The subcellular distribution of miRNA isoforms, tRNA-derived fragments, and rRNA-derived fragments depends on nucleotide sequence and cell type.

BACKGROUND: MicroRNA isoforms (isomiRs), tRNA-derived fragments (tRFs), and rRNA-derived fragments (rRFs) represent most of the small non-coding RNAs (sncRNAs) found in cells. Members of these three classes modulate messenger RNA (mRNA) and protein abundance and are dysregulated in diseases. Experimental studies to date have assumed that the subcellular distribution of these molecules is well-understood, independent of cell type, and the same for all isoforms of a sncRNA. RESULTS: We tested these assumptions by investigating the subcellular distribution of isomiRs, tRFs, and rRFs in biological replicates from three cell lines from the same tissue and same-sex donors that model the same cancer subtype. In each cell line, we profiled the isomiRs, tRFs, and rRFs in the nucleus, cytoplasm, whole mitochondrion (MT), mitoplast (MP), and whole cell. Using a rigorous mathematical model we developed, we accounted for cross-fraction contamination and technical errors and adjusted the measured abundances accordingly. Analyses of the adjusted abundances show that isomiRs, tRFs, and rRFs exhibit complex patterns of subcellular distributions. These patterns depend on each sncRNA's exact sequence and the cell type. Even in the same cell line, isoforms of the same sncRNA whose sequences differ by a few nucleotides (nts) can have different subcellular distributions. CONCLUSIONS: SncRNAs with similar sequences have different subcellular distributions within and across cell lines, suggesting that each isoform could have a different function. Future computational and experimental studies of isomiRs, tRFs, and rRFs will need to distinguish among each molecule's various isoforms and account for differences in each isoform's subcellular distribution in the cell line at hand. While the findings add to a growing body of evidence that isomiRs, tRFs, rRFs, tRNAs, and rRNAs follow complex intracellular trafficking rules, further investigation is needed to exclude alternative explanations for the observed subcellular distribution of sncRNAs.

Dual Role of microRNA-146a in Experimental Inflammation in Human Pulmonary Epithelial and Immune Cells and Expression in Inflammatory Lung Diseases.

microRNA (miR)-146a emerges as a promising post-transcriptional regulator in various inflammatory diseases with different roles for the two isoforms miR-146a-5p and miR-146a-3p. The present study aimed to examine the dual role of miR-146a-5p and miR-146a 3p in the modulation of inflammation in human pulmonary epithelial and immune cells in vitro as well as their expression in patients with inflammatory lung diseases. Experimental inflammation in human A549, HL60, and THP1 via the NF-kB pathway resulted in the major upregulation of miR-146a-5p and miR-146a-3p expression, which was partly cell-specific. Modulation by transfection with miRNA mimics and inhibitors demonstrated an anti-inflammatory effect of miR-146a-5p and a pro-inflammatory effect of miR-146a-3p, respectively. A mutual interference between miR-146a-5p and miR-146a-3p was observed, with miR-146a-5p exerting a predominant influence. In vivo NGS analyses revealed an upregulation of miR-146a-3p in the blood of patients with cystic fibrosis and bronchiolitis obliterans, while miR-146a-5p levels were downregulated or unchanged compared to controls. The reverse pattern was observed in patients with SARS-CoV-2 infection. In conclusion, miR-146a-5p and miR-146a-3p are two distinct but interconnected miRNA isoforms with opposing functions in inflammation regulation. Understanding their interaction provides important insights into the progression and persistence of inflammatory lung diseases and might provide potential therapeutic options.

MicroRNA Transcriptomes Reveal Prevalence of Rare and Species-Specific Arm Switching Events During Zebrafish Ontogenesis.

In metazoans, microRNAs (miRNAs) are essential regulators of gene expression, affecting critical cellular processes from differentiation and proliferation, to homeostasis. During miRNA biogenesis, the miRNA strand that loads onto the RNA-induced Silencing Complex (RISC) can vary, leading to changes in gene targeting and modulation of biological pathways. To investigate the impact of these "arm switching" events on gene regulation, we analyzed a diverse range of tissues and developmental stages in zebrafish by comparing 5p and 3p arms accumulation dynamics between embryonic developmental stages, adult tissues, and sexes. We also compared variable arm usage patterns observed in zebrafish to other vertebrates including arm switching data from fish, birds, and mammals. Our comprehensive analysis revealed that variable arm usage events predominantly take place during embryonic development. It is also noteworthy that isomiR occurrence correlates to changes in arm selection evidencing an important role of microRNA distinct isoforms in reinforcing and modifying gene regulation by promoting dynamics switches on miRNA 5p and 3p arms accumulation. Our results shed new light on the emergence and coordination of gene expression regulation and pave the way for future investigations in this field.

Connection between MiR-490 and CCND1 and GSK3ß genes play an effective role in Wnt signaling pathway in colorectal cancer.

The Wnt signaling pathway is identified as one of the main disrupted pathways in Colorectal cancer (CRC). Results from studies focusing on this route will aid greatly in the detection and treatment of CRC. MicroRNAs (MiRs), particularly MiR-490, has emerged as key regulator of gene expression in biological pathways, making it an attractive research target. This is notably true for the Wnt signaling pathway, which is usually disordered in CRC tissues. This study aimed to evaluate the expression level of MiR-490 isomiRs and determine some of its key target genes involved in Wnt signaling pathway in CRC tissues and cell lines, based on experimental and bioinformatics analysis. Elevated expression of GSK3ß and CCND1 indicate that the progression of CRC tumor is associated with the inhibitory effect of MiR-490 isomiRs on the Wnt/ß-catenin signaling pathway. This finding was supported by the observation of a positive connection between the expression pattern of miR-490-3p and 5p, and CCND1 and GSK3ß in CRC. The valuable results of this study provide a means of identifying biomarkers with the potential to either inhibit or activate CRC cellular pathways.

5'-Isoforms of miR-1246 Have Distinct Targets and Stronger Functional Impact Compared with Canonical miR-1246 in Colorectal Cancer Cells In Vitro.

Colorectal cancer (CRC) is a multifactorial disease involving genetic and epigenetic factors, such as miRNAs. Sequencing-based studies have revealed that miRNAs have many isoforms (isomiRs) with modifications at the 3'- and 5'-ends or in the middle, resulting in distinct targetomes and, consequently, functions. In the present study, we aimed to evaluate the putative targets and functional role of miR-1246 and its two 5'-isoforms (ISO-miR-1246_a and ISO-miR-1246_G) in vitro. Commercial Caco-2 cells of CRC origin were analyzed for the expression of WT-miR-1246 and its 5'-isoforms using small RNA sequencing data, and the overabundance of the two miR-1246 isoforms was determined in cells. The transcriptome analysis of Caco-2 cells transfected with WT-miR-1246, ISO-miR-1246_G, and ISO-miR-1246_a indicated the minor overlap of the targetomes between the studied miRNA isoforms. Consequently, an enrichment analysis showed the involvement of the potential targets of the miR-1246 isoforms in distinct signaling pathways. Cancer-related pathways were predominantly more enriched in dysregulated genes in ISO-miR-1246_G and ISO-miR-1246_a, whereas cell cycle pathways were more enriched in WT-miR-1246. The functional analysis of WT-miR-1246 and its two 5'-isoforms revealed that the inhibition of any of these molecules had a tumor-suppressive role (reduced cell viability and migration and promotion of early cell apoptosis) in CRC cells. However, the 5'-isoforms had a stronger effect on viability compared with WT-miR-1246. To conclude, this research shows that WT-miR-1246 and its two 5'-isoforms have different targetomes and are involved in distinct signaling pathways but collectively play an important role in CRC pathogenesis.

Seminal extracellular vesicle sncRNA sequencing reveals altered miRNA/isomiR profiles as sperm retrieval biomarkers for azoospermia.

BACKGROUND: Non-invasive molecular biomarkers for classifying azoospermia by origin into either obstructive or non-obstructive/secretory azoospermia, as well as for inferring the spermatogenic reserve of the testis of non-obstructive/secretory azoospermia patients, are of great interest for testicular sperm retrieval outcome prediction for assisted reproduction. Prior analyses of semen small non-coding RNA expression in azoospermia have focused on microRNAs, but there has been a lack of attention on other regulatory small RNA species. In this regard, studying more in-depth expression changes of small non-coding RNA subtypes in small extracellular vesicles from semen of azoospermic individuals could be useful to select additional non-invasive biomarkers with diagnostic/prognostic purposes. MATERIAL AND METHODS: A high-throughput small RNA profiling analysis to determine the expression pattern of seminal small extracellular vesicle microRNAs (analyzed at the isomiR level), PIWI-interacting RNAs, and transfer RNA-derived small RNAs in normozoospermic (n = 4) and azoospermic (obstructive azoospermia because of pathological occurring obstruction in the genital tract, n = 4; secretory azoospermic individuals with positive testicular sperm extraction value, n = 5; secretory azoospermic individuals with negative testicular sperm extraction value, n = 4) individuals was carried out. Reverse transcriptase-quantitative real-time polymerase chain reaction validation analysis of selected microRNAs was additionally performed in a larger number of individuals. RESULTS AND DISCUSSION: Clinically relevant quantitative changes in the small non-coding RNA levels contained in semen small extracellular vesicles can be used as biomarkers for the origin of azoospermia and for predicting the presence of residual spermatogenesis. In this regard, canonical isoform microRNAs (n = 185) but also other isomiR variants (n = 238) stand out in terms of numbers and fold-change differences in expression, underlining the need to consider isomiRs when investigating microRNA-based regulation. Conversely, although transfer RNA-derived small RNAs are shown in our study to represent a high proportion of small non-coding RNA sequences in seminal small extracellular vesicle samples, they are not able to discriminate the origin of azoospermia. PIWI-interacting RNA cluster profiles and individual PIWI-interacting RNAs with significant differential expression were also not able to discriminate. Our study demonstrated that expression values of individual and/or combined canonical isoform microRNAs (miR-10a-5p, miR-146a-5p, miR-31-5p, miR-181b-5p; area under the receiver operating characteristic curve >0.8) in small extracellular vesicles provide considerable clinical value in identifying samples with a high likelihood of sperm retrieval while discriminating azoospermia by origin. Although no individual microRNA showed sufficient discriminating power on its own to identify severe spermatogenic disorders with focal spermatogenesis, multivariate microRNA models in semen small extracellular vesicles have the potential to identify those individuals with residual spermatogenesis. Availability and adoption of such non-invasive molecular biomarkers would represent a great improvement in reproductive treatment decision protocols for azoospermia in clinical practice.

3'IsomiR Species Composition Affects Reliable Quantification of miRNA/isomiR Variants by Poly(A) RT-qPCR: Impact on Small RNA-Seq Profiling Validation.

Small RNA-sequencing (small RNA-seq) has revealed the presence of small RNA-naturally occurring variants such as microRNA (miRNA) isoforms or isomiRs. Due to their small size and the sequence similarity among miRNA isoforms, their validation by RT-qPCR is challenging. We previously identified two miR-31-5p isomiRs-the canonical and a 3'isomiR variant (3' G addition)-which were differentially expressed between individuals with azoospermia of different origin. Here, we sought to determine the discriminatory capacity between these two closely-related miRNA isoforms of three alternative poly(A) based-RT-qPCR strategies in both synthetic and real biological context. We found that these poly(A) RT-qPCR strategies exhibit a significant cross-reactivity between these miR-31-5p isomiRs which differ by a single nucleotide, compromising the reliable quantification of individual miRNA isoforms. Fortunately, in the biological context, given that the two miRNA variants show changes in the same direction, RT-qPCR results were consistent with the findings of small RNA-seq study. We suggest that miRNA selection for RT-qPCR validation should be performed with care, prioritizing those canonical miRNAs that, in small RNA-seq, show parallel/homogeneous expression behavior with their most prevalent isomiRs, to avoid confounding RT-qPCR-based results. This is suggested as the current best strategy for robust biomarker selection to develop clinically useful tests.

Characterization of all small RNAs in and comparisons across cultured megakaryocytes and platelets of healthy individuals and COVID-19 patients.

BACKGROUND: The small noncoding RNAs (sncRNAs) in megakaryocytes (MKs) and platelets are not well characterized. Neither is the impact of SARS-CoV-2 infection on the sncRNAs of platelets. OBJECTIVES: To investigate the sorting of MK sncRNAs into platelets, and the differences in the platelet sncRNAomes of healthy donors (HDs) and COVID-19 patients. METHODS: We comprehensively profiled sncRNAs from MKs cultured from cord blood-derived CD34+ cells, platelets from HDs, and platelets from patients with moderate and severe SARS-CoV-2 infection. We also comprehensively profiled Argonaute (AGO)-bound sncRNAs from the cultured MKs. RESULTS: We characterized the sncRNAs in MKs and platelets and can account for ∼95% of all sequenced reads. We found that MKs primarily comprise microRNA isoforms (isomiRs), tRNA-derived fragments (tRFs), rRNA-derived fragments (rRFs), and Y RNA-derived fragments (yRFs) in comparable abundances. The platelets of HDs showed a skewed distribution by comparison: 56.7% of all sncRNAs are yRFs, 34.4% are isomiRs, and <2.0% are tRFs and rRFs. Most isomiRs in MKs and platelets are either noncanonical, nontemplated, or both. When comparing MKs and platelets from HDs, we found numerous isomiRs, tRFs, rRFs, and yRFs showing opposite enrichments or depletions, including molecules from the same parental miRNA arm, tRNA, rRNA, or Y RNA. The sncRNAome of platelets from patients with COVID-19 is skewed compared to that of HDs with only 19.8% of all sncRNAs now being yRFs, isomiRs increasing to 63.6%, and tRFs and rRFs more than tripling their presence to 6.1%. CONCLUSION: The sncRNAomes of MKs and platelets are very rich and more complex than it has been believed. The evidence suggests complex mechanisms that sort MK sncRNAs into platelets. SARS-CoV-2 infection acutely alters the contents of platelets by changing the relative proportions of their sncRNAs.

Reassessment of miRNA variant (isomiRs) composition by small RNA sequencing.

IsomiRs, sequence variants of mature microRNAs, are usually detected and quantified using high-throughput sequencing. Many examples of their biological relevance have been reported, but sequencing artifacts identified as artificial variants might bias biological inference and therefore need to be ideally avoided. We conducted a comprehensive evaluation of 10 different small RNA sequencing protocols, exploring both a theoretically isomiR-free pool of synthetic miRNAs and HEK293T cells. We calculated that, with the exception of two protocols, less than 5% of miRNA reads can be attributed to library preparation artifacts. Randomized-end adapter protocols showed superior accuracy, with 40% of true biological isomiRs. Nevertheless, we demonstrate concordance across protocols for selected miRNAs in non-templated uridyl additions. Notably, NTA-U calling and isomiR target prediction can be inaccurate when using protocols with poor single-nucleotide resolution. Our results highlight the relevance of protocol choice for biological isomiRs detection and annotation, which has key potential implications for biomedical applications.

A Review of IsomiRs in Colorectal Cancer.

As advancements in sequencing technology rapidly continue to develop, a new classification of microRNAs has occurred with the discovery of isomiRs, which are relatively common microRNAs with sequence variations compared to their established template microRNAs. This review article seeks to compile all known information about isomiRs in colorectal cancer (CRC), which has not, to our knowledge, been gathered previously to any great extent. A brief overview is given of the history of microRNAs, their implications in colon cancer, the canonical pathway of biogenesis and isomiR classification. This is followed by a comprehensive review of the literature that is available on microRNA isoforms in CRC. The information on isomiRs presented herein shows that isomiRs hold great promise for translation into new diagnostics and therapeutics in clinical medicine.

MicroRNA 3' ends shorten during adolescent brain maturation.

MicroRNA (miRNA) dysregulation is well-documented in psychiatric disease, but miRNA dynamics remain poorly understood during adolescent and early adult brain maturation, when symptoms often first appear. Here, we use RNA sequencing to examine miRNAs and their mRNA targets in cortex and hippocampus from early-, mid-, and late-adolescent and adult mice. Furthermore, we use quantitative proteomics by tandem mass tag mass spectrometry (TMT-MS) to examine protein dynamics in cortex from the same subjects. We found that ~25% of miRNAs' 3' ends shorten with age due to increased 3' trimming and decreased U tailing. Particularly, shorter but functionally competent isoforms (isomiRs) of miR-338-3p increase up to 10-fold during adolescence and only in brain. MiRNAs that undergo 3' shortening exhibit stronger negative correlations with targets that decrease with age and stronger positive correlations with targets that increase with age, than miRNAs with stable 3' ends. Increased 3' shortening with age was also observed in available mouse and human miRNA-seq data sets, and stronger correlations between miRNAs that undergo shortening and their mRNA targets were observed in two of the three available data sets. We conclude that age-associated miRNA 3' shortening is a well-conserved feature of postnatal brain maturation.

5'­isomiR is the most abundant sequence of miR­1246, a candidate biomarker of lung cancer, in serum.

MicroRNA (miRNA/miR) 5'­isoforms (5'­isomiRs) differ from canonical sequences registered in the microRNA database in the length of their 5' ends. The 'seed sequence' of miRNAs that bind to target mRNAs is 2­8 nucleotides from the 5' end; thus, shifts at the 5' end can cause a 'seed shift'. Accumulating data from miRNA deep sequencing have revealed that, in a substantial number of miRNAs, sequences corresponding to specific isomiRs, not the canonical form, are the most abundant. Studies have so far focused on circulating miRNAs as either markers or intercellular communication factors. miR­1246 is abundant in the serum and is a candidate diagnostic and prognostic marker for esophageal squamous cell carcinoma, pancreatic cancer, hepatocellular carcinoma, colorectal adenocarcinoma and non­small cell lung cancer (NSCLC). The present study analyzed the 5'­end of serum miR­1246 by fragment analysis and found that a 5'­isomiR, which is two bases shorter than the canonical sequence, was the most abundant sequence in patients with NSCLC as well as healthy donors. To quantify the 5'­isomiR, 5'­isomiR­specific primers based on primers for allele specific­PCR were used, primarily because commercially available methods for miRNA Reverse transcription­quantitative PCR cannot discriminate among sequences, especially those located at the 5' end of miRNA. The total miR­1246 levels were significantly increased in patients with NSCLC; by contrast, the level of the canonical sequence was significantly decreased. Significant positive correlations were observed between the total miR­1246 levels and the 5'­isomiR levels, but not that of the canonical sequence. These results imply that the increase in levels of serum miR­1246 in patients with NSCLC depends on increase of the 5'­isomiR.

Versatile RNA: overlooked gems of the transcriptome.

The critical role of RNA, its use and targetability concerning different aspects of human health are gaining more attention because our understanding of the versatility of RNA has dramatically evolved over the last decades. We now appreciate that RNA is far more critical than a messenger molecule and possesses many complicated functions. As a multifunctional molecule with its sequence, flexible structures and enzymatic abilities, RNA is genuinely powerful. Mammalian transcriptomes consist of a dynamically regulated plethora of coding and noncoding RNA types. However, some aspects of RNA metabolism remain to be explored. In this Viewpoint, we focus on the transcriptome's unconventional and possibly overlooked aspects to emphasize the importance of RNA in mammalian systems.

Global profiling and annotation of templated isomiRs dynamics across Caenorhabditis elegans development.

microRNAs (miRNAs) are small non-coding RNAs that regulate gene expression through translational repression and mRNA destabilization. During canonical miRNA biogenesis, several miRNA isoforms, or isomiRs, are produced from a single precursor miRNA. Templated isomiRs are generated through Drosha or Dicer cleavage at alternate positions on either the primary or the precursor miRNAs, generating truncated or extended 5' and/or 3' miRNA ends. As changes to the mature miRNA sequence can alter miRNA gene target repertoire, we investigated the extent of templated isomiR prevalence, providing a profiling map for templated isomiRs across stages of C. elegans development. While most miRNA loci did not produce abundant templated isomiRs, a substantial number of miRNA loci produced isomiRs were just as, or more, abundant than their annotated canonical mature miRNAs. 3' end miRNA alterations were more frequent than the seed-altering 5' end extensions or truncations. However, we identified several miRNA loci that produced a considerable amount of isomiRs with 5' end alterations, predicted to target new, distinct sets of genes. Overall, the presented annotation of templated isomiR dynamics across C. elegans developmental stages provides a basis for further studies into miRNA biogenesis and the intriguing potential of functional miRNA diversification through isomiR production.

5'isomiR-183-5p|+2 elicits tumor suppressor activity in a negative feedback loop with E2F1.

BACKGROUND: MicroRNAs (miRNAs) and isomiRs play important roles in tumorigenesis as essential regulators of gene expression. 5'isomiRs exhibit a shifted seed sequence compared to the canonical miRNA, resulting in different target spectra and thereby extending the phenotypic impact of the respective common pre-miRNA. However, for most miRNAs, expression and function of 5'isomiRs have not been studied in detail yet. Therefore, this study aims to investigate the functions of miRNAs and their 5'isomiRs. METHODS: The expression of 5'isomiRs was assessed in The Cancer Genome Atlas (TCGA) breast cancer patient dataset. Phenotypic effects of miR-183 overexpression in triple-negative breast cancer (TNBC) cell lines were investigated in vitro and in vivo by quantifying migration, proliferation, tumor growth and metastasis. Direct targeting of E2F1 by miR-183-5p|+2 was validated with a 3'UTR luciferase assay and linked to the phenotypes of isomiR overexpression. RESULTS: TCGA breast cancer patient data indicated that three variants of miR-183-5p are highly expressed and upregulated, namely miR-183-5p|0, miR-183-5p|+1 and miR-183-5p|+2. However, TNBC cell lines displayed reduced proliferation and invasion upon overexpression of pre-miR-183. While invasion was reduced individually by all three isomiRs, proliferation and cell cycle progression were specifically inhibited by overexpression of miR-183-5p|+2. Proteomic analysis revealed reduced expression of E2F target genes upon overexpression of this isomiR, which could be attributed to direct targeting of E2F1, specifically by miR-183-5p|+2. Knockdown of E2F1 partially phenocopied the effect of miR-183-5p|+2 overexpression on cell proliferation and cell cycle. Gene set enrichment analysis of TCGA and METABRIC patient data indicated that the activity of E2F strongly correlated with the expression of miR-183-5p, suggesting transcriptional regulation of the miRNA by a factor of the E2F family. Indeed, in vitro, expression of miR-183-5p was regulated by E2F1. Hence, miR-183-5p|+2 directly targeting E2F1 appears to be part of a negative feedback loop potentially fine-tuning its activity. CONCLUSIONS: This study demonstrates that 5'isomiRs originating from the same arm of the same pre-miRNA (i.e. pre-miR-183-5p) may exhibit different functions and thereby collectively contribute to the same phenotype. Here, one of three isomiRs was shown to counteract expression of the pre-miRNA by negatively regulating a transcriptional activator (i.e. E2F1). We speculate that this might be part of a regulatory mechanism to prevent uncontrolled cell proliferation, which is disabled during cancer progression.

miR-155-3p: processing by-product or rising star in immunity and cancer?

MicroRNAs (miRNAs) are key players in gene regulation that target specific mRNAs for degradation or translational repression. Each miRNA is synthesized as a miRNA duplex comprising two strands (5p and 3p). However, only one of the two strands becomes active and is selectively incorporated into the RNA-induced silencing complex in a process known as miRNA strand selection. Recently, significant progress has been made in understanding the factors and processes involved in strand selection. Here, we explore the selection and functionality of the miRNA star strand (either 5p or 3p), which is generally present in the cell at low levels compared to its partner strand and, historically, has been thought to possess no biological activity. We also highlight the concepts of miRNA arm switching and miRNA isomerism. Finally, we offer insights into the impact of aberrant strand selection on immunity and cancer. Leading us through this journey is miR-155, a well-established regulator of immunity and cancer, and the increasing evidence that its 3p strand plays a role in these arenas. Interestingly, the miR-155-5p/-3p ratio appears to vary dependent on the timing of the immune response, and the 3p strand seems to play a regulatory role upon its partner 5p strand.

Flexible pri-miRNA structures enable tunable production of 5' isomiRs.

The Drosha cleavage of a pri-miRNA defines mature microRNA sequence. Drosha cleavage at alternative positions generates 5' isoforms (isomiRs) which have distinctive functions. To understand how pri-miRNA structures influence Drosha cleavage, we performed a systematic analysis of the maturation of endogenous pri-miRNAs and their variants both in vitro and in vivo. We show that in addition to previously known features, the overall structural flexibility of pri-miRNA impact Drosha cleavage fidelity. Internal loops and nearby G · U wobble pairs on the pri-miRNA stem induce the use of non-canonical cleavage sites by Drosha, resulting in 5' isomiR production. By analysing patient data deposited in the Cancer Genome Atlas, we provide evidence that alternative Drosha cleavage of pri-miRNAs is a tunable process that responds to the level of pri-miRNA-associated RNA-binding proteins. Together, our findings reveal that Drosha cleavage fidelity can be modulated by altering pri-miRNA structure, a potential mechanism underlying 5' isomiR biogenesis in tumours.[Figure: see text].

The Dynamics of miR-449a/c Expression during Uterine Cycles Are Associated with Endometrial Development.

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.

RNA uridyl transferases TUT4/7 differentially regulate miRNA variants depending on the cancer cell type.

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.

Coupling miR/isomiR and mRNA Expression Signatures Unveils New Molecular Layers of Endometrial Receptivity.

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.