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MicroRNAs (miRNAs) are highly conserved ∼22 nt small noncoding RNAs that bind partially complementary sequences in target transcripts. MicroRNAs regulate both translation and transcript stability, and play important roles in development, cellular homeostasis, and disease. There are limited approaches available to agnostically identify microRNA targets transcriptome-wide, and methods using miRNA mimics, which in principle identify direct miRNA:transcript pairs, have low sensitivity and specificity. Here, we describe a novel method to identify microRNA targets using miR-29b mimics containing 3-cyanovinylcarbazole ( CNV K), a photolabile nucleoside analog. We demonstrate that biotin-tagged, CNV K-containing miR-29b ( CNV K-miR-29b) mimics are nontoxic in cell culture, associate with endogenous mammalian Argonaute2, are sensitive for known targets and recapitulate endogenous transcript destabilization. Partnering CNV K-miR-29b with ultra-low-input RNA sequencing, we recover ∼40% of known miR-29b targets and find conservation of the focal adhesion and apoptotic target pathways in mouse and human. We also identify hundreds of novel targets, including NRAS , HOXA10 , and KLF11 , with a validation rate of 71% for a subset of 73 novel target transcripts interrogated using a high-throughput luciferase assay. Consistent with previous reports, we show that both endogenous miR-29b and CNV K-miR-29b are trafficked to the nucleus, but find no evidence of nuclear-specific miR-29b transcript binding. This may indicate that miR-29b nuclear sequestration is a regulatory mechanism in itself. We suggest that CNV K-containing small RNA mimics may find applicability in other experimental models., (© 2018 Choudhary et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Background: MicroRNAs (miRNAs) are coordinators of cellular differentiation, including granulopoiesis. Although differential expression of many miRNAs is associated with the maturation of granulocytes, analysis of differentially expressed miRNAs and their cellular localization across all stages of granulopoiesis, starting from hemopoietic stems cells, is not well characterized., Methods: We analyzed whole cell miRNA and mRNA expression during granulopoiesis using Taqman low-density and Affymetrix arrays respectively. We also performed nuclear and cytoplasmic fractionation followed by Taqman low-density array and/or quantitative PCR to identify nuclear-enriched miRNAs in hemopoietic stem/progenitor cells, promyelocytes, myelocytes, granulocytes and several hemopoietic cell lines. Anti-correlation between the expression of miRNA and target pairs was used to determine putative miRNA targets., Results: Analyses of our array data revealed distinct clusters of differentially expressed miRNAs that are specific to promyelocytes and granulocytes. While the roles of many of these miRNAs in granulopoiesis are not currently known, anti-correlation of the expression of miRNA/mRNA target pairs identified a suite of novel target genes. Clusters of miRNAs (including members of the let-7 and miR-17-92 families) are downregulated in hemopoietic stem/progenitor cells, potentially allowing the expression of target genes known to facilitate stem cell proliferation and homeostasis. Additionally, four miRNAs (miR-709, miR-706, miR-690 and miR-467a*) were found to be enriched in the nucleus of myeloid cells and multiple hemopoietic cell lines compared to other miRNAs, which are predominantly cytoplasmic-enriched. Both miR-709 and miR-706 are nuclear-enriched throughout granulopoiesis and have putative binding sites of extensive complementarity downstream of pri-miRNAs. Nuclear enrichment of miR-467a* is specific to hemopoietic stem/progenitors and promyelocytes. These miRNAs are also nuclear-enriched in other hemopoietic cell lines, where nuclear sequestering may fine-tune the expression of cytoplasmic mRNA targets., Conclusions: Overall, we have demonstrated differentially expressed miRNAs that have not previously been associated with hemopoietic differentiation and provided further evidence of regulated nuclear-enrichment of miRNAs. Further studies into miRNA function in granulocyte development may shed light on fundamental aspects of regulatory RNA biology and the role of nuclear miRNAs.

MicroRNAs are important regulators of developmental gene expression, but their contribution to fetal gonad development is not well understood. We have identified the evolutionarily conserved gonadal microRNAs miR-202-5p and miR-202-3p as having a potential role in regulating mouse embryonic gonad differentiation. These microRNAs are expressed in a sexually dimorphic pattern as the primordial XY gonad differentiates into a testis, with strong expression in Sertoli cells. In vivo, ectopic expression of pri-miR-202 in XX gonads did not result in molecular changes to the ovarian determination pathway. Expression of the primary transcript of miR-202-5p/3p remained low in XY gonads in a conditional Sox9-null mouse model, suggesting that pri-miR-202 transcription is downstream of SOX9, a transcription factor that is both necessary and sufficient for male sex determination. We identified the pri-miR-202 promoter that is sufficient to drive expression in XY but not XX fetal gonads ex vivo. Mutation of SOX9 and SF1 binding sites reduced ex vivo transactivation of the pri-miR-202 promoter, demonstrating that pri-miR-202 may be a direct transcriptional target of SOX9/SF1 during testis differentiation. Our findings indicate that expression of the conserved gonad microRNA, miR-202-5p/3p, is downstream of the testis-determining factor SOX9, suggesting an early role in testis development.

MicroRNAs (miRNAs) have been shown to play key regulatory roles in a range of biological processes, including cell differentiation and development. To identify miRNAs that participate in gonad differentiation, a fundamental and tightly regulated developmental process, we examined miRNA expression profiles at the time of sex determination and during the early fetal differentiation of mouse testes and ovaries using high-throughput sequencing. We identified several miRNAs that were expressed in a sexually dimorphic pattern, including several members of the let-7 family, miR-378, and miR-140-3p. We focused our analysis on the most highly expressed, sexually dimorphic miRNA, miR-140-3p, and found that both miR-140-3p and its more lowly expressed counterpart, the previously annotated guide strand, miR-140-5p, are testis enriched and expressed in testis cords. Analysis of the miR-140-5p/miR-140-3p-null mouse revealed a significant increase in the number of Leydig cells in the developing XY gonad, strongly suggesting an important role for miR-140-5p/miR-140-3p in testis differentiation in mouse.

Bone marrow-derived endothelial progenitor cells (EPC) contribute to the angiogenesis-dependent growth of tumors in mice and humans. EPCs regulate the angiogenic switch via paracrine secretion of proangiogenic growth factors and by direct luminal incorporation into sprouting nascent vessels. miRNAs have emerged as key regulators of several cellular processes including angiogenesis; however, whether miRNAs contribute to bone marrow-mediated angiogenesis has remained unknown. Here, we show that genetic ablation of miRNA-processing enzyme Dicer, specifically in the bone marrow, decreased the number of circulating EPCs, resulting in angiogenesis suppression and impaired tumor growth. Furthermore, genome-wide deep sequencing of small RNAs revealed tumor EPC-intrinsic miRNAs including miR-10b and miR-196b, which have been previously identified as key regulators of HOX signaling and adult stem cell differentiation. Notably, we found that both miR-10b and miR-196b are responsive to vascular endothelial growth factor stimulation and show elevated expression in human high-grade breast tumor vasculature. Strikingly, targeting miR-10b and miR-196b led to significant defects in angiogenesis-mediated tumor growth in mice. Targeting these miRNAs may constitute a novel strategy for inhibiting tumor angiogenesis.

Several recent reports have demonstrated that microRNAs (miRNAs) can exhibit heterogeneous ends and post-transcriptional nontemplate 3' end additions of uridines or adenosines. Using two small RNA deep-sequencing data sets, we show here that these miRNA isoforms (isomiRs) are differentially expressed across Drosophila melanogaster development and tissues. Specifically, we demonstrate that: (1) nontemplate nucleotide additions of adenosines to miRNA 3' ends are highly abundant in early development; (2) a subset of miRNAs with nontemplate 3' Us are expressed in adult tissues; and (3) the size of at least eight "mature" (unmodified) miRNAs varies in a life-cycle or tissue-specific manner. These results suggest that subtle variability in isomiR expression, which is widely thought to be the result of inexact Dicer processing, is regulated and biologically meaningful. Indeed, a subset of the miRNAs enriched for 3' adenosine additions during early embryonic development, including miR-282 and miR-312, show enrichment for target sites in developmental genes that are expressed during late embryogenesis, suggesting that nontemplate additions increase miRNA stability or strengthen miRNA:target interactions. This work suggests that isomiR expression is an important aspect of miRNA biology, which warrants further investigation.

MicroRNAs (miRNAs) are short (20-23 nt) RNAs that are sequence-specific mediators of transcriptional and post-transcriptional regulation of gene expression. Modern high-throughput technologies enable deep sequencing of such RNA species on an unprecedented scale. We find that the analysis of small RNA deep-sequencing libraries can be affected by cross-mapping, in which RNA sequences originating from one locus are inadvertently mapped to another. Similar to cross-hybridization on microarrays, cross-mapping is prevalent among miRNAs, as they tend to occur in families, are similar or derived from repeat or structural RNAs, or are post-transcriptionally modified. Here, we develop a strategy to correct for cross-mapping, and apply it to the analysis of RNA editing in mature miRNAs. In contrast to previous reports, our analysis suggests that RNA editing in mature miRNAs is rare in animals.

Small nucleolar RNAs (snoRNAs) guide RNA modification and are localized in nucleoli and Cajal bodies in eukaryotic cells. Components of the RNA silencing pathway associate with these structures, and two recent reports have revealed that a human and a protozoan snoRNA can be processed into miRNA-like RNAs. Here we show that small RNAs with evolutionary conservation of size and position are derived from the vast majority of snoRNA loci in animals (human, mouse, chicken, fruit fly), Arabidopsis, and fission yeast. In animals, sno-derived RNAs (sdRNAs) from H/ACA snoRNAs are predominantly 20-24 nucleotides (nt) in length and originate from the 3' end. Those derived from C/D snoRNAs show a bimodal size distribution at approximately 17-19 nt and >27 nt and predominantly originate from the 5' end. SdRNAs are associated with AGO7 in Arabidopsis and Ago1 in fission yeast with characteristic 5' nucleotide biases and show altered expression patterns in fly loquacious and Dicer-2 and mouse Dicer1 and Dgcr8 mutants. These findings indicate that there is interplay between the RNA silencing and snoRNA-mediated RNA processing systems, and that sdRNAs comprise a novel and ancient class of small RNAs in eukaryotes.

MicroRNAs (miRNAs) are small ( approximately 22 nucleotides) noncoding RNAs which play an essential role in gene regulation and affect a wide range of processes, including development, differentiation, and oncogenesis. Here we report the identification of the first miRNA from an insect virus, derived from the major capsid protein (MCP) gene in Heliothis virescens ascovirus (HvAV) (HvAV-miR-1). Although MCP was abundantly expressed at all time points 24 h after infection, HvAV-miR-1 expression was strictly regulated and specifically detected from 96 h postinfection. HvAV-miR-1 expression coincided with a marked reduction of the expression of HvAV DNA polymerase I, which is a predicted target. Ectopic expression of full-length and truncated versions of MCP retaining the miRNA sequence significantly reduced DNA polymerase I transcript levels and inhibited viral replication. Our results indicate that HvAV-miR-1 directs transcriptional degradation of DNA polymerase I and regulates HvAV replication. These findings are congruent with recent reports that miR-BART-2 regulates Epstein-Barr virus DNA polymerase expression and suggest that virus-encoded miRNA regulation of virus replication may be a general phenomenon.