Complexity of murine cardiomyocyte miRNA biogenesis, sequence variant expression and function

microRNAs (miRNAs) are critical to heart development and disease. Emerging research indicates that regulated precursor processing can give rise to an unexpected diversity of miRNA variants. We subjected small RNA from murine HL-1 cardiomyocyte cells to next generation sequencing to investigate the relevance of such diversity to cardiac biology. ～40 million tags were mapped to known miRNA hairpin sequences as deposited in miRBase version 16, calling 403 generic miRNAs as appreciably expressed. Hairpin arm bias broadly agreed with miRBase annotation, although 44 miR* were unexpectedly abundant (>20% of tags); conversely, 33 -5p/-3p annotated hairpins were asymmetrically expressed. Overall, variability was infrequent at the 5' start but common at the 3' end of miRNAs (5.2% and 52.3% of tags, respectively). Nevertheless, 105 miRNAs showed marked 5' isomiR expression (>20% of tags). Among these was miR-133a, a miRNA with important cardiac functions, and we demonstrated differential mRNA targeting by two of its prevalent 5' isomiRs. Analyses of miRNA termini and base-pairing patterns around Drosha and Dicer cleavage regions confirmed the known bias towards uridine at the 5' most position of miRNAs, as well as supporting the thermodynamic asymmetry rule for miRNA strand selection and a role for local structural distortions in fine tuning miRNA processing. We further recorded appreciable expression of 5 novel miR*, 38 extreme variants and 8 antisense miRNAs. Analysis of genome-mapped tags revealed 147 novel candidate miRNAs. In summary, we revealed pronounced sequence diversity among cardiomyocyte miRNAs, knowledge of which will underpin future research into the mechanisms involved in miRNA biogenesis and, importantly, cardiac function, disease and therapy.     

The miRNA pathway intrinsically controls self-renewal of Drosophila germline stem cells

Stem cells uniquely self-renew and maintain tissue homoeostasis by differentiating into different cell types to replace aged or damaged cells [1]. During oogenesis of Drosophila melanogaster, self-renewal of germline stem cells (GSCs) requires both intrinsic signaling mechanisms and extrinsic signals from neighboring niche cells [2]. Emerging evidence suggests that microRNA (miRNA)-mediated translational regulation may also control Drosophila GSC self-renewal [3, 4]. It is unclear, however, whether the miRNA pathway functions within stem cells or niche cells to maintain GSCs. In Drosophila, Dicer-1 (Dcr-1) and the double-stranded RNA binding protein Loquacious (Loqs) catalyze miRNA biogenesis [3-5]. Here, we generate loqs knockout (loqs(KO)) flies by ends-out homologous recombination and show that loqs is essential for embryonic viability and ovarian GSC maintenance. Both developmental and miRNA processing defects are rescued by transgenic expression of Loqs-PB, but not Loqs-PA. Furthermore, mosaic germline analysis indicates that Loqs is required intrinsically for GSC maintenance. Consistently, GSCs are restored in loqs mutant ovaries by germline expression, but not somatic expression, of Loqs-PB. Together, these results demonstrate that Loqs-PB, but not Loqs-PA, is necessary and sufficient for Drosophila development and the miRNA pathway. Our study strongly suggests that miRNAs play an intrinsic, but not extrinsic, role in Drosophila female GSC self-renewal.     

Characterization of the mammalian miRNA turnover landscape

Steady state cellular microRNA (miRNA) levels represent the balance between miRNA biogenesis and turnover. The kinetics and sequence determinants of mammalian miRNA turnover during and after miRNA maturation are not fully understood. Through a large-scale study on mammalian miRNA turnover, we report the co-existence of multiple cellular miRNA pools with distinct turnover kinetics and biogenesis properties and reveal previously unrecognized sequence features for fast turnover miRNAs. We measured miRNA turnover rates in eight mammalian cell types with a combination of expression profiling and deep sequencing. While most miRNAs are stable, a subset of miRNAs, mostly miRNA*s, turnovers quickly, many of which display a two-step turnover kinetics. Moreover, different sequence isoforms of the same miRNA can possess vastly different turnover rates. Fast turnover miRNA isoforms are enriched for 5′ nucleotide bias against Argonaute-(AGO)-loading, but also additional 3′ and central sequence features. Modeling based on two fast turnover miRNA*s miR-222-5p and miR-125b-1-3p, we unexpectedly found that while both miRNA*s are associated with AGO, they strongly differ in HSP90 association and sensitivity to HSP90 inhibition. Our data characterize the landscape of genome-wide miRNA turnover in cultured mammalian cells and reveal differential HSP90 requirements for different miRNA*s. Our findings also implicate rules for designing stable small RNAs, such as siRNAs.     

TDP-43 regulates cancer-associated microRNAs

Aberrant regulation of miRNA genes contributes to pathogenesis of a wide range of human diseases, including cancer. The TAR DNA binding protein 43 (TDP-43), a RNA/DNA binding protein associated with neurodegeneration, is involved in miRNA biogenesis. Here, we systematically examined miRNAs regulated by TDP-43 using RNA-Seq coupled with an siRNA-mediated knockdown approach. TDP-43 knockdown affected the expression of a number of miRNAs. In addition, TDP-43 down-regulation led to alterations in the patterns of different isoforms of miRNAs (isomiRs) and miRNA arm selection, suggesting a previously unknown role of TDP-43 in miRNA processing. A number of TDP-43 associated miRNAs, and their candidate target genes, are associated with human cancers. Our data reveal highly complex roles of TDP-43 in regulating different miRNAs and their target genes. Our results suggest that TDP-43 may promote migration of lung cancer cells by regulating miR-423-3p. In contrast, TDP-43 increases miR-500a-3p expression and binds to the mature miR-500a-3p sequence. Reduced expression of miR-500a-3p is associated with poor survival of lung cancer patients, suggesting that TDP-43 may have a suppressive role in cancer by regulating miR-500a-3p. Cancer-associated genes LIF and PAPPA are possible targets of miR-500a-3p. Our work suggests that TDP-43-regulated miRNAs may play multifaceted roles in the pathogenesis of cancer.     

The Arabidopsis nucleotidyl transferase HESO1 uridylates unmethylated small RNAs to trigger their degradation

MicroRNAs (miRNAs), small interfering RNAs (siRNAs), and piwi-interacting RNAs (piRNAs) impact numerous biological processes in eukaryotes. In addition to biogenesis, turnover contributes to the steady-state levels of small RNAs. One major factor that stabilizes miRNAs and siRNAs in plants as well as siRNAs and piRNAs in animals is 2'-O-methylation on the 3' terminal ribose by the methyltransferase HUA ENHANCER1 (HEN1) [1-6]. Genetic studies with Arabidopsis, Drosophila, and zebrafish hen1 mutants show that 2'-O-methylation protects small RNAs from 3'-to-5' truncation and 3' uridylation, the addition of nontemplated nucleotides, predominantly uridine [2, 7, 8]. Uridylation is a widespread phenomenon that is not restricted to small RNAs in hen1 mutants and is often associated with their reduced accumulation ([7, 9, 10]; reviewed in [11]). The enzymes responsible for 3' uridylation of small RNAs when they lack methylation in plants or animals have remained elusive. Here, we identify the Arabidopsis HEN1 SUPPRESSOR1 (HESO1) gene as responsible for small RNA uridylation in hen1 mutants. HESO1 exhibits terminal nucleotidyl transferase activity, prefers uridine as the substrate nucleotide, and is completely inhibited by 2'-O-methylation. We show that uridylation leads to miRNA degradation, and the degradation is most likely through an enzyme that is distinct from that causing the 3' truncation in hen1 mutants.     

miRNA profiling of naïve, effector and memory CD8 T cells

microRNAs have recently emerged as master regulators of gene expression during development and cell differentiation. Although profound changes in gene expression also occur during antigen-induced T cell differentiation, the role of miRNAs in the process is not known. We compared the miRNA expression profiles between antigen-specific na?ve, effector and memory CD8+ T cells using 3 different methods--small RNA cloning, miRNA microarray analysis and real-time PCR. Although many miRNAs were expressed in all the T cell subsets, the frequency of 7 miRNAs (miR-16, miR-21, miR-142-3p, miR-142-5p, miR-150, miR-15b and let-7f) alone accounted for approximately 60% of all miRNAs, and their expression was several fold higher than the other expressed miRNAs. Global downregulation of miRNAs (including 6/7 dominantly expressed miRNAs) was observed in effector T cells compared to na?ve cells and the miRNA expression levels tended to come back up in memory T cells. However, a few miRNAs, notably miR-21 were higher in effector and memory T cells compared to na?ve T cells. These results suggest that concomitant with profound changes in gene expression, miRNA profile also changes dynamically during T cell differentiation. Sequence analysis of the cloned mature miRNAs revealed an extensive degree of end polymorphism. While 3'end polymorphisms dominated, heterogeneity at both ends, resembling drosha/dicer processing shift was also seen in miR-142, suggesting a possible novel mechanism to generate new miRNA and/or to diversify miRNA target selection. Overall, our results suggest that dynamic changes in the expression of miRNAs may be important for the regulation of gene expression during antigen-induced T cell differentiation. Our study also suggests possible novel mechanisms for miRNA biogenesis and function.     

Molecular mechanisms and microRNAs in osteosarcoma pathogenesis

This review summarizes data on microRNA (miRNA) genomic organization, biogenesis, and functions in carcinogenesis. The roles of key genes and regulatory miRNAs in molecular mechanisms and signaling pathways involved in the development of osteosarcoma, the most aggressive type of bone tumor striking mainly in adolescence and early adulthood, are discussed in detail. The most critical pathways in osteosarcoma pathogenesis are the Notch, Wnt, NF-κB, p53, PI3K/Akt, and MAPK pathways. The balance between cell survival and apoptosis is determined by the Wnt and NF-κB pathways, as well as by the ratio between the activities of the MAPK and PI3K/Akt pathways. Several miRNAs (miR-21, -34a, -143, -148a, -195a, -199a-3p, -382) regulate multiple target genes, pathways, and processes essential for osteosarcoma pathogenesis. Data on the key genes and regulatory miRNAs involved in metastasis and tumor cell response to drug treatment are presented. Possible applications of miRNA in osteosarcoma diagnostics and treatment are discussed.     

Deep Sequencing of RNA from Three Different Extracellular Vesicle (EV) Subtypes Released from the Human LIM1863 Colon Cancer Cell Line Uncovers Distinct Mirna-Enrichment Signatures

Secreted microRNAs (miRNAs) enclosed within extracellular vesicles (EVs) play a pivotal role in intercellular communication by regulating recipient cell gene expression and affecting target cell function. Here, we report the isolation of three distinct EV subtypes from the human colon carcinoma cell line LIM1863 – shed microvesicles (sMVs) and two exosome populations (immunoaffinity isolated A33-exosomes and EpCAM-exosomes). Deep sequencing of miRNA libraries prepared from parental LIM1863 cells/derived EV subtype RNA yielded 254 miRNA identifications, of which 63 are selectively enriched in the EVs - miR-19a/b-3p, miR-378a/c/d, and miR-577 and members of the let-7 and miR-8 families being the most prominent. Let-7a-3p*, let-7f-1-3p*, miR-451a, miR-574-5p*, miR-4454 and miR-7641 are common to all EV subtypes, and 6 miRNAs (miR-320a/b/c/d, miR-221-3p, and miR-200c-3p) discern LIM1863 exosomes from sMVs; miR-98-5p was selectively represented only in sMVs. Notably, A33-Exos contained the largest number (32) of exclusively-enriched miRNAs; 14 of these miRNAs have not been reported in the context of CRC tissue/biofluid analyses and warrant further examination as potential diagnostic markers of CRC. Surprisingly, miRNA passenger strands (star miRNAs) for miR-3613-3p*, -362-3p*, -625-3p*, -6842-3p* were the dominant strand in A33-Exos, the converse to that observed in parental cells. This finding suggests miRNA biogenesis may be interlinked with endosomal/exosomal processing.     

The Drosophila RNA methyltransferase, DmHen1, modifies germline piRNAs and single-stranded siRNAs in RISC

Small silencing RNAs repress gene expression by a set of related mechanisms collectively called RNA-silencing pathways [1, 2]. In the RNA interference (RNAi) pathway [3], small interfering mRNA (siRNAs) defend cells from invasion by foreign nucleic acids, such as those produced by viruses. In contrast, microRNAs (miRNAs) sculpt endogenous mRNA expression [4]. A third class of small RNAs, Piwi-interacting RNAs (piRNAs), defends the genome from transposons [5-9]. Here, we report that Drosophila piRNAs contain a 2'-O-methyl group on their 3' termini; this is a modification previously reported for plant miRNAs and siRNAs [10] and mouse and rat piRNAs [11, 12, 13]. Plant small-RNA methylation is catalyzed by the protein HEN1 [10, 14, 15]. We find that DmHen1, the Drosophila homolog of HEN1, methylates the termini of siRNAs and piRNAs. Without DmHen1, the length and abundance of piRNAs are decreased, and piRNA function is perturbed. Unlike plant HEN1, DmHen1 acts on single strands, not duplexes, explaining how it can use as substrates both siRNAs-which derive from double-stranded precursors-and piRNAs-which do not [8, 13]. 2'-O-methylation of siRNAs may be the final step in assembly of the RNAi-enzyme complex, RISC, occurring after an Argonaute-bound siRNA duplex is converted to single-stranded RNA.