells. We provided a complete set of experimental validation of novel candidate snoRNAs by evaluating the prerequisites for their biogenesis and functionality, leading to their validation as genuine snoRNAs. Interestingly, we also found intergenic snoRNAs, which we showed are in fact integrated into candidate introns of unannotated transcripts or degraded by the Nonsense Mediated Decay pathway. Hence, intergenic snoRNAs represent a new type of landmark for the identification of new transcripts that have gone undetected because of low abundance or degradation after the release of the snoRNA. Full article

Pituitary adenomas (PAs) are one of the most common lesions of intracranial neoplasms, occurring in approximately 15% of the general population. They are typically benign, although some adenomas show aggressive behavior, exhibiting rapid growth, drug resistance, and invasion of surrounding tissues. Despite ongoing improvements in diagnostic and therapeutic strategies, late first diagnosis is common, and patients with PAs are prone to relapse. Therefore, earlier diagnosis and prevention of recurrence are of importance to improve patient care. MicroRNAs (miRNAs) are short non-coding single stranded RNAs that regulate gene expression at the post-transcriptional level. An increasing number of studies indicate that a deregulation of their expression patterns is related with pituitary tumorigenesis, suggesting that these small molecules could play a critical role in contributing to tumorigenesis and the onset of these tumors by acting either as oncosuppressors or as oncogenes, depending on the biological context. This paper provides an overview of miRNAs involved in PA tumorigenesis, which might serve as novel potential diagnostic and prognostic non-invasive biomarkers, and for the future development of miRNA-based therapeutic strategies for PAs. Full article

The highly infectious severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged as the causative agent of coronavirus disease 2019 (COVID-19) in late 2019, igniting an unprecedented pandemic. A mechanistic picture characterising the acute immunopathological disease in severe COVID-19 is developing. Non-coding RNAs (ncRNAs) constitute the transcribed but un-translated portion of the genome and, until recent decades, have been undiscovered or overlooked. A growing body of research continues to demonstrate their interconnected involvement in the immune response to SARS-CoV-2 and COVID-19 development by regulating several of its pathological hallmarks: cytokine storm syndrome, haemostatic alterations, immune cell recruitment, and vascular dysregulation. There is also keen interest in exploring the possibility of host–virus RNA–RNA and RNA–RBP interactions. Here, we discuss and evaluate evidence demonstrating the involvement of short and long ncRNAs in COVID-19 and use this information to propose hypotheses for future mechanistic and clinical studies. Full article

Clinical studies demonstrated that the ovarian hormone 17β-estradiol (E₂) is neuroprotective within a narrow window of time following menopause, suggesting that there is a biological switch in E₂ action that is temporally dependent. However, the molecular mechanisms mediating this temporal switch have not been determined. Our previous studies focused on microRNAs (miRNA) as one potential molecular mediator and showed that E₂ differentially regulated a subset of mature miRNAs which was dependent on age and the length of time following E₂ deprivation. Notably, E₂ significantly increased both strands of the miR-9 duplex (miR-9-5p and miR-9-3p) in the hypothalamus, raising the possibility that E₂ could regulate miRNA stability/degradation. We tested this hypothesis using a biochemical approach to measure miRNA decay in a hypothalamic neuronal cell line and in hypothalamic brain tissue from a rat model of surgical menopause. Notably, we found that E₂ treatment stabilized both miRNAs in neuronal cells and in the rat hypothalamus. We also used polysome profiling as a proxy for miR-9-5p and miR-9-3p function and found that E₂ was able to shift polysome loading of the miRNAs, which repressed the translation of a predicted miR-9-3p target. Moreover, miR-9-5p and miR-9-3p transcripts appeared to occupy different fractions of the polysome profile, indicating differential subcellular. localization. Together, these studies reveal a novel role for E₂ in modulating mature miRNA behavior, independent of its effects at regulating the primary and/or precursor form of miRNAs. Full article

Long non-coding RNA (lncRNA) TP53 target 1 (TP53TG1) was discovered as a TP53 target gene. TP53TG1 has been reported as having dual roles by exerting tumor-suppressive and oncogenic activities that vary depending on the cancer type. Yet, the role of TP53TG1 in hepatocellular carcinoma (HCC) is not fully understood. In this study, we performed both gain- and loss-of-function studies to determine the biological role of TP53TG1 in HCC. We found that the knockdown of TP53 in HCC cells caused the upregulation of TP53TG1. Furthermore, we found that the knockdown of TP53TG1 not only suppressed HCC cell proliferation and migration, but also reduced intrinsic ERK signaling. In contrast, the overexpression of TP53TG1 increased ERK activation and enhanced HCC proliferation. In conclusion, our study reveals an oncogenic role of TP53TG1 in HCC, which provides a novel insight into the cell-type-specific function of TP53TG1 in HCC. Full article

RNA modifications play an essential role in determining RNA fate. Recent studies have revealed the effects of such modifications on all steps of RNA metabolism. These modifications range from the addition of simple groups, such as methyl groups, to the addition of highly complex structures, such as sugars. Their consequences for translation fidelity are not always well documented. Unlike the well-known m⁶A modification, they are thought to have direct effects on either the folding of the molecule or the ability of tRNAs to bind their codons. Here we describe how modifications found in tRNAs anticodon-loop, rRNA, and mRNA can affect translation fidelity, and how approaches based on direct manipulations of the level of RNA modification could potentially be used to modulate translation for the treatment of human genetic diseases. Full article

Ribosomal intergenic spacer (rIGS), located between the 45S rRNA coding arrays in humans, is a deep, unexplored source of small and long non-coding RNA molecules transcribed in certain conditions to help a cell generate a stress response, pass through a differentiation state or fine tune the functioning of the nucleolus as a ribosome biogenesis center of the cell. Many of the non-coding transcripts originating from the rIGS are not characterized to date. Here, we confirm the transcriptional activity of the region laying a 2 kb upstream of the rRNA promoter, and demonstrate its altered expression under transcriptional stress, induced by a wide range of known transcription inhibitors. We managed to show an increased variability of anti-sense transcripts in alpha-amanitin treated cells by applying the low-molecular RNA fraction extracted from agarose gel to PAGE-northern. Also, the fractioning of RNA by size using agarose gel slices occurred, being applicable for determining the sizes of target transcripts via RT-PCR. Full article

Long non-coding RNAs are nucleotide molecules that regulate transcription in numerous cellular processes and are related to the occurrence of many diseases, including cancer. In this regard, we recently discovered a polyadenylated long non-coding RNA (named TG2-lncRNA) encoded within the first intron of the Transglutaminase type 2 gene (TGM2), which is related to tumour proliferation in human cancer cell lines. To better characterize this new biological player, we investigated the effects of its suppression in MCF-7 breast cancer cells, using siRNA treatment and RNA-sequencing. In this way, we found modifications in several networks associated to biological functions relevant for tumorigenesis (apoptosis, chronic inflammation, angiogenesis, immunomodulation, cell mobility, and epithelial–mesenchymal transition) that were originally attributed only to Transglutaminase type 2 protein but that could be regulated also by TG2-lncRNA. Moreover, our experiments strongly suggest the ability of TG2-lncRNA to directly interact with important transcription factors, such as RXRα and TP53, paving the way for several regulatory loops that can potentially influence the phenotypic behaviour of MCF-7 cells. These considerations imply the need to further investigate the relative relevance of the TG2 protein itself and/or other gene products as key regulators in the organization of breast cancer program. Full article

Circular RNAs (circRNAs) are emerging as novel regulators of gene expression in various biological processes. CircRNAs regulate gene expression by interacting with cellular regulators such as microRNAs and RNA binding proteins (RBPs) to regulate downstream gene expression. The accumulation of high-throughput RNA–protein interaction data revealed the interaction of RBPs with the coding and noncoding RNAs, including recently discovered circRNAs. RBPs are a large family of proteins known to play a critical role in gene expression by modulating RNA splicing, nuclear export, mRNA stability, localization, and translation. However, the interaction of RBPs with circRNAs and their implications on circRNA biogenesis and function has been emerging in the last few years. Recent studies suggest that circRNA interaction with target proteins modulates the interaction of the protein with downstream target mRNAs or proteins. This review outlines the emerging mechanisms of circRNA–protein interactions and their functional role in cell physiology. Full article

The cancer genome is characterized by extensive variability, in the form of Single Nucleotide Polymorphisms (SNPs) or structural variations such as Copy Number Alterations (CNAs) across wider genomic areas. At the molecular level, most SNPs and/or CNAs reside in non-coding sequences, ultimately affecting the regulation of oncogenes and/or tumor-suppressors in a cancer-specific manner. Notably, inherited non-coding variants can predispose for cancer decades prior to disease onset. Furthermore, accumulation of additional non-coding driver mutations during progression of the disease, gives rise to genomic instability, acting as the driving force of neoplastic development and malignant evolution. Therefore, detection and characterization of such mutations can improve risk assessment for healthy carriers and expand the diagnostic and therapeutic toolbox for the patient. This review focuses on functional variants that reside in transcribed or not transcribed non-coding regions of the cancer genome and presents a collection of appropriate state-of-the-art methodologies to study them. Full article

Carotenoid biosynthesis in the fungus Fusarium fujikuroi is regulated by environmental factors, with light being the main stimulating signal. The CarS RING-finger protein plays an important role in the downregulation of structural genes of the carotenoid pathway. A recent transcriptomic analysis on the effect of carS mutation identified a gene for a long non-coding RNA (lncRNA) upstream of carS, called carP, the deletion of which results in increased carS mRNA levels and lack of carotenoid production. We have investigated the function of carP by studying the transcriptomic effect of its deletion and the phenotypes resulting from the reintroduction of carP to a deletion strain. The RNA-seq data showed that the loss of carP affected the mRNA levels of hundreds of genes, especially after illumination. Many of these changes appeared to be cascade effects as a result of changes in carS expression, as suggested by the comparison with differentially expressed genes in a carS mutant. Carotenoid production only recovered when carP was integrated upstream of carS, but not at other genomic locations, indicating a cis-acting mechanism on carS. However, some genes hardly affected by CarS were strongly upregulated in the carP mutant, indicating that carP may have other regulatory functions as an independent regulatory element. Full article

Triple-negative breast cancers affect thousands of women in the United States and disproportionately drive mortality from breast cancer. MicroRNAs are small, non-coding RNAs that negatively regulate gene expression post-transcriptionally by inhibiting target mRNA translation or by promoting mRNA degradation. We have identified that miRNA-203, silenced by epithelial–mesenchymal transition (EMT), is a tumor suppressor and can promote differentiation of breast cancer stem cells. In this study, we tested the ability of liposomal delivery of miR-203 to reverse aspects of breast cancer pathogenesis using breast cancer and EMT cell lines. We show that translationally relevant methods for increasing miR-203 abundance within a target tissue affects cellular properties associated with cancer progression. While stable miR-203 expression suppresses LASP1 and survivin, nanoliposomal delivery suppresses BMI1, indicating that suppression of distinct mRNA target profiles can lead to loss of cancer cell migration. Full article

The Nonsense-Mediated mRNA Decay (NMD) has been classically viewed as a translation-dependent RNA surveillance pathway degrading aberrant mRNAs containing premature stop codons. However, it is now clear that mRNA quality control represents only one face of the multiple functions of NMD. Indeed, NMD also regulates the physiological expression of normal mRNAs, and more surprisingly, of long non-coding (lnc)RNAs. Here, we review the different mechanisms of NMD activation in yeast and mammals, and we discuss the molecular bases of the NMD sensitivity of lncRNAs, considering the functional roles of NMD and of translation in the metabolism of these transcripts. In this regard, we describe several examples of functional micropeptides produced from lncRNAs. We propose that translation and NMD provide potent means to regulate the expression of lncRNAs, which might be critical for the cell to respond to environmental changes. Full article

Nuclear group I introns are restricted to the ribosomal DNA locus where they interrupt genes for small subunit and large subunit ribosomal RNAs at conserved sites in some eukaryotic microorganisms. Here, the myxomycete protists are a frequent source of nuclear group I introns due to their unique life strategy and a billion years of separate evolution. The ribosomal DNA of the myxomycete Mucilago crustacea was investigated and found to contain seven group I introns, including a direct repeat-containing intron at insertion site S1389 in the small subunit ribosomal RNA gene. We collected, analyzed, and compared 72 S1389 group IC1 introns representing diverse myxomycete taxa. The consensus secondary structure revealed a conserved ribozyme core, but with surprising sequence variations in the guanosine binding site in segment P7. Some S1389 introns harbored large extension sequences in the peripheral region of segment P9 containing direct repeat arrays. These repeats contained up to 52 copies of a putative internal guide sequence motif. Other S1389 introns harbored homing endonuclease genes in segment P1 encoding His-Cys proteins. Homing endonuclease genes were further interrupted by small spliceosomal introns that have to be removed in order to generate the open reading frames. Phylogenetic analyses of S1389 intron and host gene indicated both vertical and horizontal intron transfer during evolution, and revealed sporadic appearances of direct repeats, homing endonuclease genes, and guanosine binding site variants among the myxomycete taxa. Full article

Gene expression is an essential process for cellular growth, proliferation, and differentiation. The transcription of protein-coding genes and non-coding loci depends on RNA polymerases. Interestingly, numerous loci encode long non-coding (lnc)RNA transcripts that are transcribed by RNA polymerase II (RNAPII) and fine-tune the RNA metabolism. The nucleolus is a prime example of how different lncRNA species concomitantly regulate gene expression by facilitating the production and processing of ribosomal (r)RNA for ribosome biogenesis. Here, we summarise the current findings on how RNAPII influences nucleolar structure and function. We describe how RNAPII-dependent lncRNA can both promote nucleolar integrity and inhibit ribosomal (r)RNA synthesis by modulating the availability of rRNA synthesis factors in trans. Surprisingly, some lncRNA transcripts can directly originate from nucleolar loci and function in cis. The nucleolar intergenic spacer (IGS), for example, encodes nucleolar transcripts that counteract spurious rRNA synthesis in unperturbed cells. In response to DNA damage, RNAPII-dependent lncRNA originates directly at broken ribosomal (r)DNA loci and is processed into small ncRNA, possibly to modulate DNA repair. Thus, lncRNA-mediated regulation of nucleolar biology occurs by several modes of action and is more direct than anticipated, pointing to an intimate crosstalk of RNA metabolic events. Full article

Pervasive transcription is widespread in eukaryotes, generating large families of non-coding RNAs. Such pervasive transcription is a key player in the regulatory pathways controlling chromatin state and gene expression. Here, we describe long non-coding RNAs generated from the ribosomal RNA gene promoter called UPStream-initiating transcripts (UPS). In yeast, rDNA genes are organized in tandem repeats in at least two different chromatin states, either transcribed and largely depleted of nucleosomes (open) or assembled in regular arrays of nucleosomes (closed). The production of UPS transcripts by RNA Polymerase II from endogenous rDNA genes was initially documented in mutants defective for rRNA production by RNA polymerase I. We show here that UPS are produced in wild-type cells from closed rDNA genes but are hidden within the enormous production of rRNA. UPS levels are increased when rDNA chromatin states are modified at high temperatures or entering/leaving quiescence. We discuss their role in the regulation of rDNA chromatin states and rRNA production. Full article

Exosomes are a class of small, secreted extracellular vesicles (EV) that have recently gained considerable attention for their role in normal cellular function, disease processes and potential as biomarkers. Exosomes serve as intercellular messengers and carry molecular cargo that can alter gene expression and the phenotype of recipient cells. Here, we investigated alterations of microRNA cargo in exosomes secreted by epileptogenic tissue in tuberous sclerosis complex (TSC), a multi-system genetic disorder that includes brain lesions known as tubers. Approximately 90% of TSC patients suffer from seizures that originate from tubers, and ~60% are resistant to antiseizure drugs. It is unknown why some tubers cause seizures while others do not, and the molecular basis of drug-resistant epilepsy is not well understood. It is believed that neuroinflammation is involved, and characterization of this mechanism may be key to disrupting the “vicious cycle” between seizures, neuroinflammation, and increased seizure susceptibility. We isolated exosomes from epileptogenic and non-epileptogenic TSC tubers, and we identified differences in their microRNA cargo using small RNA-seq. We identified 12 microRNAs (including miR-142-3p, miR-223-3p and miR-21-5p) that are significantly increased in epileptogenic tubers and contain nucleic acid motifs that activate toll-like receptors (TLR7/8), initiating a neuroinflammatory cascade. Exosomes from epileptogenic tissue caused induction of key pathways in cultured cells, including innate immune signaling (TLR), inflammatory response and key signaling nodes SQSTM1 (p62) and CDKN1A (p21). Genes induced in vitro were also significantly upregulated in epileptogenic tissue. These results provide new evidence on the role of exosomes and non-coding RNA cargo in the neuroinflammatory cascade of epilepsy and may help advance the development of novel biomarkers and therapeutic approaches for the treatment of drug-resistant epilepsy. Full article

Substantial evidence indicates that microRNA-155 (miR-155) plays a crucial role in the pathogenesis of diabetes mellitus (DM) and its complications. A number of clinical studies reported low serum levels of miR-155 in patients with type 2 diabetes (T2D). Preclinical studies revealed that miR-155 partakes in the phenotypic switch of cells within the islets of Langerhans under metabolic stress. Moreover, miR-155 was shown to regulate insulin sensitivity in liver, adipose tissue, and skeletal muscle. Dysregulation of miR-155 expression was also shown to predict the development of nephropathy, neuropathy, and retinopathy in DM. Here, we systematically describe the reports investigating the role of miR-155 in DM and its complications. We also discuss the recent results from in vivo and in vitro models of type 1 diabetes (T1D) and T2D, discussing the differences between clinical and preclinical studies and shedding light on the molecular pathways mediated by miR-155 in different tissues affected by DM. Full article

Neuroendocrine neoplasms (NEN) are infrequent malignant tumors of a neuroendocrine nature that arise in various organs. They occur most frequently in the lungs, intestines, stomach and pancreas. Molecular diagnostics and prognosis of NEN development are highly relevant. The role of clinical biomarkers can be played by microRNAs (miRNAs). This work is devoted to the analysis of data on miRNA expression in NENs. For the first time, a search for specificity or a community of their functional characteristics in different types of NEN was carried out. Their properties as biomarkers were also analyzed. To date, more than 100 miRNAs have been characterized as differentially expressed and significant for the development of NEN tumors. Only about 10% of the studied miRNAs are expressed in several types of NEN; differential expression of the remaining 90% was found only in tumors of specific localizations. A significant number of miRNAs have been identified as potential biomarkers. However, only a few miRNAs have values that characterized their quality as markers. The analysis demonstrates the predominant specific expression of miRNA in each studied type of NEN. This indicates that miRNA’s functional features are predominantly influenced by the tissue in which they are formed. Full article