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The human endometrium is a highly dynamic tissue. Increasing evidence has shown that microRNAs (miRs) play essential roles in human endometrium development. Our previous assay, based on small RNA-sequencing (sRNA-seq) indicated the complexity and dynamics of numerous sequence variants of miRs (isomiRs) that can act together to control genes of functional relevance to the receptive endometrium (RE). Here, we used a greater average depth of sRNA-seq to detect poorly expressed small RNAs. The sequencing data confirmed the up-regulation of miR-449c and uncovered other members of the miR-449 family up-regulated in RE—among them miR-449a, as well as several isoforms of both miR-449a and miR-449c, while the third family member, miR-449b, was not identified. Stem-looped RT-qPCR analysis of miR expression at four-time points of the endometrial cycle verified the increased expression of the miR-449a/c family members in RE, among which the 5′ isoform of miR-449c–miR-449c.1 was the most strongly up-regulated. Moreover, we found in a case study that the expression of miR-449c.1 and its precursor correlated with the histological assessment of the endometrial phase and patient age. We believe this study will promote the clinical investigation and application of the miR-449 family in the diagnosis and prognosis of human reproductive diseases.

Beyond the most crucial roles of RNA molecules as a messenger, ribosomal, and transfer RNAs, the regulatory role of many non-coding RNAs (ncRNAs) in plant biology has been recognized. ncRNAs act as riboregulators by recognizing specific nucleic acid targets through homologous sequence interactions to regulate plant growth, development, and stress responses. Regulatory ncRNAs, ranging from small to long ncRNAs (lncRNAs), exert their control over a vast array of biological processes. Based on the mode of biogenesis and their function, ncRNAs evolved into different forms that include microRNAs (miRNAs), small interfering RNAs (siRNAs), miRNA variants (isomiRs), lncRNAs, circular RNAs (circRNAs), and derived ncRNAs. This article explains the different classes of ncRNAs and their role in plant development and stress responses. Furthermore, the applications of regulatory ncRNAs in crop improvement, targeting agriculturally important traits, have been discussed. Supplementary Information The online version contains supplementary material available at 10.1007/s10142-021-00787-8.

Background The advent of next generation sequencing (NGS) has allowed the discovery of short and long non-coding RNAs (ncRNAs) in an unbiased manner using reverse genetics approaches, enabling the discovery of multiple categories of ncRNAs and characterization of the way their expression is regulated. We previously showed that the identities and abundances of microRNA isoforms (isomiRs) and transfer RNA-derived fragments (tRFs) are tightly regulated, and that they depend on a person’s sex and population origin, as well as on tissue type, tissue state, and disease type. Here, we characterize the regulation and distribution of fragments derived from ribosomal RNAs (rRNAs). rRNAs form a group that includes four (5S, 5.8S, 18S, 28S) rRNAs encoded by the human nuclear genome and two (12S, 16S) by the mitochondrial genome. rRNAs constitute the most abundant RNA type in eukaryotic cells. Results We analyzed rRNA-derived fragments (rRFs) across 434 transcriptomic datasets obtained from lymphoblastoid cell lines (LCLs) derived from healthy participants of the 1000 Genomes Project. The 434 datasets represent five human populations and both sexes. We examined each of the six rRNAs and their respective rRFs, and did so separately for each population and sex. Our analysis shows that all six rRNAs produce rRFs with unique identities, normalized abundances, and lengths. The rRFs arise from the 5′-end (5′-rRFs), the interior (i-rRFs), and the 3′-end (3′-rRFs) or straddle the 5′ or 3′ terminus of the parental rRNA (x-rRFs). Notably, a large number of rRFs are produced in a population-specific or sex-specific manner. Preliminary evidence suggests that rRF production is also tissue-dependent. Of note, we find that rRF production is not affected by the identity of the processing laboratory or the library preparation kit. Conclusions Our findings suggest that rRFs are produced in a regimented manner by currently unknown processes that are influenced by both ubiquitous as well as population-specific and sex-specific factors. The properties of rRFs mirror the previously reported properties of isomiRs and tRFs and have implications for the study of homeostasis and disease.

MicroRNAs are ~20 nt long small noncoding RNAs that are processed from stem-looped precursors and function mainly as posttranscriptional regulators of protein coding genes through binding to 3′-untranslated regions of messenger RNAs to inhibit the translation or cause RNA degradation. It is predicted microRNAs could regulate up to half of all human genes and are proved to play important roles in human diseases including cancer. They bind to target mRNAs based on complementary binding which is dominated by the so-called “seed” region which are the 5′ 2–8 bases of the microRNA. Due to the small size in nature, even a single nucleotide variation in the precursor region especially those located in the seed regions could show big influence. Here, I summarized and reviewed the current knowledge of these single nucleotide alterations in microRNAs in human cancer including (i) common SNPs in the precursor region, (ii) isomiRs, (iii) somatic mutations of microRNAs. Briefly, this is an underexploited field and clearly, warrants further studies to reveal their biological and clinical significances. I believe they will be key to advancing personalized medicine. Keywords: microRNAs, Single nucleotide alterations, SNPs, Isomirs, Somatic mutations

Evidence suggests that miRNA signatures could have clinical applications as biomarkers for diagnostic and prognostic purposes. In standard analyses, miRNAs are considered to exist as well-defined features with a precise start and stop positions. In reality, they exist as a population of similar but slightly different isoforms - isomiRs. The project has developed methods to investigate isomiR populations and studied their variation between healthy and cancer patients. The methods presented here have been implemented in an analysis pipeline that allows standardised and scalable analysis of raw NGS datasets. The value of this approach has been demonstrated by investigating publicly available datasets to identify statistically significant changes in isomiR populations in various cancers. Forskning tilsier at miRNA-signaturer kan ha kliniske applikasjoner i diagnostisk- og prognostisk sammenheng. I konvensjonelle analyser antas miRNA å være veldefinerte enheter med nøyaktige start- og slutt-posisjoner. I virkeligheten eksisterer de som en variert populasjon av lignende, men noe ulike, isomerer. Gjennom dette prosjektet har det blitt utviklet metoder for å undersøke hvordan miRNA-populasjoner varierer mellom friske og syke. Metodene har blitt implementert i eksisterende programvare som muliggjør standardisert og skalerbar analyse av sekvenseringsdata. Verdien av denne tilnærmingen har blitt demonstrert ved å analysere offentlig tilgjengelige datasett for å identifisere statistisk signifikante endringer i miRNA-populasjoner i ulike krefttyper. M-KB

Simple Summary MicroRNAs are essential regulators of gene expression and potential non-invasive biomarker candidates for various human cancers as they can be detected in bodily fluids. Several tools have been developed to analyze small RNA-sequencing data; however, they have limitations and restrictions such as lack of optimal configuration, parameterization, and interoperability with other tools and platforms. miRGalaxy is an open-source, Galaxy-based framework for analyzing NGS data focusing on microRNAs and their sequence variants—isomiRs. Galaxy is a web-based platform for data-intensive biomedical research, allowing user-friendly analysis and accessibility to hundreds of tools. miRGalaxy is designed specifically for identifying and classifying human microRNAs and isomiRs, as well as detecting deregulated microRNAs and isomiRs between two test groups, summarized by output visualization. By examining the differential expression of individual isomiR species across samples, miRGalaxy can help discover novel biomarkers. Abstract Tools for microRNA (miR) sequencing data analyses are broadly used in biomedical research. However, the complexity of computational approaches still remains a challenge for biologists with scarce experience in data analytics and bioinformatics. Here, we present miRGalaxy, a Galaxy-based framework for comprehensive analysis of miRs and their sequence variants—miR isoforms (isomiRs). Though isomiRs are commonly reported in deep-sequencing experiments, their detailed structure complexity and specific differential expression (DE) remain not fully examined by the majority of the available analysis tools. miRGalaxy encompasses biologist-user-friendly tools and workflows dedicated to the analysis of the isomiR-ome and its complex behavior in various biological samples. miRGalaxy is developed as a modular, accessible, redistributable, shareable, and user-friendly framework for scientists working with small RNA (sRNA)-seq data. Due to its modular workflow, advanced users can customize the steps and tools for their needs. In addition, the framework provides an analysis report where the significant output results are summarized in charts and visualizations. miRGalaxy can be accessed via preconfigured Docker image flavor and a Toolshed installation if the user already has a running Galaxy instance. Over the last decade, studies on the expression of miRs and isomiRs in normal and deregulated tissues have led to the discovery of their potential as diagnostic biomarkers. The detection of miRs in biofluids further expanded the exploration of the miR repertoire as a source of liquid biopsy biomarkers. Here we show the miRGalaxy framework application for in-depth analysis of the sRNA-seq data from two different biofluids, milk and plasma, to identify, annotate, and discover specific differentially expressed miRs and isomiRs.

The human terminal uridyl transferases TUT4 and TUT7 (TUT4/7) catalyze the additions of uridines at the 3′ end of RNAs, including the precursors of the tumor suppressor miRNA let-7 upon recruitment by the oncoprotein LIN28A. As a consequence, let-7 family miRNAs are down-regulated. Disruption of this TUT4/7 activity inhibits tumorigenesis. Hence, targeting TUT4/7 could be a potential anticancer therapy. In this study, we investigate TUT4/7-mediated RNA regulation in two cancer cell lines by establishing catalytic knockout models. Upon TUT4/7 mutation, we observe a significant reduction in miRNA uridylation, which results in defects in cancer cell properties such as cell proliferation and migration. With the loss of TUT4/7-mediated miRNA uridylation, the uridylated miRNA variants are replaced by adenylated isomiRs. Changes in miRNA modification profiles are accompanied by deregulation of expression levels in specific cases. Unlike let-7s, most miRNAs do not depend on LIN28A for TUT4/7-mediated regulation. Additionally, we identify TUT4/7-regulated cell-type-specific miRNA clusters and deregulation in their corresponding mRNA targets. Expression levels of miR-200c-3p and miR-141-3p are regulated by TUT4/7 in a cancer cell-type-specific manner. Subsequently, BCL2, which is a well-established target of miR-200c is up-regulated. Therefore, TUT4/7 loss causes deregulation of miRNA–mRNA networks in a cell-type-specific manner. Understanding of the underlying biology of such cell-type-specific deregulation will be an important aspect of targeting TUT4/7 for potential cancer therapies.

Objective MicroRNA-140-3p is the most prevalent form of canonical miR-140 in native chondrocytes. IsomiRs are sequence variants of microRNAs with potentially distinct functionalities. Here we present functional studies of canonical microRNA-140-3p and two of its most prevalent isomiRs, a 5′ isomiR and a 3′ isomiR, in an inflammation-induced model of osteoarthritis (OA). Method Canonical miR-140-3p, the 5′ isomiR and the 3′ isomiR were overexpressed separately in chondrocytes from three donors and subsequently subjected to an inflammatory milieu mediated by interleukin 1 beta and tumor necrosis factor alpha. RNA sequencing was performed on the cells to investigate the altered transcriptomes, RT-qPCR was performed to validate important observations, and western blot analysis was carried out to further study key inflammatory molecules. Results The three microRNAs downregulated many of the same genes. However, the 5′ isomiR showed a much greater target spectrum compared to the other two miRNAs, and downregulated cascades of genes downstream of interferon beta, interferon gamma and interleukin 1 beta as well as genes involved in several other inflammatory and antiviral pathways. In addition the 5′ isomiR downregulated practically all HLA class II and class I genes. Conclusion Introduction of the 5′ isomiR led to downregulation of genes essential for some of the most important inflammation cascades and virtual silencing of genes responsible for antigen presentation. These observations may indicate a very promising therapeutic potential for the 5′ isomiR for OA and several inflammatory conditions, particularly HLA associated immune conditions including many arthritic diseases.

microRNAs (miRNAs) are small, non-protein coding RNAs which post-transcriptionally downregulate gene expression. Circulating miRNAs are promising clinical biomarkers, and circulate either within extracellular vesicles (ECVs) or bound to proteins, both of which protect miRNAs against degradation. Recently, small RNA sequencing has demonstrated the existence of isomiRs, of which vary in length and/or sequence when compared to the parent (canonical) miRNA. microRNA-122 (miR-122) is a liver-specific, circulating biomarker of drug-induced liver injury (DILI), notably that caused by paracetamol (APAP; acetaminophen). It has been previously demonstrated by our group that miR-122 is present in circulating exosomes, a type of ECV. IsomiRs of miR-122 were identified in serum samples obtained from patients with paracetamol-DILI by small RNA sequencing. We observed that certain isomiRs are DILI-specific. Clinically relevant isomiRs of miR-122 underwent reverse transcription quantitative PCR (RT-qPCR) using two commercially available assays for miR-122. We demonstrated that these systems are not specific for canonical miR-122, and additionally that the presence of different isomiRs interferes with the accurate quantification of miR-122. A previously published PCR-based assay termed 3’-Dumbbell-PCR (3’-Db-PCR) was adapted and optimised for the selective detection of a DILI-specific miR-122 isomiR. The 3’-Db-PCR assay was demonstrated to be more selective for the DILI-specific isomiR than standard RT-qPCR, and was applied in the subsequent in vivo study. Using a wild-type mouse model of paracetamol-DILI, we demonstrated that liver miR-122 decreased and, concurrently, circulating miR-122 increased. Over time, miR-122 increased in the renal cortex, renal medulla, and spleen. Fluorescence-activated cell sorting (FACS) of the kidney revealed that levels of miR-122 increased specifically in renal tubular cells. 3’-Db-PCR produced results which correlated to standard RT-qPCR of blood plasma, but these correlations were found to be less significant or completely insignificant in the tissues. Validation of PCR products by Sanger sequencing was inconclusive. Following this study, we utilised a genetically-modified mouse model containing a double-fluorescent Cre reporter allele in order to investigate the release of miR-122 in ECVs from the liver. These mice were administered a liver specific adeno-associated viral (AAV) vector expressing Cre recombinase, with the aim of causing the liver to produce enhanced green fluorescent protein (eGFP) tagged ECVs which could be identified in the blood, kidney, and spleen. Additionally, some of these mice were also administered paracetamol to increase ECV release. After paracetamol administration, miR-122 decreased in the liver and increased in the blood, kidney and spleen, as previously observed. Western blotting and confocal microscopy confirmed the expression of eGFP in the liver, however the expression of eGFP in the blood, kidney and spleen, was not detected. These results therefore confirm that certain isomiRs of miR-122 are DILI-specific, but current ‘gold standard’ RT-qPCR assays are unable to discriminate between canonical miR-122 and its isomiRs. Therefore, miR-122 cannot be quantified by RT-qPCR due to isomiR interference, which may also translate to other miRNAs under clinical investigation. 3’-Db-PCR is more selective than RT-qPCR assays for miR-122, but further investigations are required to validate its PCR products and therefore what the assay actually detects. miR-122 is released from the liver in paracetamol-DILI into the circulation, and is then taken up by both the spleen and renal tubular cells. The confirmation of ECV-dependent or independent miR-122 release and uptake into these organs in liver-derived ECVs must be investigated further by refining our current model, using a different model, and/or by use of different techniques.

Blood circulating microRNAs (c‐miRs) are potential biomarkers to trace aging and longevity trajectories to identify molecular targets for anti‐aging therapies. Based on a cross‐sectional study, a discovery phase was performed on 12 donors divided into four groups: young, old, healthy, and unhealthy centenarians. The identification of healthy and unhealthy phenotype was based on cognitive performance and capabilities to perform daily activities. Small RNA sequencing identified 79 differentially expressed c‐miRs when comparing young, old, healthy centenarians, and unhealthy centenarians. Two miRs, that is, miR‐19a‐3p and miR‐19b‐3p, were found increased at old age but decreased at extreme age, as confirmed by RT‐qPCR in 49 donors of validation phase. The significant decrease of those miR levels in healthy compared to unhealthy centenarians appears to be due to the presence of isomiRs, not detectable with RT‐qPCR, but only with a high‐resolution technique such as deep sequencing. Bioinformatically, three main common targets of miR‐19a/b‐3p were identified, that is, SMAD4, PTEN, and BCL2L11, converging into the FoxO signaling pathway, known to have a significant role in aging mechanisms. For the first time, this study shows the age‐related increase of plasma miR‐19a/b‐3p in old subjects but a decrease in centenarians. This decrease is more pronounced in healthy centenarians and was confirmed by the modified pattern of isomiRs comparing healthy and unhealthy centenarians. Thus, our study paves the way for functional studies using c‐miRs and isomiRs as additional parameter to track the onset of aging and age‐related diseases using new potential biomarkers., Small RNA deep sequencing on plasma‐derived RNA from 12 donors (young and old subjects, healthy, and unhealthy centenarians) was performed: 79 differentially expressed c‐miRs were identified. MiR‐19a/b‐3p increased at old age but decreased at extreme age, as confirmed by RT‐qPCR in 49 subjects. The decrease was more relevant in healthy centenarians, also confirmed by the modified pattern of isomiRs. Three common targets of miR‐19a/b‐3p were identified converging into the FoxO signaling pathway.

MicroRNAs (miRNAs or miRs) are the most characterized class of non-coding RNAs and are engaged in many cellular processes, including cell differentiation, development, and homeostasis. MicroRNA dysregulation was observed in several diseases, cancer included. Epitranscriptomics is a branch of epigenomics that embraces all RNA modifications occurring after DNA transcription and RNA synthesis and involving coding and non-coding RNAs. The development of new high-throughput technologies, especially deep RNA sequencing, has facilitated the discovery of miRNA isoforms (named isomiRs) resulting from RNA modifications mediated by enzymes, such as deaminases and exonucleases, and differing from the canonical ones in length, sequence, or both. In this review, we summarize the distinct classes of isomiRs, their regulation and biogenesis, and the active role of these newly discovered molecules in cancer and other diseases.

The advent of Next Generation Sequencing technologies brought with it the discovery of several microRNA (miRNA) variants of heterogeneous lengths and/or sequences. Initially ascribed to sequencing errors/artifacts, these isoforms, named isomiRs, are now considered non-canonical variants that originate from physiological processes affecting the canonical miRNA biogenesis. To date, accurate IsomiRs abundance, biological activity, and functions are not completely understood; however, the study of isomiR biology is an area of great interest due to their high frequency in the human miRNome, their putative functions in cooperating with the canonical miRNAs, and potential for exhibiting novel functional roles. The discovery of isomiRs highlighted the complexity of the small RNA transcriptional landscape in several diseases, including cancer. In this field, the study of isomiRs could provide further insights into the miRNA biology and its implication in oncogenesis, possibly providing putative new cancer diagnostic, prognostic, and predictive biomarkers as well. In this review, a comprehensive overview of the state of research on isomiRs in different cancer types, including the most common tumors such as breast cancer, colorectal cancer, melanoma, and prostate cancer, as well as in the less frequent tumors, as for example brain tumors and hematological malignancies, will be summarized and discussed.

Embryo implantation depends on endometrial receptivity (ER). To achieve ER, the preparation of the uterine lining requires controlled priming by ovarian hormones and the expression of numerous genes in the endometrial tissue. microRNAs (miRs) have emerged as critical genetic regulators of ER in fertility and of the diseases that are associated with infertility. With the rapid development of next-generation sequencing technologies, it has become clear that miR genes can produce canonical miRs and variants—isomiRs. Here, we describe miR/isomiR expression dynamics across the four time points of natural chorionic gonadotropin (hCG)-administered cycles. Sequencing of the small RNAs (sRNA-seq) revealed that the most significant expression changes during the transition from the pre-receptive to the receptive phase occurred in the isomiR families of miR-125a, miR-125b, miR-10a, miR-10b, miR-449c, miR-92a, miR-92b, and miR-99a. Pairing the analysis of the differentially expressed (DE) miRs/isomiRs and their predicted DE mRNA targets uncovered 280 negatively correlating pairs. In the receptive endometrium, the 5′3′-isomiRs of miR-449c, which were among the most highly up-regulated isomiRs, showed a negative correlation with their target, transcription factor (TF) MYCN, which was down-regulated. Joint analysis of the miR/isomiR and TF expression identified several regulatory interactions. Based on these data, a regulatory TF-miR/isomiR gene-target circuit including let7g-5p and miR-345; the isomiR families of miR-10a, miR-10b, miR-92a, and miR-449c; and MYCN and TWIST1 was proposed to play a key role in the establishment of ER. Our work uncovers the complexity and dynamics of the endometrial isomiRs that can act cooperatively with miRs to control the functionally important genes that are critical to ER. Further studies of miR/isomiR expression patterns that are paired with those of their target mRNAs may provide a more in-depth picture of the endometrial pathologies that are associated with implantation failure.

Embryonic stem cells (ESCs) have the extraordinary properties to indefinitely proliferate and self-renew in culture to produce different cell progeny through differentiation. This latter process recapitulates embryonic development and requires rounds of the epithelial–mesenchymal transition (EMT). EMT is characterized by the loss of the epithelial features and the acquisition of the typical phenotype of the mesenchymal cells. In pathological conditions, EMT can confer stemness or stem-like phenotypes, playing a role in the tumorigenic process. Cancer stem cells (CSCs) represent a subpopulation, found in the tumor tissues, with stem-like properties such as uncontrolled proliferation, self-renewal, and ability to differentiate into different cell types. ESCs and CSCs share numerous features (pluripotency, self-renewal, expression of stemness genes, and acquisition of epithelial–mesenchymal features), and most of them are under the control of microRNAs (miRNAs). These small molecules have relevant roles during both embryogenesis and cancer development. The aim of this review was to recapitulate molecular mechanisms shared by ESCs and CSCs, with a special focus on the recently identified classes of microRNAs (noncanonical miRNAs, mirtrons, isomiRs, and competitive endogenous miRNAs) and their complex functions during embryogenesis and cancer development.

Numerous studies on microRNAs (miRNA) in cancer and other diseases have been accompanied by diverse computational approaches and experimental methods to predict and validate miRNA biological and clinical significance as easily accessible disease biomarkers. In recent years, the application of the next-generation deep sequencing for the analysis and discovery of novel RNA biomarkers has clearly shown an expanding repertoire of diverse sequence variants of mature miRNAs, or isomiRs, resulting from alternative post-transcriptional processing events, and affected by (patho)physiological changes, population origin, individual’s gender, and age. Here, we provide an in-depth overview of currently available bioinformatics approaches for the detection and visualization of both mature miRNA and cognate isomiR sequences. An attempt has been made to present in a systematic way the advantages and downsides of in silico approaches in terms of their sensitivity and accuracy performance, as well as used methods, workflows, and processing steps, and end output dataset overlapping issues. The focus is given to the challenges and pitfalls of isomiR expression analysis. Specifically, we address the availability of tools enabling research without extensive bioinformatics background to explore this fascinating corner of the small RNAome universe that may facilitate the discovery of new and more reliable disease biomarkers.

Cardiovascular diseases (CVDs) remain the leading cause of death worldwide, and thus, novel therapies are required. CVDs generally result in local shortages in the blood supply, known as ischemia. Neovascularization is the body's innate response mechanism that stimulates the restoration of blood flow to ischemic tissues. During the last decade, microRNAs have emerged as critical regulators of both CVD and neovascularization. Recent studies demonstrated that microRNAs are altered in many ways; however, whether these microRNA modifications could be physiologically relevant remained unclear. We examined whether specific microRNAs with a known cardiovascular function are subject to particular microRNA-alterations and if they could be relevant in cardiovascular disease. Our experiments demonstrated that the level of specific microRNA alterations, including isomiR formation, adenosine-to-inosine editing, and N6-adenosine methylation, changed in response to cardiovascular pathology. Many of these alterations changed the microRNAs function, which had a direct effect on processes like neovascularization. For example, microRNA adenosine-to-inosine editing increased after ischemia in both mice and humans and promoted neovascularization. These findings suggest that microRNA modifications can potentially be harnessed as a biomarker for cardiovascular disease, or even a novel therapeutic target.

MicroRNA-offset RNAs (moRNAs) are microRNA-like small RNAs generated by microRNA precursors. To date, little is known about moRNAs and bioinformatics tools to inspect their expression are still missing. We developed miR&amp, moRe2, the first bioinformatics method to consistently characterize microRNAs, moRNAs, and their isoforms from small RNA sequencing data. To illustrate miR&amp, moRe2 discovery power, we applied it to several published datasets. MoRNAs identified by miR&amp, moRe2 were in agreement with previous research findings. Moreover, we observed that moRNAs and new microRNAs predicted by miR&amp, moRe2 were downregulated upon the silencing of the microRNA-biogenesis pathway. Further, in a sizeable dataset of human blood cell populations, tens of novel miRNAs and moRNAs were discovered, some of them with significantly varied expression levels among the cell types. Results demonstrate that miR&amp, moRe2 is a valid tool for a comprehensive study of small RNAs generated from microRNA precursors and could help to investigate their biogenesis and function.

Therapeutic neovascularization can facilitate blood flow recovery in patients with ischemic cardiovascular disease, the leading cause of death worldwide. Neovascularization encompasses both angiogenesis, the sprouting of new capillaries from existing vessels, and arteriogenesis, the maturation of preexisting collateral arterioles into fully functional arteries. Both angiogenesis and arteriogenesis are highly multifactorial processes that require a multifactorial regulator to be stimulated simultaneously. MicroRNAs can regulate both angiogenesis and arteriogenesis due to their ability to modulate expression of many genes simultaneously. Recent studies have revealed that many microRNAs have variants with altered terminal sequences, known as isomiRs. Additionally, endogenous microRNAs have been identified that carry biochemically modified nucleotides, revealing a dynamic microRNA epitranscriptome. Both types of microRNA alterations were shown to be dynamically regulated in response to ischemia and are able to influence neovascularization by affecting the microRNA’s biogenesis, or even its silencing activity. Therefore, these novel regulatory layers influence microRNA functioning and could provide new opportunities to stimulate neovascularization. In this review we will highlight the formation and function of isomiRs and various forms of microRNA modifications, and discuss recent findings that demonstrate that both isomiRs and microRNA modifications directly affect neovascularization and vascular remodeling.

Neuroblastoma (NB) originates from neural crest-derived precursors and represents the most common childhood extracranial solid tumour. MicroRNAs (miRNAs), a class of small non-coding RNAs that participate in a wide variety of biological processes by regulating gene expression, appear to play an essential role within the NB context. High-throughput next generation sequencing (NGS) was applied to study the miRNA transcriptome in a cohort of NB tumours with and without MYCN-amplification (MNA and MNnA, respectively) and in dorsal root ganglia (DRG), as a control. Out of the 128 miRNAs differentially expressed in the NB vs. DRG comparison, 47 were expressed at higher levels, while 81 were expressed at lower levels in the NB tumours. We also found that 23 miRNAs were differentially expressed in NB with or without MYCN-amplification, with 17 miRNAs being upregulated and 6 being downregulated in the MNA subtypes. Functional annotation analysis of the target genes of these differentially expressed miRNAs demonstrated that many mRNAs were involved in cancer-related pathways, such as DNA-repair and apoptosis as well as FGFR and EGFR signalling. In particular, we found that miR-628-3p negatively affects MYCN gene expression. Furthermore, we identified a novel miRNA candidate with variable expression in MNA vs. MNnA tumours, whose putative target genes are implicated in the mTOR pathway. The present study provides further insight into the molecular mechanisms that correlate miRNA dysregulation to NB development and progression.

Next-generation sequencing is an increasingly popular and efficient approach to characterize the full set of microRNAs (miRNAs) present in human biosamples. MiRNAs’ detection and quantification still remain a challenge as they can undergo different post transcriptional modifications and might harbor genetic variations (polymiRs) that may impact on the alignment step. We present a novel algorithm, OPTIMIR, that incorporates biological knowledge on miRNA editing and genome-wide genotype data available in the processed samples to improve alignment accuracy.OPTIMIR was applied to 391 human plasma samples that had been typed with genome-wide genotyping arrays. OPTIMIR was able to detect genotyping errors, suggested the existence of novel miRNAs and highlighted the allelic imbalance expression of polymiRs in heterozygous carriers.OPTIMIR is written in python, and freely available on the GENMED website (http://www.genmed.fr/index.php/fr/) and on Github (github.com/FlorianThibord/OptimiR).

Background MicroRNA isoforms are the variants of a canonical miRNA-sequence with alteration at the 3` or 5` termini due to addition, deletion and/or substitution of nucleotide. The present study aims at identifying the isomiRs of the expressed miRNAs in peripheral blood mononuclear cells (PBMCs) of diseased vis-à-vis healthy buffaloes, vis-à-vis exploring the molecular pathways of the predicted target genes of the isomiRs/miRNAs. Four groups of experimental animals (adult, male or female) were included in the study: a) Brucellosis (Murrah breed), b) Paratuberculosis or Johne’s disease (Murrah breed); c) Brucellosis (Nili-Ravi breed) and d) control group of healthy buffaloes of the Murrah breed. The small RNA (sRNA) samples, extracted from PBMCs of each of the four groups, pooled into four samples and then were subjected to next-generation sequencing (Ion-Torrent PGM™ platform). Results The NGS data were analyzed using the miRanalyzer tool and R-programming to identify the differentially expressed (up and down-regulated (fold change ratio > 2)) miRNAs (exhibiting isomiRs and uniquely expressed miRNAs having isomiRs) of each of the three disease-groups as compared to the healthy-Murrah group. The target genes of these selected differentially and uniquely expressed miRNA & isomiRs were predicted using three different online tools (TargetScan, PicTar, and miRDB). These target genes were analyzed to determine their role in systems biology. We identified 153, 125 and 139 isomiR-exhibiting miRNAs that were common in those three experimental groups (healthy vs. Brucellosis-Murrah, Johne’s disease-Murrah, and Brucellosis-Nili-Ravi, respectively). Gene ontology and pathway analyses of the target genes (Panther classification system) yielded several biological hits. Functional classification of the target genes indicated that these target genes were involved in different systems biology related functions like molecular binding, enzyme modulation, signal modulation, etc. The specificity of function was varying in the three experimental groups. Conclusion We are presenting the first report on the identification of isomiRs and functional classification of the target genes in water buffaloes. The results revealed that isomiRs may be involved in the biological processes and can be used in disease diagnosis.

microRNAs (miRNAs) play an important role as key regulators controlling the post-transcriptional events in plants across development, abiotic and biotic stress, tissue polarity and also in defining the evolutionary basis of the origin of the post-transcriptional machinery. Identifying patterns of regulated and co-regulated small RNAs, in particular miRNAs and their sequence variants with the availability of next generation sequencing approaches has widely demonstrated the role of miRNAs and their temporal regulation in maintaining plant development and their response to stress conditions. Although the role of canonical miRNAs has been widely explored and functional diversity is revealed, those works for isomiRs are still limited and urgent to be carried out across plants. This relative lack of information with respect to isomiRs might be attributed to the non-availability of large-scale detection of isomiRs across wide plant species. In the present research, we addressed this by developing Plant isomiR Atlas, which provides large-scale detection of isomiRs across 23 plant species utilizing 677 smallRNAs datasets and reveals a total of 98,374 templated and non-templated isomiRs from 6,167 precursors. Plant isomiR Atlas provides several visualization features such as species specific isomiRs, isomiRs and canonical miRNAs overlap, terminal modification classifications, target identification using psRNATarget and TargetFinder and also canonical miRNAs:target interactions. Plant isomiR Atlas will play a key role in understanding the regulatory nature of miRNAome and will accelerate to understand the functional role of isomiRs. Plant isomiR Atlas is available at www.mcr.org.in/isomir. One Sentence Summary Plant isomiR Atlas will play a key role in understanding the regulatory nature of miRNAome and will accelerate the understanding and diversity of functional targets of plants isomiRs.

50 p.-6 fig.-1 tab., The increase in male idiopathic infertility has been associated with daily exposure to endocrine disruptors chemicals (EDCs). Nevertheless, the mechanisms of action in relation to dysregulating proteins and regulatory microRNAs are unknown.We combined proteomic and miRNome analyses of mouse testis chronically exposed to low doses of a define mixture of EDCs [phthalates: bis (2-ethylhexyl), dibutyl and benzyl-butyl; 4-nonylphenol and 4-tert-octylphenol], administered in the drinking water from conception until adulthood (post-natal day 60/75) and compared them with no-exposed control mice.We analysed fertility parameters and global changes in the patterns of mice testis proteome by 2D-electrophoresis/mass spectrometry, along with bioinformatic analyses of dysregulated microRNAs, and their association with published data in human infertile patients.We detected a decrease in the potential fertility of exposed mice associated with changes in the expression of 18 proteins (10 up-regulated, 8 down-regulated). Functional analysis showed that 89% were involved in cell death. Furthermore, we found a group of 23 microRNAs/isomiRs (down-regulated) correlated with six of the up-regulated target proteins (DIABLO, PGAM1, RTRAF, EIF4E, IVD and CNDP2). Regarding this, PGAM1 up-regulation was validated by Western blot and mainly detected in Sertoli cells. Some of these microRNA/protein dysregulations were reported in human testis with spermatogenic failure.Overall, a chronic exposure to EDCs mixture in human males could potentially lead to spermatogenic failure through changes in microRNA expression, which could post-transcriptionally dysregulate mRNA targets that encode proteins participating in cell death in testicular cells. Finally, these microRNA/protein dysregulations need to be validated with other EDCs mixtures and concentrations., This work was supported by grants from FONDECYT 1150352 to R.D.M., FONDECYT 3160273 to P.U-M and CONICYT 21120505 to J.B., Chile, and MINECO BFU2013-42164-R and BFU2017-87095-R to J. d. M., Spain

Inflammatory bowel disease (IBD) is a chronic relapsing disorder of the alimentary tract, encompassing Crohn's disease (CD) and ulcerative colitis (UC) as its two major subtypes. The diagnosis of inflammatory bowel disease remains a clinical challenge and involves the assessment of numerous parameters. A multitude of biomarkers has been proposed to complement the diagnostic process. However, none of them can be recommended for routine clinical practice. MicroRNAs (miRNAs) represent a class of short, non-coding RNAs that act as post-transcriptional regulators of gene expression. In the scope of this work we evaluate whether systematic miRNA or miRNA variant expression profiling, in conjunction with state-of-the-art machine learning techniques, is suitable as a non-invasive tool for diagnostics of IBD. In a first study we employed microarray technology to determine expression levels of 863 miRNAs for whole blood samples drawn from a cohort comprising 314 individuals, to establish miRNA signature being informative for the highly accurate distinction of CD and UC among each other as well as from healthy and inflammatory controls. In another study we extended this approach by additionally incorporating expression profiles of miRNA variants. We employed next generation sequencing technology to examine isomiR expression profiles drawn from a cohort of 515 individuals, comprising IBD cases as well as healthy and symptomatic controls. Incorporating distinctive isomiR signatures, we generated classifiers performing with median balanced accuracies of 78.57%/78.83% (untreated/treated CD vs. UC) and 100.00%/98.28% (untreated/treated CD or UC vs. HC), respectively. Hence, we provide sampling-based evidence for our models' superiority over established biomarkers. Bei entzündlichen Darmerkrankungen (IBD) handelt es sich um eine Gruppe chronischer, rezidivierender Krankheiten des Verdauungstraktes, die die Hauptformen Morbus Crohn (CD) und Colitis ulcerosa (UC) umfasst. Nach wie vor stellt die Diagnostik von IBD eine klinische Herausforderung dar und schließt die Bewertung zahlreicher Parameter ein. Als Ergänzung des diagnostischen Prozesses wurden zahlreiche Biomarker vorgeschlagen. Für die routinemäßige klinische Praxis jedoch kann keiner dieser Biomarker empfohlen werden. Bei microRNAs (miRNAs) handelt es sich um eine Klasse kurzer, nicht-kodierender RNAs, die als post-transkriptionelle Regulatoren der Genexpression fungieren. Unter Verwendung globaler Expressionprofile sowie Methoden des maschinellen Lernens, wird im Rahmen der vorliegenden Arbeit die Eignung dieser Moleküle als nicht-invasives Werkzeug für die Diagnose von IBD evaluiert. In einer ersten Studie verwendeten wir Microarray-Technologie zur Quantifizierung blutbasierter Expressionslevel von 863 miRNAs, generiert auf Grundlage einer 314 Individuen umfassenden Kohorte. Die resultierenden Expressionsprofile bildeten die Grundlage für die Etablierung von miRNA-Signaturen, hilfreich für die Unterscheidung zwischen CD and UC, sowie deren Abgrenzung von gesunden/inflammatorischen Kontrollen. In einer weiteren Studie wurde dieser Ansatz um die Berücksichtung von miRNA-Varianten erweitert. Wir verwendeten Sequenzier-Technologie zur Bestimmung von isomiR-Expressionsprofilen, generiert auf Grundlage einer Kohorte von 515 Individuen, einschließlich IBD-Patienten sowie gesunder/symptomatischer Kontrollen. Unter Verwendung von isomiR-Signaturen generierten wir Modelle, deren Klassifikationgüte wir mit medianen balanced accuracies von 78.57%/78.83% (CD vs. UC, unbehandelt/behandelt) bzw. 100.00%/98.28% (CD oder UC vs. HC, unbehandelt/behandelt) bewerten konnten. Damit liefern wir Belege für eine Überlegenheit unserer Modelle bezüglich etablierter Biomarker.

Non-coding RNAs (ncRNA) are regulators of cell functions and circulating ncRNAs from the majority of RNA classes, such as miRNA, tRNA, piRNAs, lncRNA, snoRNA, snRNA and miscRNAs, are potential non-invasive biomarkers. Understanding how non-disease traits influence ncRNA expression is essential for assessing their biomarker potential.We studied associations of common traits (sex, age, smoking, body mass, physical activity, and technical factors such as sample storage and processing) with serum ncRNAs. We used RNAseq data from 526 donors from the Janus Serum Bank and traits from health examination surveys. We identified associations between all RNA classes and traits. Ageing showed the strongest association with ncRNA expression, both in terms of statistical significance and number of RNAs, regardless of RNA class. Serum processing modifications and storage times significantly altered expression levels of a number of ncRNAs. Interestingly, smoking cessation generally restored RNA expression to non-smoking levels, although for some isomiRs, mRNA fragments and tRNAs smoking-related expression levels persisted.Our results show that common traits influence circulating ncRNA expression. Therefore it is clear that ncRNA biomarker analyses should be adjusted for age and sex. In addition, for specific ncRNAs identified in our study, analyses should also be adjusted for body mass, smoking, physical activity and serum processing and storage.

Los microARNs son reguladores post-transcripcionales de la expresión génica que participanen la regulación de distintos procesos celulares como por ejemplo en el desarrollo embrionario. En el presente trabajo estudiamos el perfil de expresión de los microARNs durante la diferenciacióna mesodermo temprano y al linaje cardíaco mediante secuenciación masiva de los ARNpequeños. A partir de estos resultados determinamos que hay aproximadamente 700 micro- ARNs que se expresan diferencialmente en las células pluripotentes humanas, un progenitortemprano de mesodermo y cardiomiocitos. A su vez, analizamos la presencia de variantes demicroARNs llamados isomiRs y determinamos la abundancia de las distintas isoformas en lasdistintas poblaciones celulares. A partir del análisis in silico de los genes target determinamosque estas isoformas amplían el repertorio de genes regulados por el microARN canónico, locual sugiere un rol funcional de los isomiRs durante el proceso de diferenciación celular. Porotro lado, analizamos el miRNoma de cada una de las tres poblaciones celulares en base a lasfamilias y clusters de microARNs e identificamos que los microARNs de dichos grupos compartenun perfil de expresión y colaboran entre sí en la regulación de procesos relacionados conel desarrollo embrionario. Además, establecimos el miRNoma de los cardiomiocitos mediantela comparación de los microARNs que se expresan en otros tipos celulares. Finalmente, estudiamosel cluster C19MC, up-regulado en las células pluripotentes, y analizamos de manerain silico y experimental los genes target de dicho cluster. De esta manera, determinamos queel C19MC participa en la regulación del ciclo celular y de distintos procesos de diferenciaciónen el desarrollo embrionario. Por último, estudiamos el miR-205 en la diferenciación a mesodermoe identificamos que su sobreexpresión aumenta al doble el porcentaje del progenitortemprano de mesodermo. Los microARNs ejercen su función formando complejas redes deregulación, lo cual requiere de un análisis más global para poder entender mejor el rol queestos pequeños ARN tienen en las células. Nuestros resultados expanden el conocimiento delos microARNs en relación con el desarrollo cardíaco. MicroRNAs are small non-coding RNAs involved in post-transcriptional regulation of geneexpression related to many cellular functions, including human embryonic development. Inthis work we study the microRNAs involved in the cardiac differentiation by small RNA highthroughputsequencing of three specific cell populations: Pluripotent stem cells, an early mesodermprogenitor and cardiomyocytes.We identified 700 microRNAs involved in the differentiationprocess where some of them are clustered according to their expression level. We alsostudied the expression level of isomiRs and determined their abundance and predicted targetsby an in silico analysis. These results suggest that these isoforms might have an important rolethat should be considered when studying the microARNs involved the differentiation process. Moreover, we identified the microRNAs families and clusters involved in the establishment ofcardiac lineage and define the mirRNome based on these groups. Furthermore, we were ableto determine a more accurate miRNome associated with cardiomyocytes by comparing theexpressed microRNAs with other mature cells. On the other hand, we studied the microRNAcluster C19MC which is highly expressed in pluripotent stem cells. Based on the predicted targets,as well as experimental targets and functional analysis, we determined that this clusteris involved in the pluripotent stem cells in the regulation of the cell cycle and in the regulationof different differation processes during early development. Finally, we studied the role of themiR-205 during differentiation towards mesoderm and determined that its overexpression increasetwice the percentage of the early mesoderm progenitor population. MicroRNAs exert their effect in a complex and interconnected way, making necessary a globalanalysis to better understand their role. Our data expands the knowledge of microRNAs andtheir implications in cardiomyogenesis. Fil: Garate, Ximena. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.

Background The expression of microRNAs (miRNAs) is essential for the proper development of the mammalian embryo. A maternal exposure to endocrine disrupting chemicals during preimplantation bears the potential for transgenerational inheritance of disease through the epigenetic perturbation of the developing embryo. A comprehensive assembly of embryo-specific miRNAs and respective isoforms (isomiR) is lacking to date. We aimed at revealing the sex-specific miRNA expression profile of single porcine blastocysts developing in gilts orally exposed to exogenous estradiol-17 β (E2). Therefore we analyzed the miRNA profile specifically focusing on isomiRs and potentially embryo-specific miRNAs. Results Deep sequencing of small RNA (small RNA-seq) result in the detection of miRNA sequences mapping to known and predicted porcine miRNAs as well as novel miRNAs highly conserved in human and cattle. A set of highly abundant miRNAs and a large number of rarely expressed miRNAs were identified by using a small RNA analysis pipeline, which was integrated into a novel Galaxy workflow specifically benefits incompletely annotated species. In particular, orthologue species information increased the total number of annotated miRNAs, while mapping to other non-coding RNAs avoided falsely annotated miRNAs. Neither the low nor the high dose of E2 treatment (10 and 1000 µ E2/kg body weight daily, respectively) affected the miRNA profile in blastocysts despite the distinct differential mRNA expression and DNA methylation found in previous studies. The high number of generated sequence reads enabled a comprehensive analysis of the isomiR repertoire showing various templated and non-templated modifications. Furthermore, potentially blastocyst-specific miRNAs were identified. Conclusions In pre-implantation embryos, numerous distinct isomiRs were discovered indicating a high complexity of miRNA expression. Neither the sex of the embryo nor a maternal E2 exposure affected the miRNA expression profile of developing porcine blastocysts. The adaptation to the continuous duration of the E2 treatment might explain the lack of an effect., BMC Genomics, 19

// Lyndsay V. Rhodes 4, * , Elizabeth C. Martin 1, * , H. Chris Segar 1 , David F. B. Miller 3 , Aaron Buechlein 5 , Douglas B. Rusch 5 , Kenneth P. Nephew 3 , Matthew E. Burow 1, 2 , Bridgette M. Collins-Burow 1 1 Department of Medicine, Section of Hematology and Medical Oncology, Tulane University, New Orleans, LA, USA 2 Department of Pharmacology, Tulane University, New Orleans, LA, USA 3 Medical Sciences and Department of Cellular and Integrative Physiology, Indiana University School of Medicine, Bloomington, IN, USA 4 Department of Biological Sciences, Florida Gulf Coast University, Fort Myers, FL, USA 5 Indiana University Center for Genomics and Bioinformatics, Bloomington, IN, USA * These authors have contributed equally to this work Correspondence to: Bridgette M. Collins-Burow, e-mail: bcollin1@tulane.edu Keywords: triple negative breast cancer, RHOGDI, miRNA biogenesis, star strand, isomiRs Received: November 20, 2014 Accepted: January 23, 2015 Published: March 21, 2015 ABSTRACT Epithelial to mesenchymal transition (EMT) involves loss of an epithelial phenotype and activation of a mesenchymal one. Enhanced expression of genes associated with a mesenchymal transition includes ZEB1/2, TWIST, and FOXC1. miRNAs are known regulators of gene expression and altered miRNA expression is known to enhance EMT in breast cancer. Here we demonstrate that the tumor suppressive miRNA family, miR-200, is not expressed in triple negative breast cancer (TNBC) cell lines and that miR-200b-3p over-expression represses EMT, which is evident through decreased migration and increased CDH1 expression. Despite the loss of migratory capacity following re-expression of miR-200b-3p, no subsequent loss of the conventional miR-200 family targets and EMT markers ZEB1/2 was observed. Next generation RNA-sequencing analysis showed that enhanced expression of pri-miR-200b lead to ectopic expression of both miR-200b-3p and miR-200b-5p with multiple isomiRs expressed for each of these miRNAs. Furthermore, miR-200b-5p was expressed in the receptor positive, epithelial breast cancer cell lines but not in the TNBC (mesenchymal) cell lines. In addition, a compensatory mechanism for miR-200b-3p/200b-5p targeting, where both miRNAs target the RHOGDI pathway leading to non-canonical repression of EMT, was demonstrated. Collectively, these data are the first to demonstrate dual targeting by miR-200b-3p and miR-200b-5p and a previously undescribed role for microRNA processing and strand expression in EMT and TNBC, the most aggressive breast cancer subtype.

Increase in soil salt causes osmotic and ionic stress to plants, which inhibits their growth and productivity. Rice production is also hampered by salinity and the effect of salt is most severe at the seedling and reproductive stages. Salainity tolerance is a quantitative property controlled by multiple genes coding for signaling molecules, ion transporters, metabolic enzymes and transcription regulators. MicroRNAs are key modulators of gene-expression that act at the post-transcriptional level by translation repression or transcript cleavage. They also play an important role in regulating plant’s response to salt-stress. In this work we adopted the approach of comparative and integrated data-mining to understand the miRNA-mediated regulation of salt-stress in rice. We profiled and compared the miRNA regulations using natural varieties and transgenic lines with contrasting behaviors in response to salt-stress. The information obtained from sRNAseq, RNAseq and degradome datasets was integrated to identify the salt-deregulated miRNAs, their targets and the associated metabolic pathways. The analysis revealed the modulation of many biological pathways, which are involved in salt-tolerance and play an important role in plant phenotype and physiology. The end modifications of the miRNAs were also studied in our analysis and isomiRs having a dynamic role in salt-tolerance mechanism were identified.

Activation of T lymphocytes requires a tight regulation of microRNA (miRNA) expression. Terminal uridyltransferases (TUTases) catalyze 3' nontemplated nucleotide addition (3'NTA) to miRNAs, which may influence miRNA stability and function. Here, we investigated 3'NTA to mature miRNA in CD4 T lymphocytes by deep sequencing. Upon T-cell activation, miRNA sequences bearing terminal uridines are specifically decreased, concomitantly with down-regulation of TUT4 and TUT7 enzymes. Analyzing TUT4-deficient T lymphocytes, we proved that this terminal uridyltransferase is essential for the maintenance of miRNA uridylation in the steady state of T lymphocytes. Analysis of synthetic uridylated miRNAs shows that 3' addition of uridine promotes degradation of these uridylated miRNAs after T-cell activation. Our data underline post-transcriptional uridylation as a mechanism to fine-tune miRNA levels during T-cell activation.

// Phillipe Loher 1 , Eric R. Londin 1 , and Isidore Rigoutsos 1 1 Computational Medicine Center, Sidney Kimmel Medical College at Thomas Jefferson University, Philadelphia, PA Correspondence: Isidore Rigoutsos, email: // Keywords : microRNAs, miRNAs, isomiRs, 1000 Genomes Received : July 16, 2014 Accepted : August 22, 2014 Published : August 27, 2014 Abstract For many years it was believed that each mature microRNA (miRNA) existed as a single entity with fixed endpoints and a ‘static’ and unchangeable primary sequence. However, recent evidence suggests that mature miRNAs are more ‘dynamic’ and that each miRNA precursor arm gives rise to multiple isoforms, the isomiRs. Here we report on our identification of numerous and abundant isomiRs in the lymphoblastoid cell lines (LCLs) of 452 men and women from five different population groups. Unexpectedly, we find that these isomiRs exhibit an expression profile that is population-dependent and gender-dependent. This is important as it indicates that the LCLs of each gender/population combination have their own unique collection of mature miRNA transcripts. Moreover, each identified isomiR has its own characteristic abundance that remains consistent across biological replicates indicating that these are not degradation products. The primary sequences of identified isomiRs differ from the known miRBase miRNA either at their 5´-endpoint (leads to a different ‘seed’ sequence and suggests a different targetome), their 3´-endpoint, or both simultaneously. Our analysis of Argonaute PAR-CLIP data from LCLs supports the association of many of these newly identified isomiRs with the Argonaute silencing complex and thus their functional roles through participation in the RNA interference pathway.

In the recent years, glyoxalase pathway has been an active area of research in both human and plants. This pathway is reported to confer stress tolerance in plants, by modulating the glutathione homeostasis to achieve detoxification of a potent cytotoxic and mutagenic compound, methylglyoxal. The microRNAs (miRNAs) are also reported to play significant role in stress tolerance for plants. However, the cross-talk of miRNAs with the metabolism regulated by glyoxalase in the salinity-tolerance is unexplored. We therefore investigated whether expression profiles of miRNAs are altered in response to glyoxalase overexpression, and if any of these are also responsible for modulating the stress responses of plants. In this study, the Next Generation Sequencing (NGS) was employed to profile miRNA expression levels from glyoxalase overexpressing transgenic lines. The associated targets of differentially expressed miRNAs were predicted and their functional annotation was carried out using Gene Ontology (GO) and KEGG Orthology (KO), which showed their involvement in several crucial biological pathways. The analysis of NGS datasets also identified other isoforms or isomiRs of selected miRNAs, which may have an active role in developing tolerance against salt stress. Different aspects of miRNA modifications were also studied in glyoxalase overexpressing lines.

Background MicroRNAs (miRNAs), which comprise a large family of endogenous small non-coding RNA molecules, play important roles in the regulation of gene expression in various biological processes. The Chinese rare minnow (Gobiocypris rarus) is a Chinese native fish species and is used extensively as an experimental fish in China; however, relevant biological data, especially miRNA transcriptome data, have not been well documented. To discover conserved and potential novel miRNAs in Chinese rare minnows, a pool of equal amounts of RNA obtained from 6 different adult rare minnow tissues (brain, eye, gill, liver, muscle and heart) was sequenced using illumina deep sequencing technology. Results In the present study, 26,930,553 raw reads, representing 2,118,439 unique high-quality reads, were obtained from the pooled small RNA library. Using bioinformatics analysis, 352 conserved and 112 novel Chinese rare minnow miRNAs were first discovered and characterized in this study. Moreover, we found extensive sequence variations (isomiRs) in rare minnow miRNAs, including internal miRNA isomiRs and terminal isomiRs at both the 5′ and 3′ ends and nucleotide variants. Six conserved and 4 novel miRNAs were selected and validated in 6 different adult rare minnow tissues using quantitative real-time PCR (qPCR). The results showed that miR-30a, miR-30b, and Novel-37 are ubiquitously expressed in a variety of tissues. miR-16a, miR-9, miR-125b, miR-34a, and Novel-69 were predominantly expressed in the brain. Novel-115 and Novel-7 were highly expressed in gills, but were relatively weakly expressed in other tissues. These results provided the expression patterns of miRNA genes in Chinese rare minnow. Finally, based on bioinformatics predictions, we mainly found that Novel-94 and Novel-1b-5p were simultaneously targeted to the 3′UTR of Dmrt1, which controls sex determination and/or sexual differentiation in a variety of metazoans at different sites. Novel-29b targeted the 3′UTR of Foxl2, which is involved in the maintenance of ovarian function and the transcriptional regulation of gonadal differentiation-related genes. Novel-62 and Novel-53 targeted the 3′UTR of ERbeta1 and ERbeta2 (which regulate the transcription of target genes), respectively. Conclusions Rare minnow is a widely used model for assessing the risk of environmental pollution in China. Identifying and characterizing rare minnow miRNA genes is necessary to discover the biological function of miRNAs and to screen for new molecule biomarkers to assess the risk of environmental pollution in the future. Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-2606-5) contains supplementary material, which is available to authorized users.

The authors thank J. Bossenga for collecting urine and P.P. Eijk for their technical assistance in smallRNA sequencing library preparations., In many cancer types, the expression and function of ~22 nucleotide‑long microRNAs (miRNA) is deregulated. Mature miRNAs can be stably detected in extracellular vesicles (EVs) in biofluids, therefore they are considered to have great potential as biomarkers. In the present study, we investigated whether miRNAs have a distinct expression pattern in urine‑EVs of prostate cancer (PCa) patients compared to control males. By next generation sequencing, we determined the miRNA expression in a discovery cohort of 4 control men and 9 PCa patients. miRNAs were validated by using a stemloop RT‑PCR in an independent cohort of 74 patients (26 control and 48 PCa‑patients). Whereas standard mapping protocols identified > 10 PCa associated miRNAs in urinary EVs, miR‑21, miR‑375 and miR‑204 failed to robustly discriminate for disease in a validation study with RT‑PCR‑detection of mature miRNA sequences. In contrast, we observed that miRNA isoforms (isomiRs) with 3′ end modifications were highly discriminatory between samples from control men and PCa patients. Highly differentially expressed isomiRs of miR‑21, miR‑204 and miR‑375 were subsequently validated in an independent group of 74 patients. Receiver‑operating characteristic analysis was performed to evaluate the diagnostic performance of three isomiRs, resulting in a 72.9% sensitivity with a high (88%) specificity and an area under the curve (AUC) of 0.866. In comparison, prostate specific antigen had an AUC of 0.707 and measuring the mature form of these miRNAs yielded a lower 70.8% sensitivity and 72% specificity (AUC 0.766). We propose that isomiRs may carry discriminatory information which is useful to generate stronger biomarkers., Stichting Urologie 1973, VUmc-CCA, Worldwide Cancer Research (AICR) 15-1005, KWF-VU-5510, Worldwide Cancer Research 15-1005

Two decades after the discovery of the first animal microRNA (miRNA), the number of miRNAs in animal genomes remains a vexing question. Here, we report findings from analyzing 1,323 short RNA sequencing samples (RNA-seq) from 13 different human tissue types. Using stringent thresholding criteria, we identified 3,707 statistically significant novel mature miRNAs at a false discovery rate of ≤0.05 arising from 3,494 novel precursors; 91.5% of these novel miRNAs were identified independently in 10 or more of the processed samples. Analysis of these novel miRNAs revealed tissue-specific dependencies and a commensurate low Jaccard similarity index in intertissue comparisons. Of these novel miRNAs, 1,657 (45%) were identified in 43 datasets that were generated by cross-linking followed by Argonaute immunoprecipitation and sequencing (Ago CLIP-seq) and represented 3 of the 13 tissues, indicating that these miRNAs are active in the RNA interference pathway. Moreover, experimental investigation through stem-loop PCR of a random collection of newly discovered miRNAs in 12 cell lines representing 5 tissues confirmed their presence and tissue dependence. Among the newly identified miRNAs are many novel miRNA clusters, new members of known miRNA clusters, previously unreported products from uncharacterized arms of miRNA precursors, and previously unrecognized paralogues of functionally important miRNA families (e.g., miR-15/107). Examination of the sequence conservation across vertebrate and invertebrate organisms showed 56.7% of the newly discovered miRNAs to be human-specific whereas the majority (94.4%) are primate lineage-specific. Our findings suggest that the repertoire of human miRNAs is far more extensive than currently represented by public repositories and that there is a significant number of lineage- and/or tissue-specific miRNAs that are uncharacterized.

Along with the canonical miRNA, distinct miRNA-like sequences called sibling miRNAs (sib-miRs) are generated from the same pre-miRNA. Among them, isomeric sequences featuring slight variations at the terminals, relative to the canonical miRNA, constitute a pool of isomeric sibling miRNAs (isomiRs). Despite the high prevalence of isomiRs in eukaryotes, their features and relevance remain elusive. In this study, we performed a comprehensive analysis of mature precursor miRNA (pre-miRNA) sequences from Arabidopsis to understand their features and regulatory targets. The influence of isomiR terminal heterogeneity in target binding was examined comprehensively. Our comprehensive analyses suggested a novel computational strategy that utilizes miRNA and its isomiRs to enhance the accuracy of their regulatory target prediction in Arabidopsis. A few targets are shared by several members of isomiRs; however, this phenomenon was not typical. Gene Ontology (GO) enrichment analysis showed that commonly targeted mRNAs were enriched for certain GO terms. Moreover, comparison of these commonly targeted genes with validated targets from published data demonstrated that the validated targets are bound by most isomiRs and not only the canonical miRNA. Furthermore, the biological role of isomiRs in target cleavage was supported by degradome data. Incorporating this finding, we predicted potential target genes of several miRNAs and confirmed them by experimental assays. This study proposes a novel strategy to improve the accuracy of predicting miRNA targets through combined use of miRNA with its isomiRs.

As more small RNA sequencing libraries are becoming available, it clearly emerges that microRNAs (miRNAs) are highly heterogeneous both in length and sequence. In comparison to canonical miRNAs, miRNA isoforms (termed as “isomiRs”) might exhibit different biological properties, such as a different target repertoire, or enhanced/reduced stability. Nonetheless, this layer of information has remained largely unexplored due to the scarcity of small RNA NGS-datasets and the absence of proper analytical tools. Here, we present a workflow for the characterization and analysis of miRNAs and their variants in next-generation sequencing datasets. IsomiRs can originate from an alternative dicing event (“templated” forms) or from the addition of nucleotides through an enzymatic activity or target-dependent mechanisms (“non-templated” forms). Our pipeline allows distinguishing canonical miRNAs from templated and non-templated isomiRs by alignment to a custom database, which comprises all possible 3′-, 5′-, and trimmed variants. Functionally equivalent isomiRs can be grouped together according to the type of modification (e.g., uridylation, adenylation, trimming …) to assess which miRNAs are more intensively modified in a given biological context. When applied to the analysis of primary epithelial breast cancer cells, our methodology provided a 40% increase in the number of detected miRNA species and allowed to easily identify and classify more than 1000 variants. Most modifications were compatible with templated IsomiRs, as a consequence of imprecise Drosha or Dicer cleavage. However, some non-templated variants were consistently found either in the normal or in the cancer cells, with the 3′-end adenylation and uridylation as the most frequent events, suggesting that miRNA post-transcriptional modification frequently occurs. In conclusion, our analytical tool permits the deconvolution of miRNA heterogeneity and could be used to explore the functional role of miRNA isoforms.

As more small RNA sequencing libraries are becoming available, it clearly emerges that microRNAs (miRNAs) are highly heterogeneous both in length and sequence. In comparison to canonical miRNAs, miRNA isoforms (termed as isomiRs) might exhibit different biological properties, such as a different target repertoire, or enhanced/reduced stability. Nonetheless, this layer of information has remained largely unexplored due to the scarcity of small RNA NGS-datasets and the absence of proper analytical tools. Here, we present a workflow for the characterization and analysis of miRNAs and their variants in next-generation sequencing datasets. IsomiRs can originate from an alternative dicing event (templated forms) or from the addition of nucleotides through an enzymatic activity or target-dependent mechanisms (non-templated forms). Our pipeline allows distinguishing canonical miRNAs from templated and non-templated isomiRs by alignment to a custom database, which comprises all possible 3’-, 5’- and trimmed variants. Functionally equivalent isomiRs can be grouped together according to the type of modification (e.g. uridylation, adenylation, trimming…) to assess which miRNAs are more intensively modified in a given biological context. When applied to the analysis of primary epithelial breast cancer cells, our methodology provided a 40% increase in the number of detected miRNA species and allowed to easily identify and classify more than 1000 variants. Most modifications were compatible with templated isomiRs, as a consequence of imprecise Drosha or Dicer cleavage. However, some non-templated variants were consistently found either in the normal or in the cancer cells, with the 3’-end adenylation and uridylation as the most frequent events, suggesting that miRNA post-transcriptional modification frequently occurs. In conclusion, our analytical tool permits the deconvolution of miRNA heterogeneity and could be used to explore the functional role of miRNA isoforms.

41 p.-2 fig., MicroRNAs (miRNAs) are cell-endogenous small noncoding RNAs that, through RNA interference, are involved in the posttranscriptional regulation of mRNAs. The biogenesis and function of miRNAs entail multiple elements with different alternative pathways. These confer a high versatility of regulation and a high variability to generate different miRNAs and hence possess a broad potential to regulate gene expression. Here we review the different mechanisms, both canonical and noncanonical, that generate miRNAs in animals. The ' miRNome ' panorama enhances our knowledge regarding the fine regulation of gene expression and provides new insights concerning normal, as opposed to pathological, cell differentiation and development., Research on microRNAs carried out in del Mazo´s laboratory was supported by the following grants:, CEFIC-LRi,; MEDDTL (11-MRES-PNRPE-9-CVS-072), France; and CSIC (PIE 201020E016), Spain.

Since microRNAs (miRNAs) were discovered, their impact on regulating various biological activities has been a surprising and exciting field. Knowing the entire repertoire of these small molecules is the first step to gain a better understanding of their function. High throughput discovery tools such as next-generation sequencing significantly increased the number of miRNAs in different organisms in recent years. However, the process of being able to accurately identify miRNA is still a complex and difficult task, requiring the integration of experimental approaches with computational methods. A number of prediction algorisms based on characteristics of miRNA molecules have been developed to identify new miRNA species. Different approaches have certain strengths and weaknesses and, in this review, we aim to summarize several commonly used tools in miRNA discovery and provide some prospects on future developments.

We present an open-access web platform isomiRex, to identify isomiRs and on the fly graphical visualization of the differentially expressed miRNAs in control as well as treated library. The open-access web-platform is not restricted only to NGS sequence dataset from animals and potentially analyzes a wider dataset for plants, animals and viral NGS dataset supporting miRBase (version 19 supporting 193 species). The platform can handle the bloated amount of the read counts and reports the annotated microRNAs from plant, animal and viral NGS datasets. isomiRex also provides an estimation of the the isomiRs, of miRNAs with higher copy number relative to their mature reference sequences indexed in miRBase (version 19 supporting 193 species). Visually enhanced graphs potentially display differentially expressed isomiRs, which will help the user to demonstrate and correlate the abundance of the isomiR as a signature event to the specific condition. An additional module for estimating the differential expression has been implemented allowing the users to postulate the differential expression across the user input samples. The developed web-platform can be accessed at http://bioinfo1.uni-plovdiv.bg/isomiRex/.

MicroRNAs (miRNAs) are short non-coding RNA molecules produced from hairpin structures and involved in gene expression regulation with major roles in plant development and stress response. Although each annotated miRNA in miRBase (www.mirbase.org) is a single defined sequence with no further details on possible variable sequence length, isomiRs - namely the population of variants of miRNAs coming from the same precursors - have been identified in several species and could represent a way of broadening the regulatory network of the cell. Next-gen-based sequencing makes it possible to comprehensively and accurately assess the entire miRNA repertoire including isomiRs. The aim of this work was to survey the complexity of the peach miRNome by carrying out Illumina high-throughput sequencing of miRNAs in three replicates of five biological samples arising from a set of different peach organs and/or phenological stages. Three hundred-ninety-two isomiRs (miRNA and miRNA*-related) corresponding to 26 putative miRNA coding loci, have been highlighted by mirDeep-P and analyzed. The presence of the same isomiRs in different biological replicates of a sample and in different tissues demonstrates that the generation of most of the detected isomiRs is not random. The degree of mature sequence heterogeneity is very different for each individual locus. Results obtained in the present work can thus contribute to a deeper view of the miRNome complexity and to better explore the mechanism of action of these tiny regulators.

Background miRNAs are small noncoding RNA molecules that play an important role in post-transcriptional regulation of gene expression. Length and/or sequence variants of the same miRNA are termed isomiRs. While most isomiRs are functionally redundant compared to their canonical counterparts, the so-called 5’isomiRs exhibit a shifted 5’ end and therefore a shifted seed sequence resulting in a different target spectrum. However, not much is known about the functional relevance of these isoforms. Results Analysis of miRNA-seq data from breast cancer cell lines identified six pairs of highly expressed miRNAs and associated 5’isomiRs. Among them, hsa-miR-140-3p was of particular interest because its 5’isomiR showed higher expression compared to the canonical miRNA annotated in miRbase. This miRNA has previously been shown to control stemness of breast cancer cells. miRNAseq data of breast cancer patients (TCGA dataset) showed that both the canonical hsa-miR-140-3p and its 5’isomiR-140-3p were highly expressed in patients’ tumors compared to normal breast tissue. In the current work, we present the functional characterization of 5’isomiR-140-3p and the cellular phenotypes associated with its overexpression in MCF10A, MDA-MB-468 and MDA-MB-231 cell lines in comparison to the canonical hsa-miR-140-3p. Contrary to the effect of the canonical hsa-miR-140-3p, overexpression of the 5’isomiR-140-3p led to a decrease in cell viability. The latter observation was supported by cell cycle analysis, where the 5’isomiR-140-3p but not the hsa-miR-140-3p caused cell cycle arrest in G0/G1-phase. Additionally, 5’ismoiR-140-3p overexpression was found to cause a decrease in cell migration in the three cell lines. We identified three novel direct target genes of the 5’isomiR-140-3p; COL4A1, ITGA6 and MARCKSL1. Finally, we have shown that knocking down these genes partially phenocopied the effects of the 5’isomiR-140-4p overexpression, where COL4A1 and ITGA6 knockdown led to reduced cell viability and cell cycle arrest, while MARCKSL1 knockdown resulted in a decrease in the migratory potential of cells. Conclusions In summary, this work presents evidence that there is functional synergy between the canonical hsa-miR-140-3p and the newly identified 5’isomiR-140-3p in suppressing growth and progression of breast cancer by simultaneously targeting genes related to differentiation, proliferation, and migration. Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-2869-x) contains supplementary material, which is available to authorized users.

Tese de mestrado, Bioinformática e Biologia Computacional (Bioinformática), Universidade de Lisboa, Faculdade de Ciências, 2017 Submitted by Teresa Boa (tdboa@fc.ul.pt) on 2018-01-12T16:02:18Z No. of bitstreams: 1 ulfc121581_tm_Beatriz_Ferreira.pdf: 2285523 bytes, checksum: 540bd6faa0557a2c7fb879655a7f0c2f (MD5) Made available in DSpace on 2018-01-12T16:02:28Z (GMT). No. of bitstreams: 1 ulfc121581_tm_Beatriz_Ferreira.pdf: 2285523 bytes, checksum: 540bd6faa0557a2c7fb879655a7f0c2f (MD5) Previous issue date: 2017