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Although Argonaute (AGO) proteins have been the focus of microRNA (miRNA) studies, we observed AGO-free mature miRNAs directly interacting with RNA-binding proteins, implying the sophisticated nature of fine-tuning gene regulation by miRNAs. To investigate microRNA-binding proteins (miRBPs) globally, we analyzed PAR-CLIP data sets to identify RBP quaking (QKI) as a novel miRBP for let-7b. Potential existence of AGO-free miRNAs were further verified by measuring miRNA levels in genetically engineered AGO-depleted human and mouse cells. We have shown that QKI regulates miRNA-mediated gene silencing at multiple steps, and collectively serves as an auxiliary factor empowering AGO2/let-7b-mediated gene silencing. Depletion of QKI decreases interaction of AGO2 with let-7b and target mRNA, consequently controlling target mRNA decay. This finding indicates that QKI is a complementary factor in miRNA-mediated mRNA decay. QKI, however, also suppresses the dissociation of let-7b from AGO2, and slows the assembly of AGO2/miRNA/target mRNA complexes at the single-molecule level. We also revealed that QKI overexpression suppresses cMYC expression at post-transcriptional level, and decreases proliferation and migration of HeLa cells, demonstrating that QKI is a tumour suppressor gene by in part augmenting let-7b activity. Our data show that QKI is a new type of RBP implicated in the versatile regulation of miRNA-mediated gene silencing.

Glioblastoma (GBM) is the most frequent and lethal primary malignant brain tumor. Despite decades of research, therapeutic advances that significantly prolong life are non-existent. In recent years, microRNAs (miRNAs) have been a focus of study in the pathobiology of cancer because of their ability to simultaneously regulate multiple genes. The aim of this study was to determine the functional and mechanistic effects of miR-3928 in GBM both in vitro and in vivo. To the best of our knowledge, this is the first article investigating the role of miR-3928 in GBM. We measured endogenous miR-3928 expression levels in a panel of patient-derived GBM tissue samples and cell lines. We found that GBM tissue samples and cell lines express lower levels of miR-3928 than normal brain cortex and astrocytes, respectively. Therefore, we hypothesized that miR-3928 is a tumor suppressive microRNA. We verified this hypothesis by showing that exogenous expression of miR-3928 has a strong inhibitory effect on both cell growth and invasiveness of GBM cells. Stable ex vivo overexpression of miR-3928 in GBM cells led to a reduction in tumor size in nude mice xenografts. We identified many targets (MDM2, CD44, DDX3X, HMGA2, CCND1, BRAF, ATOH8, and BMI1) of miR-3928. Interestingly, inhibition of the oncogene MDM2 also led to an upregulation of wild-type p53 expression and phosphorylation. In conclusion, we find that miR-3928, through the downregulation of several oncogenes and upregulation and activation of wild-type p53, is a strong tumor suppressor in GBM. Furthermore, the fact that miR-3928 can target many important dysregulated proteins in GBM suggests it might be a "master" regulatory microRNA that could be therapeutically exploited.

Repressor element 1-silencing transcription factor (REST) plays a crucial role in the differentiation of neural progenitor cells (NPCs). C-terminal domain small phosphatases (CTDSPs) are REST effector proteins that reduce RNA polymerase II activity on genes required for neurogenesis. miR-26b regulates neurogenesis in zebrafish by targeting ctdsp2 mRNA, but the molecular events triggered by this microRNA (miR) remain unknown. Here, we show in a murine embryonic stem cell differentiation paradigm that inactivation of miR-26 family members disrupts the formation of neurons and astroglia and arrests neurogenesis at the neural progenitor level. Furthermore, we show that miR-26 directly targets Rest, thereby inducing the expression of a large set of REST complex-repressed neuronal genes, including miRs required for induction of the neuronal gene expression program. Our data identify the miR-26 family as the trigger of a self-amplifying system required for neural differentiation that acts upstream of REST-controlled miRs., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

Lung carcinoids are variably aggressive and mechanistically understudied neuroendocrine neoplasms (NENs). Here, we identified and elucidated the function of a miR-375/yes-associated protein (YAP) axis in lung carcinoid (H727) cells. miR-375 and YAP are respectively high and low expressed in wild-type H727 cells. Following lentiviral CRISPR/Cas9-mediated miR-375 depletion, we identified distinct transcriptomic changes including dramatic YAP upregulation. We also observed a significant decrease in neuroendocrine differentiation and substantial reductions in cell proliferation, transformation, and tumor growth in cell culture and xenograft mouse disease models. Similarly, YAP overexpression resulted in distinct and partially overlapping transcriptomic changes, phenocopying the effects of miR-375 depletion in the same models as above. Transient YAP knockdown in miR-375-depleted cells reversed the effects of miR-375 on neuroendocrine differentiation and cell proliferation. Pathways analysis and confirmatory real-time PCR studies of shared dysregulated target genes indicate that this axis controls neuroendocrine related functions such as neural differentiation, exocytosis, and secretion. Taken together, we provide compelling evidence that a miR-375/YAP axis is a critical mediator of neuroendocrine differentiation and tumorigenesis in lung carcinoid cells.

MicroRNAs are important regulators of immune responses. Here, we show miR-221 and miR-222 modulate the intestinal Th17 cell response. Expression of miR-221 and miR-222 was induced by proinflammatory cytokines and repressed by the cytokine TGF-β. Molecular targets of miR-221 and miR-222 included Maf and Il23r, and loss of miR-221 and miR-222 expression shifted the transcriptomic spectrum of intestinal Th17 cells to a proinflammatory signature. Although the loss of miR-221 and miR-222 was tolerated for maintaining intestinal Th17 cell homeostasis in healthy mice, Th17 cells lacking miR-221 and miR-222 expanded more efficiently in response to IL-23. Both global and T cell-specific deletion of miR-221 and miR-222 rendered mice prone to mucosal barrier damage. Collectively, these findings demonstrate that miR-221 and miR-222 are an integral part of intestinal Th17 cell response that are induced after IL-23 stimulation to constrain the magnitude of proinflammatory response., Competing Interests: Declaration of interests The authors declare no competing financial interests. J.J.O. is a member of the advisory board of Immunity., (Published by Elsevier Inc.)

During the course of their life cycle, most RNAs move between several cellular environments where they associate with different RNA binding proteins (RBPs). Reciprocally, a significant portion of RBPs reside in more than a single cellular compartment, where they can interact with discrete RNAs and even exert distinct biological roles. Proximity-CLIP combines proximity biotinylation of proteins with photoactivatable ribonucleoside-enhanced protein-RNA crosslinking to simultaneously profile the proteome, including RBPs and the RBP-bound transcriptome, in any given subcellular compartment. Here we provide a detailed experimental protocol for Proximity-CLIP along with a simplified non-radioactive, small-RNA cDNA library preparation protocol. Published 2020 U.S. Government. Basic Protocol 1: Cell culture, 4SU labeling, proximity biotinylation, and crosslinking Basic Protocol 2: Cell extraction, streptavidin affinity purification, and on-beads trypsinization Basic Protocol 3: RNA footprints cDNA library preparation Support Protocol: Preparation of RNA-seq libraries from intact RNA., (Published 2020. This article is a US Government work and is in the public domain in the USA.)

Dysregulation of miRNA expression is associated with multiple diseases, including cancers, in which small RNAs can have either oncogenic or tumor suppressive functions. Here we investigated the potential tumor suppressive function of miR-450a, one of the most significantly downregulated miRNAs in ovarian cancer. RNA-seq analysis of the ovarian cancer cell line A2780 revealed that overexpression of miR-450a suppressed multiple genes involved in the epithelial-to-mesenchymal transition (EMT). Overexpression of miR-450a reduced tumor migration and invasion and increased anoikis in A2780 and SKOV-3 cell lines and reduced tumor growth in an ovarian tumor xenographic model. Combined AGO-PAR-CLIP and RNA-seq analysis identified a panel of potential miR-450a targets, of which many, including TIMMDC1, MT-ND2, ACO2, and ATP5B, regulate energetic metabolism. Following glutamine withdrawal, miR-450a overexpression decreased mitochondrial membrane potential but increased glucose uptake and viability, characteristics of less invasive ovarian cancer cell lines. In summary, we propose that miR-450a acts as a tumor suppressor in ovarian cancer cells by modulating targets associated with glutaminolysis, which leads to decreased production of lipids, amino acids, and nucleic acids, as well as inhibition of signaling pathways associated with EMT. SIGNIFICANCE: miR-450a limits the metastatic potential of ovarian cancer cells by targeting a set of mitochondrial mRNAs to reduce glycolysis and glutaminolysis. Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/13/3294/F1.large.jpg., (©2019 American Association for Cancer Research.)

Many small-interfering (si)RNAs are toxic to cancer cells through a 6mer seed sequence (positions 2-7 of the guide strand). Here we performed an siRNA screen with all 4096 6mer seeds revealing a preference for guanine in positions 1 and 2 and a high overall G or C content in the seed of the most toxic siRNAs for four tested human and mouse cell lines. Toxicity of these siRNAs stems from targeting survival genes with C-rich 3'UTRs. The master tumor suppressor miRNA miR-34a-5p is toxic through such a G-rich 6mer seed and is upregulated in cells subjected to genotoxic stress. An analysis of all mature miRNAs suggests that during evolution most miRNAs evolved to avoid guanine at the 5' end of the 6mer seed sequence of the guide strand. In contrast, for certain tumor-suppressive miRNAs the guide strand contains a G-rich toxic 6mer seed, presumably to eliminate cancer cells.

In mammals, gene silencing by the RNA-induced silencing complex (RISC) is a well-understood cytoplasmic posttranscriptional gene regulatory mechanism. Here, we show that embryonic stem cells (ESCs) contain high levels of nuclear AGO proteins and that in ESCs nuclear AGO protein activity allows for the onset of differentiation. In the nucleus, AGO proteins interact with core RISC components, including the TNRC6 proteins and the CCR4-NOT deadenylase complex. In contrast to cytoplasmic miRNA-mediated gene silencing that mainly operates on cis-acting elements in mRNA 3' untranslated (UTR) sequences, in the nucleus AGO binding in the coding sequence and potentially introns also contributed to post-transcriptional gene silencing. Thus, nuclear localization of AGO proteins in specific cell types leads to a previously unappreciated expansion of the miRNA-regulated transcriptome., (Published by Elsevier Inc.)

We describe a novel approach for the detection of small non-coding RNAs in single cells by Single-Molecule Localization Microscopy (SMLM). We used a modified SMLM-setup and applied this instrument in a first proof-of-principle concept to human cancer cell lines. Our method is able to visualize single microRNA (miR)-molecules in fixed cells with a localization accuracy of 10-15 nm, and is able to quantify and analyse clustering and localization in particular subcellular sites, including exosomes. We compared the metastasis-site derived (SW620) and primary site derived (SW480) human colorectal cancer (CRC) cell lines, and (as a proof of principle) evaluated the metastasis relevant miR-31 as a first example. We observed that the subcellular distribution of miR-31 molecules in both cell lines was very heterogeneous with the largest subpopulation of optically acquired weakly metastatic cells characterized by a low number of miR-31 molecules, as opposed to a significantly higher number in the majority of the highly metastatic cells. Furthermore, the highly metastatic cells had significantly more miR-31-molecules in the extracellular space, which were visualized to co-localize with exosomes in significantly higher numbers. From this study, we conclude that miRs are not only aberrantly expressed and regulated, but also differentially compartmentalized in cells with different metastatic potential. Taken together, this novel approach, by providing single molecule images of miRNAs in cellulo can be used as a powerful supplementary tool in the analysis of miRNA function and behaviour and has far reaching potential in defining metastasis-critical subpopulations within a given heterogeneous cancer cell population.

Eukaryotic gene expression is tightly regulated post-transcriptionally by RNA-binding proteins (RBPs) and microRNAs. The RBP AU-rich-binding factor 1 (AUF1) isoform p37 was found to have high affinity for the microRNA let-7b in vitro (Kd = ∼ 6 nM) in cells. Ribonucleoprotein immunoprecipitation, in vitro association, and single-molecule-binding analyses revealed that AUF1 promoted let-7b loading onto Argonaute 2 (AGO2), the catalytic component of the RNA-induced silencing complex (RISC). In turn, AGO2-let-7 triggered target mRNA decay. Our findings uncover a novel mechanism by which AUF1 binding and transfer of microRNA let-7 to AGO2 facilitates let-7-elicited gene silencing., (© 2015 Yoon et al.; Published by Cold Spring Harbor Laboratory Press.)

Psoriasis is a systemic disease with cutaneous manifestations. MicroRNAs (miRNAs) are small non-coding RNA molecules that are differentially expressed in psoriatic skin; however, only few cell- and region-specific miRNAs have been identified in psoriatic lesions. We used laser capture microdissection (LCM) and next-generation sequencing (NGS) to study the specific miRNA expression profiles in the epidermis (Epi) and dermal inflammatory infiltrates (RD) of psoriatic skin (N = 6). We identified 24 deregulated miRNAs in the Epi and 37 deregulated miRNAs in the RD of psoriatic plaque compared with normal psoriatic skin (FCH > 2, FDR < 0.05). Interestingly, 9 of the 37 miRNAs in RD, including miR-193b and miR-223, were recently described as deregulated in circulating peripheral blood mononuclear cells (PBMCs) from patients with psoriasis. Using flow cytometry and qRT-PCR, we found that miR-193b and miR-223 were expressed in Th17 cells. In conclusion, we demonstrate that LCM combined with NGS provides a robust approach to explore the global miRNA expression in the epidermal and dermal compartments of psoriatic skin. Furthermore, our results indicate that the altered local miRNA changes seen in the RD are reflected in the circulating immune cells, suggesting that miRNAs may contribute to the pathogenesis of psoriasis., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Posttranscriptional gene regulation by miRNAs and RNA binding proteins (RBP) is important in development, physiology, and disease. To examine the interplay between miRNAs and the RBP ELAVL1 (HuR), we mapped miRNA binding sites at the transcriptome-wide scale in wild-type and Elavl1 knockout murine bone-marrow-derived macrophages. Proximity of ELAVL1 binding sites attenuated miRNA binding to transcripts and promoted gene expression. Transcripts that regulate angiogenesis and macrophage/endothelial crosstalk were preferentially targeted by miRNAs, suggesting that ELAVL1 promotes angiogenesis, at least in part by antagonism of miRNA function. We found that ELAVL1 antagonized binding of miR-27 to the 3' UTR of Zfp36 mRNA and alleviated miR-27-mediated suppression of the RBP ZFP36 (Tristetraprolin). Thus, the miR-27-regulated mechanism synchronizes the expression of ELAVL1 and ZFP36. This study provides a resource for systems-level interrogation of posttranscriptional gene regulation in macrophages, a key cell type in inflammation, angiogenesis, and tissue homeostasis., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

Cellular and viral microRNAs (miRNAs) are the transcriptional products of RNA polymerase II and are regulated by transcriptional factors for their differential expression. The altered expression of miRNAs in many cancer types has been explored as a marker for possible diagnosis and therapy. We report in this study that oncogenic human papillomaviruses (HPVs) induce aberrant expression of many cellular miRNAs and that HPV18 infection produces no detectable viral miRNA. Thirteen abundant host miRNAs were specifically regulated by HPV16 and HPV18 in organotypic raft cultures of foreskin and vaginal keratinocytes as determined by miRNA array in combination with small RNA sequencing. The increase of miR-16, miR-25, miR-92a, and miR-378 and the decrease of miR-22, miR-27a, miR-29a, and miR-100 could be attributed to viral oncoprotein E6, E7, or both, all of which are known to target many cellular transcription factors. The examination of 158 cervical specimens, including 38 normal, 52 cervical intraepithelial neoplasia (CIN), and 68 cervical cancer (CC) tissues, for the expression of these eight miRNAs showed a remarkable increase of miR-25, miR-92a, and miR-378 with lesion progression but no obvious change of miR-22, miR-29a, and miR-100 among the HPV-infected tissues. Further analyses indicate that an expression ratio ≥1.5 of miR-25/92a group over miR-22/29a group could serve as a cutoff value to distinguish normal cervix from CIN and from CIN to CC.

MicroRNAs (miRNAs) are excellent tumor biomarkers because of their cell-type specificity and abundance. However, many miRNA detection methods, such as real-time PCR, obliterate valuable visuospatial information in tissue samples. To enable miRNA visualization in formalin-fixed paraffin-embedded (FFPE) tissues, we developed multicolor miRNA FISH. As a proof of concept, we used this method to differentiate two skin tumors, basal cell carcinoma (BCC) and Merkel cell carcinoma (MCC), with overlapping histologic features but distinct cellular origins. Using sequencing-based miRNA profiling and discriminant analysis, we identified the tumor-specific miRNAs miR-205 and miR-375 in BCC and MCC, respectively. We addressed three major shortcomings in miRNA FISH, identifying optimal conditions for miRNA fixation and ribosomal RNA (rRNA) retention using model compounds and high-pressure liquid chromatography (HPLC) analyses, enhancing signal amplification and detection by increasing probe-hapten linker lengths, and improving probe specificity using shortened probes with minimal rRNA sequence complementarity. We validated our method on 4 BCC and 12 MCC tumors. Amplified miR-205 and miR-375 signals were normalized against directly detectable reference rRNA signals. Tumors were classified using predefined cutoff values, and all were correctly identified in blinded analysis. Our study establishes a reliable miRNA FISH technique for parallel visualization of differentially expressed miRNAs in FFPE tumor tissues.

Human LIN28A and LIN28B are RNA-binding proteins (RBPs) conserved in animals with important roles during development and stem cell reprogramming. We used Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation (PAR-CLIP) in HEK293 cells and identified a largely overlapping set of ∼3000 mRNAs at ∼9500 sites located in the 3' UTR and CDS. In vitro and in vivo, LIN28 preferentially bound single-stranded RNA containing a uridine-rich element and one or more flanking guanosines and appeared to be able to disrupt base-pairing to access these elements when embedded in predicted secondary structure. In HEK293 cells, LIN28 protein binding mildly stabilized target mRNAs and increased protein abundance. The top targets were its own mRNAs and those of other RBPs and cell cycle regulators. Alteration of LIN28 protein levels also negatively regulated the abundance of some but not all let-7 miRNA family members, indicating sequence-specific binding of let-7 precursors to LIN28 proteins and competition with cytoplasmic miRNA biogenesis factors.

Background: MicroRNAs are a population of short non-coding RNAs with widespread negative regulatory impact on mRNA translation. Unrestricted somatic stem cells (USSC) are a rare population in human cord blood that can be induced into cells representative of all three germinal layers. Here we analyzed the functional impact of miRNAs on the osteogenic differentiation in USSC., Results: Gene expression profiling identified 20 microRNAs that were consistently upregulated during osteogenic differentiation of two different USSC cell lines (SA5/73 and SA8/25). Bioinformatic target gene prediction indicated that among these microRNAs, miR-10a, -22, -26a, -26b, and -29b recognize transcripts that encode a set of proteins inhibiting osteogenesis. We subsequently verified osteo-inhibitory CDK6, CTNNBIP1, HDAC4, and TOB1 and osteo-promoting SMAD1 as targets of these microRNAs. In Western blot analyses demonstrated that endogenous levels of CDK6 and HDAC4 were downregulated during osteogenic differentiation of USSC and reduced following ectopic expression of miR-26a/b and miR-29b. In contrast, endogenous expression of SMAD1, targeted by miR-26a/b, was unaltered during osteogenic differentiation of USSC or following ectopic expression of miR-26a/b. Functional overexpression analyses using microRNA mimics revealed that miR-26a/b, as well as miR-29b strongly accelerated osteogenic differentiation of USSC as assessed by Alizarin-Red staining and calcium-release assays., Conclusions: miR-26a/b and miR-29b are upregulated during osteogenic differentiation of USSC and share target genes inhibiting osteogenesis. Furthermore, these microRNAs accelerate osteogenic differentiation, likely mediated by osteo-inhibitory proteins such as CDK6 and HDAC4.

Background: MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression post-transcriptionally in a wide range of biological processes. The zebra finch (Taeniopygia guttata), an oscine songbird with characteristic learned vocal behavior, provides biologists a unique model system for studying vocal behavior, sexually dimorphic brain development and functions, and comparative genomics., Results: We deep sequenced small RNA libraries made from the brain, heart, liver, and muscle tissues of adult male and female zebra finches. By mapping the sequence reads to the zebra finch genome and to known miRNAs in miRBase, we annotated a total of 193 miRNAs. Among them, 29 (15%) are avian specific, including three novel zebra finch specific miRNAs. Many of the miRNAs exhibit sequence heterogeneity including length variations, untemplated terminal nucleotide additions, and internal substitution events occurring at the uridine nucleotide within a GGU motif. We also identified seven Z chromosome-encoded miRNAs. Among them, miR-2954, an avian specific miRNA, is expressed at significantly higher levels in males than in females in all tissues examined. Target prediction analysis reveals that miR-2954, but not other Z-linked miRNAs, preferentially targets Z chromosome-encoded genes, including several genes known to be expressed in a sexually dimorphic manner in the zebra finch brain., Conclusions: Our genome-wide systematic analysis of mature sequences, genomic locations, evolutionary sequence conservation, and tissue expression profiles of the zebra finch miRNA repertoire provides a valuable resource to the research community. Our analysis also reveals a miRNA-mediated mechanism that potentially regulates sex-biased gene expression in avian species.

The characterization of post-transcriptional gene regulation by small regulatory RNAs of 20-30 nt length, particularly miRNAs and piRNAs, has become a major focus of research in recent years. A prerequisite for the characterization of small RNAs is their identification and quantification across different developmental stages, normal and diseased tissues, as well as model cell lines. Here we present a step-by-step protocol for the bioinformatic analysis of barcoded cDNA libraries for small RNA profiling generated by Illumina sequencing, thereby facilitating miRNA and other small RNA profiling of large sample collections., (Copyright © 2012 Elsevier Inc. All rights reserved.)

miRNAs are short (20-23 nt) RNAs that are loaded into proteins of the Argonaute (AGO) family and guide them to partially complementary target sites on mRNAs, resulting in mRNA destabilization and/or translational repression. It is estimated that about 60% of the mammalian genes are potentially regulated by miRNAs, and therefore methods for experimental miRNA target determination have become valuable tools for the characterization of posttranscriptional gene regulation. Here we present a step-by-step protocol and guidelines for the computational analysis for the large-scale identification of miRNA target sites in cultured cells by photoactivatable ribonucleoside enhanced crosslinking and immunoprecipitation (PAR-CLIP) of AGO proteins., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The characterization of post-transcriptional gene regulation by small regulatory (20-30 nt) RNAs, particularly miRNAs and piRNAs, has become a major focus of research in recent years. A prerequisite for characterizing small RNAs is their identification and quantification across different developmental stages, and in normal and disease tissues, as well as model cell lines. Here we present a step-by-step protocol for generating barcoded small RNA cDNA libraries compatible with Illumina HiSeq sequencing, thereby facilitating miRNA and other small RNA profiling of large sample collections., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Angiosarcomas (ASs) represent a heterogeneous group of malignant vascular tumors that may occur spontaneously as primary tumors or secondarily after radiation therapy or in the context of chronic lymphedema. Most secondary ASs have been associated with MYC oncogene amplification, whereas the role of MYC abnormalities in primary AS is not well defined. Twenty-two primary and secondary ASs were analyzed by array-comparative genomic hybridization (aCGH) and by deep sequencing of small RNA libraries. By aCGH and subsequently confirmed by fluorescence in situ hybridization, MYC amplification was identified in three out of six primary tumors and in 8 out of 12 secondary AS. We have also found MAML1 as a new potential oncogene in MYC-amplified AS. Significant upregulation of the miR-17-92 cluster was observed in MYC-amplified AS compared to AS lacking MYC amplification and the control group (other vascular tumors, nonvascular sarcomas). Moreover, MYC-amplified ASs were associated with a significantly lower expression of thrombospondin-1 (THBS1) than AS without MYC amplification or controls. Altogether, our study implicates MYC amplification not only in the pathogenesis of secondary AS but also in a subset of primary AS. Thus, MYC amplification may play a crucial role in the angiogenic phenotype of AS through upregulation of the miR-17-92 cluster, which subsequently downregulates THBS1, a potent endogenous inhibitor of angiogenesis., (Copyright © 2012 Wiley Periodicals, Inc.)

Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus linked to a number of B cell cancers and lymphoproliferative disorders. During latent infection, EBV expresses 25 viral pre-microRNAs (miRNAs) and induces the expression of specific host miRNAs, such as miR-155 and miR-21, which potentially play a role in viral oncogenesis. To date, only a limited number of EBV miRNA targets have been identified; thus, the role of EBV miRNAs in viral pathogenesis and/or lymphomagenesis is not well defined. Here, we used photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) combined with deep sequencing and computational analysis to comprehensively examine the viral and cellular miRNA targetome in EBV strain B95-8-infected lymphoblastoid cell lines (LCLs). We identified 7,827 miRNA-interaction sites in 3,492 cellular 3'UTRs. 531 of these sites contained seed matches to viral miRNAs. 24 PAR-CLIP-identified miRNA:3'UTR interactions were confirmed by reporter assays. Our results reveal that EBV miRNAs predominantly target cellular transcripts during latent infection, thereby manipulating the host environment. Furthermore, targets of EBV miRNAs are involved in multiple cellular processes that are directly relevant to viral infection, including innate immunity, cell survival, and cell proliferation. Finally, we present evidence that myc-regulated host miRNAs from the miR-17/92 cluster can regulate latent viral gene expression. This comprehensive survey of the miRNA targetome in EBV-infected B cells represents a key step towards defining the functions of EBV-encoded miRNAs, and potentially, identifying novel therapeutic targets for EBV-associated malignancies.

Primary effusion lymphoma (PEL) is caused by Kaposi's sarcoma-associated herpesvirus (KSHV) and frequently also harbors Epstein-Barr virus (EBV). The expression of KSHV- and EBV-encoded microRNAs (miRNAs) in PELs suggests a role for these miRNAs in latency and lymphomagenesis. Using PAR-CLIP, a technology which allows the direct and transcriptome-wide identification of miRNA targets, we delineate the target sites for all viral and cellular miRNAs expressed in PEL cell lines. The resulting data set revealed that KSHV miRNAs directly target more than 2000 cellular mRNAs, including many involved in pathways relevant to KSHV pathogenesis. Moreover, 58% of these mRNAs are also targeted by EBV miRNAs, via distinct binding sites. In addition to a known viral analog of cellular miR-155, we show that KSHV encodes a viral miRNA that mimics cellular miR-142-3p function. In summary, this study identifies an extensive list of KSHV miRNA targets, which are likely to influence viral replication and pathogenesis., (Copyright © 2011 Elsevier Inc. All rights reserved.)

MicroRNAs are important regulators in many cellular processes, including stem cell self-renewal. Recent studies demonstrated their function as pluripotency factors with the capacity for somatic cell reprogramming. However, their role in human embryonic stem (ES) cells (hESCs) remains poorly understood, partially due to the lack of genome-wide strategies to identify their targets. Here, we performed comprehensive microRNA profiling in hESCs and in purified neural and mesenchymal derivatives. Using a combination of AGO cross-linking and microRNA perturbation experiments, together with computational prediction, we identified the targets of the miR-302/367 cluster, the most abundant microRNAs in hESCs. Functional studies identified novel roles of miR-302/367 in maintaining pluripotency and regulating hESC differentiation. We show that in addition to its role in TGF-β signaling, miR-302/367 promotes bone morphogenetic protein (BMP) signaling by targeting BMP inhibitors TOB2, DAZAP2, and SLAIN1. This study broadens our understanding of microRNA function in hESCs and is a valuable resource for future studies in this area.

Liposarcoma remains the most common mesenchymal cancer, with a mortality rate of 60% among patients with this disease. To address the present lack of therapeutic options, we embarked upon a study of microRNA (miRNA) expression alterations associated with liposarcomagenesis with the goal of exploiting differentially expressed miRNAs and the gene products they regulate as potential therapeutic targets. MicroRNA expression was profiled in samples of normal adipose tissue, well-differentiated liposarcoma, and dedifferentiated liposarcoma by both deep sequencing of small RNA libraries and hybridization-based Agilent microarrays. The expression profiles discriminated liposarcoma from normal adipose tissue and well differentiated from dedifferentiated disease. We defined over 40 miRNAs that were dysregulated in dedifferentiated liposarcomas in both the sequencing and the microarray analysis. The upregulated miRNAs included two cancer-associated species (miR-21 and miR-26a), and the downregulated miRNAs included two species that were highly abundant in adipose tissue (miR-143 and miR-145). Restoring miR-143 expression in dedifferentiated liposarcoma cells inhibited proliferation, induced apoptosis, and decreased expression of BCL2, topoisomerase 2A, protein regulator of cytokinesis 1 (PRC1), and polo-like kinase 1 (PLK1). The downregulation of PRC1 and its docking partner PLK1 suggests that miR-143 inhibits cytokinesis in these cells. In support of this idea, treatment with a PLK1 inhibitor potently induced G(2)-M growth arrest and apoptosis in liposarcoma cells. Taken together, our findings suggest that miR-143 re-expression vectors or selective agents directed at miR-143 or its targets may have therapeutic value in dedifferentiated liposarcoma., (©2011 AACR.)

Sequencing of small RNA cDNA libraries is an important tool for the discovery of new RNAs and the analysis of their mutational status as well as expression changes across samples. It requires multiple enzyme-catalyzed steps, including sequential oligonucleotide adapter ligations to the 3' and 5' ends of the small RNAs, reverse transcription (RT), and PCR. We assessed biases in representation of miRNAs relative to their input concentration, using a pool of 770 synthetic miRNAs and 45 calibrator oligoribonucleotides, and tested the influence of Rnl1 and two variants of Rnl2, Rnl2(1-249) and Rnl2(1-249)K227Q, for 3'-adapter ligation. The use of the Rnl2 variants for adapter ligations yielded substantially fewer side products compared with Rnl1; however, the benefits of using Rnl2 remained largely obscured by additional biases in the 5'-adapter ligation step; RT and PCR steps did not have a significant impact on read frequencies. Intramolecular secondary structures of miRNA and/or miRNA/3'-adapter products contributed to these biases, which were highly reproducible under defined experimental conditions. We used the synthetic miRNA cocktail to derive correction factors for approximation of the absolute levels of individual miRNAs in biological samples. Finally, we evaluated the influence of 5'-terminal 5-nt barcode extensions for a set of 20 barcoded 3' adapters and observed similar biases in miRNA read distribution, thereby enabling cost-saving multiplex analysis for large-scale miRNA profiling.

MicroRNAs (miRNA) regulate many genes critical for tumorigenesis. We profiled miRNAs from 11 normal breast tissues, 17 noninvasive, 151 invasive breast carcinomas, and 6 cell lines by in-house-developed barcoded Solexa sequencing. miRNAs were organized in genomic clusters representing promoter-controlled miRNA expression and sequence families representing seed sequence-dependent miRNA target regulation. Unsupervised clustering of samples by miRNA sequence families best reflected the clustering based on mRNA expression available for this sample set. Clustering and comparative analysis of miRNA read frequencies showed that normal breast samples were separated from most noninvasive ductal carcinoma in situ and invasive carcinomas by increased miR-21 (the most abundant miRNA in carcinomas) and multiple decreased miRNA families (including miR-98/let-7), with most miRNA changes apparent already in the noninvasive carcinomas. In addition, patients that went on to develop metastasis showed increased expression of mir-423, and triple-negative breast carcinomas were most distinct from other tumor subtypes due to upregulation of the mir~17-92 cluster. However, absolute miRNA levels between normal breast and carcinomas did not reveal any significant differences. We also discovered two polymorphic nucleotide variations among the more abundant miRNAs miR-181a (T19G) and miR-185 (T16G), but we did not identify nucleotide variations expected for classical tumor suppressor function associated with miRNAs. The differentiation of tumor subtypes and prediction of metastasis based on miRNA levels is statistically possible but is not driven by deregulation of abundant miRNAs, implicating far fewer miRNAs in tumorigenic processes than previously suggested., (©2011 AACR.)

MicroRNAs (miRNAs) have been shown to play an important role in hematopoiesis. To elucidate the role of miRNAs in the early steps of hematopoiesis, we directly compared donor-matched CD133(+) cells with the more differentiated CD34(+) CD133(-) and CD34(-) CD133(-) cells from bone marrow on the miRNA and mRNA level. Using quantitative whole genome miRNA microarray and sequencing-based profiling, we found that between 109 (CD133(+) ) and 216 (CD34(-) CD133(-) ) miRNAs were expressed. Quantification revealed that the 25 highest expressed miRNAs accounted for 73% of the total miRNA pool. miR-142-3p was the highest expressed miRNA with up to 2,000 copies per cell in CD34(+) CD133(-) cells. Eighteen miRNAs were significantly differentially expressed between CD133(+) and CD34(+) CD133(-) cells. We analyzed their biological role by examining the coexpression of miRNAs and its bioinformatically predicted mRNA targets and luciferase-based reporter assays. We provide the first evidence for a direct regulation of CD133 by miR-142-3p as well as tropomyosin 1 and frizzled homolog 5 by miR-29a. Overexpression of miRNAs in CD133(+) cells demonstrated that miR-142-3p has a negative influence on the overall colony-forming ability. In conclusion, the miRNAs expressed differentially between the CD133(+) and CD34(+) CD133(-) cells are involved in inhibition of differentiation, prevention of apoptosis, and cytoskeletal remodeling. These results are highly relevant for stem cell-based therapies with CD133(+) cells and delineate for the first time how the stem cell character of CD133(+) cells is defined by the expression of specific miRNAs., (Copyright © 2011 AlphaMed Press.)

Background: MicroRNAs are short (∼22 nt) non-coding regulatory RNAs that control gene expression at the post-transcriptional level. Here the functional impact of microRNAs on cell cycle arrest during neuronal lineage differentiation of unrestricted somatic stem cells from human cord blood (USSC) was analyzed., Methodology/principal Findings: Expression profiling revealed downregulation of microRNAs miR-17, -20a, and -106b in USSC differentiated into neuronal lineage but not in USSC differentiated into osteogenic lineage. Transfection experiments followed by Ki67 immunostainings demonstrated that each of these microRNAs was able to promote proliferation of native USSC and to prevent in part cell cycle arrest during neuronal lineage differentiation of USSC. Bioinformatic target gene predictions followed by experimental target gene validations revealed that miR-17, -20a, and -106b act in a common manner by downregulating an overlapping set of target genes mostly involved in regulation and execution of G(1)/S transition. Pro-proliferative target genes cyclinD1 (CCND1) and E2F1 as well as anti-proliferative targets CDKN1A (p21), PTEN, RB1, RBL1 (p107), RBL2 (p130) were shown as common targets for miR-17, -20a, and -106b. Furthermore, these microRNAs also downregulate WEE1 which is involved in G(2)/M transition. Most strikingly, miR-17, -20a, and -106b were found to promote cell proliferation by increasing the intracellular activity of E2F transcription factors, despite the fact that miR-17, -20a, and -106b directly target the transcripts that encode for this protein family., Conclusions/significance: Mir-17, -20a, and -106b downregulate a common set of pro- and anti-proliferative target genes to impact cell cycle progression of USSC and increase intracellular activity of E2F transcription factors to govern G(1)/S transition.

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs) that are often expressed in a cell-type dependently. To understand how the interplay of these RNA-binding factors affects the regulation of individual transcripts, high resolution maps of in vivo protein-RNA interactions are necessary. A combination of genetic, biochemical and computational approaches are typically applied to identify RNA-RBP or RNA-RNP interactions. Microarray profiling of RNAs associated with immunopurified RBPs (RIP-Chip) defines targets at a transcriptome level, but its application is limited to the characterization of kinetically stable interactions and only in rare cases allows to identify the RBP recognition element (RRE) within the long target RNA. More direct RBP target site information is obtained by combining in vivo UV crosslinking with immunoprecipitation followed by the isolation of crosslinked RNA segments and cDNA sequencing (CLIP). CLIP was used to identify targets of a number of RBPs. However, CLIP is limited by the low efficiency of UV 254 nm RNA-protein crosslinking, and the location of the crosslink is not readily identifiable within the sequenced crosslinked fragments, making it difficult to separate UV-crosslinked target RNA segments from background non-crosslinked RNA fragments also present in the sample. We developed a powerful cell-based crosslinking approach to determine at high resolution and transcriptome-wide the binding sites of cellular RBPs and miRNPs that we term PAR-CliP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) (see Fig. 1A for an outline of the method). The method relies on the incorporation of photoreactive ribonucleoside analogs, such as 4-thiouridine (4-SU) and 6-thioguanosine (6-SG) into nascent RNA transcripts by living cells. Irradiation of the cells by UV light of 365 nm induces efficient crosslinking of photoreactive nucleoside-labeled cellular RNAs to interacting RBPs. Immunoprecipitation of the RBP of interest is followed by isolation of the crosslinked and coimmunoprecipitated RNA. The isolated RNA is converted into a cDNA library and deep sequenced using Solexa technology. One characteristic feature of cDNA libraries prepared by PAR-CliP is that the precise position of crosslinking can be identified by mutations residing in the sequenced cDNA. When using 4-SU, crosslinked sequences thymidine to cytidine transition, whereas using 6-SG results in guanosine to adenosine mutations. The presence of the mutations in crosslinked sequences makes it possible to separate them from the background of sequences derived from abundant cellular RNAs. Application of the method to a number of diverse RNA binding proteins was reported in Hafner et al.

RNA transcripts are subject to posttranscriptional gene regulation involving hundreds of RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs) expressed in a cell-type dependent fashion. We developed a cell-based crosslinking approach to determine at high resolution and transcriptome-wide the binding sites of cellular RBPs and miRNPs. The crosslinked sites are revealed by thymidine to cytidine transitions in the cDNAs prepared from immunopurified RNPs of 4-thiouridine-treated cells. We determined the binding sites and regulatory consequences for several intensely studied RBPs and miRNPs, including PUM2, QKI, IGF2BP1-3, AGO/EIF2C1-4 and TNRC6A-C. Our study revealed that these factors bind thousands of sites containing defined sequence motifs and have distinct preferences for exonic versus intronic or coding versus untranslated transcript regions. The precise mapping of binding sites across the transcriptome will be critical to the interpretation of the rapidly emerging data on genetic variation between individuals and how these variations contribute to complex genetic diseases., (Copyright 2010 Elsevier Inc. All rights reserved.)

MicroRNAs (miRNAs) are a species of small RNAs approximately 21-23-nucleotides long that have been shown to play an important role in many different cellular, developmental, and physiological processes. Accordingly, numerous PCR-, sequencing-, or hybridization-based methods have been established to identify and quantify miRNAs. Their short length results in a high dynamic range of melting temperatures and therefore impedes a proper selection of detection probes or optimized PCR primers. While miRNA microarrays allow for massive parallel and accurate relative measurement of all known miRNAs, they have so far been less useful as an assay for absolute quantification. Here, we present a microarray-based approach for global and absolute quantification of miRNAs. The method relies on the parallel hybridization of the sample of interest labeled with Cy5 and a universal reference of 954 synthetic miRNAs in equimolar concentrations that are labeled with Cy3 on a microarray slide containing probes for all human, mouse, rat, and viral miRNAs (miRBase 12.0). Each single miRNA is quantified with respect to the universal reference canceling biases related to sequence, labeling, or hybridization. We demonstrate the accuracy of the method by various spike-in experiments. Furthermore, we quantified miRNA copy numbers in liver samples and CD34(+)/CD133(-) hematopoietic progenitor cells.

Background: In humans and mice naturally occurring CD4(+)CD25(+) regulatory T cells (nTregs) are a thymus-derived subset of T cells, crucial for the maintenance of peripheral tolerance by controlling not only potentially autoreactive T cells but virtually all cells of the adaptive and innate immune system. Recent work using Dicer-deficient mice irrevocably demonstrated the importance of miRNAs for nTreg cell-mediated tolerance., Principal Findings: DNA-Microarray analyses of human as well as murine conventional CD4(+) Th cells and nTregs revealed a strong up-regulation of mature miR-155 (microRNA-155) upon activation in both populations. Studying miR-155 expression in FoxP3-deficient scurfy mice and performing FoxP3 ChIP-Seq experiments using activated human T lymphocytes, we show that the expression and maturation of miR-155 seem to be not necessarily regulated by FoxP3. In order to address the functional relevance of elevated miR-155 levels, we transfected miR-155 inhibitors or mature miR-155 RNAs into freshly-isolated human and mouse primary CD4(+) Th cells and nTregs and investigated the resulting phenotype in nTreg suppression assays. Whereas miR-155 inhibition in conventional CD4(+) Th cells strengthened nTreg cell-mediated suppression, overexpression of mature miR-155 rendered these cells unresponsive to nTreg cell-mediated suppression., Conclusion: Investigation of FoxP3 downstream targets, certainly of bound and regulated miRNAs revealed the associated function between the master regulator FoxP3 and miRNAs as regulators itself. miR-155 is shown to be crucially involved in nTreg cell mediated tolerance by regulating the susceptibility of conventional human as well as murine CD4(+) Th cells to nTreg cell-mediated suppression.

MicroRNAs are small regulatory RNAs with many biological functions and disease associations. We showed that in situ hybridization (ISH) using conventional formaldehyde fixation results in substantial microRNA loss from mouse tissue sections, which can be prevented by fixation with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide that irreversibly immobilizes the microRNA at its 5' phosphate. We determined optimal hybridization parameters for 130 locked nucleic acid probes by recording nucleic acid melting temperature during ISH.

MicroRNAs play important roles in animal development. Numerous conditional knockout (cKO) studies of Dicer have been performed to interrogate the functions of microRNA during mammalian development. However, because Dicer was recently implicated in the biogenesis of endogenous siRNAs in mammals, it raises the question whether the Dicer cKO defects can be attributable to the loss of microRNAs. Previously, we and others conditionally targeted Dicer and identified its critical roles in embryonic skin morphogenesis. Here, we focus explicitly on microRNAs by taking a parallel strategy with Dgcr8, encoding an essential component of the microprocessor complex that is exclusively required for microRNA biogenesis. With this comparative analysis, we show definitively that the Dicer- and Dgcr8-null skin defects are both striking and indistinguishable. By deep sequencing analysis of microRNA depletion in both Dicer- and Dgcr8-null skin, we demonstrate that most abundantly expressed skin microRNAs are dependent on both Dicer and DGCR8. Our results underscore a specific importance of microRNAs in controlling mammalian skin development.

Distinct classes of small RNAs, 20-32 nucleotides long, play important regulatory roles for diverse cellular processes. It is therefore important to identify and quantify small RNAs as a function of development, tissue and cell type, in normal and disease states. Here we describe methods to prepare cDNA libraries from pools of small RNAs isolated from organisms, tissues or cells. These methods enable the identification of new members or new classes of small RNAs, and they are also suitable to obtain miRNA expression profiles based on clone count frequencies. This protocol includes the use of new deep sequencing methods (454/Roche and Solexa) to facilitate the characterization of diverse sequence pools of small RNAs.