Large-Scale Identification of Mirtrons in Arabidopsis and Rice

A new catalog of microRNA (miRNA) species called mirtrons has been discovered in animals recently, which originate from spliced introns of the gene transcripts. However, only one putative mirtron, osa-MIR1429, has been identified in rice (Oryza sativa). We employed a high-throughput sequencing (HTS) data- and structure-based approach to do a genome-wide search for the mirtron candidate in both Arabidopsis (Arabidopsis thaliana) and rice. Five and eighteen candidates were discovered in the two plants respectively. To investigate their biological roles, the targets of these mirtrons were predicted and validated based on degradome sequencing data. The result indicates that the mirtrons could guide target cleavages to exert their regulatory roles post-transcriptionally, which needs further experimental validation

Detection and Verification of Mammalian Mirtrons by Northern Blotting

microRNAs (miRNAs) have vital roles in regulating gene expression—contributing to major diseases like cancer and heart disease. Over the last decade, thousands of miRNAs have been discovered through high throughput sequencing-based annotation. Different classes have been described, as well as a great dynamic range of expression levels. While sequencing approaches provide insight into biogenesis and allow confident identification, there is a need for additional methods for validation and characterization. Northern blotting was one of the first techniques used for studying miRNAs, and remains one of the most valuable as it avoids enzymatic manipulation of miRNA transcripts. Blotting can also provide insight into biogenesis by revealing RNA processing intermediates. Compared to sequencing, however, northern blotting is a relatively insensitive technology. This creates a challenge for detecting low expressed miRNAs, particularly those produced by inefficient, non-canonical pathways. In this chapter, we describe a strategy to study such miRNAs by northern blotting that involves ectopic expression of both miRNAs and miRNA-binding Argonaute (Ago) proteins. Through use of epitope tags, this strategy also provides a convenient method for verification of small RNA competency to be loaded into regulatory complexes

Knock-Down of Core Proteins Regulating MicroRNA Biogenesis Has No Effect on Sensitivity of Lung Cancer Cells to Ionizing Radiation

Recent studies underline the important role of microRNAs (miRNA) in the development of lung cancer. The main regulators of miRNA biogenesis are the ribonucleases Drosha, Dicer and Ago2. Here the role of core proteins of miRNA biogenesis machinery in the response of human non-small and small cell lung carcinoma cell lines to treatment with ionizing radiation was assessed. We found that Drosha and Dicer were expressed at higher levels in radioresistant but not in sensitive cell lines. However, down-regulation of either Dicer or Drosha had no effect on the sensitivity of cells to irradiation. Elimination of components of the RNA-induced silencing complex Ago2 and Tudor staphylococcal nuclease also did not sensitize cells to the same treatment. Thus, modulation of miRNA biogenesis machinery is not sufficient to increase the radiosensitivity of lung tumors and other strategies are required to combat lung cancer

Cross-Mapping Events in miRNAs Reveal Potential miRNA-Mimics and Evolutionary Implications

MicroRNAs (miRNAs) have important roles in various biological processes. miRNA cross-mapping is a prevalent phenomenon where miRNA sequence originating from one genomic region is mapped to another location. To have a better understanding of this phenomenon in the human genome, we performed a detailed analysis in this paper using public miRNA high-throughput sequencing data and all known human miRNAs. We observed widespread cross-mapping events between miRNA precursors (pre-miRNAs), other non-coding RNAs (ncRNAs) and the opposite strands of pre-miRNAs by analyzing the high-throughput sequencing data. Computational analysis on all known human miRNAs also confirmed that many of them could be involved in cross-mapping events. The processing or decay of both ncRNAs and pre-miRNA opposite strand transcripts may contribute to miRNA enrichment, although some might be miRNA-mimics due to miRNA mis-annotation. Comparing to canonical miRNAs, miRNAs involved in cross-mapping events between pre-miRNAs and other ncRNAs normally had shorter lengths (17–19 nt), lower prediction scores and were classified as pseudo miRNA precursors. Notably, 4.9% of all human miRNAs could be accurately mapped to the opposite strands of pre-miRNAs, which showed that both strands of the same genomic region had the potential to produce mature miRNAs and simultaneously implied some potential miRNA precursors. We proposed that the cross-mapping events are more complex than we previously thought. Sequence similarity between other ncRNAs and pre-miRNAs and the specific stem-loop structures of pre-miRNAs may provide evolutionary implications

Shortcomings of short hairpin RNA-based transgenic RNA interference in mouse oocytes

<p>Abstract</p> <p>Background</p> <p>RNA interference (RNAi) is a powerful approach to study a gene function. Transgenic RNAi is an adaptation of this approach where suppression of a specific gene is achieved by expression of an RNA hairpin from a transgene. In somatic cells, where a long double-stranded RNA (dsRNA) longer than 30 base-pairs can induce a sequence-independent interferon response, short hairpin RNA (shRNA) expression is used to induce RNAi. In contrast, transgenic RNAi in the oocyte routinely employs a long RNA hairpin. Transgenic RNAi based on long hairpin RNA, although robust and successful, is restricted to a few cell types, where long double-stranded RNA does not induce sequence-independent responses. Transgenic RNAi in mouse oocytes based on a shRNA offers several potential advantages, including simple cloning of the transgenic vector and an ability to use the same targeting construct in any cell type.</p> <p>Results</p> <p>Here we report our experience with shRNA-based transgenic RNAi in mouse oocytes. Despite optimal starting conditions for this experiment, we experienced several setbacks, which outweigh potential benefits of the shRNA system. First, obtaining an efficient shRNA is potentially a time-consuming and expensive task. Second, we observed that our transgene, which was based on a common commercial vector, was readily silenced in transgenic animals.</p> <p>Conclusions</p> <p>We conclude that, the long RNA hairpin-based RNAi is more reliable and cost-effective and we recommend it as a method-of-choice when a gene is studied selectively in the oocyte.</p

OsPIE1, the Rice Ortholog of Arabidopsis PHOTOPERIOD-INDEPENDENT EARLY FLOWERING1, Is Essential for Embryo Development

orthologs in monocots remains unknown., respectively).Taken together, our results suggest that OsPIE1 is the rice ortholog of Arabidopsis PIE1 and plays an essential role in rice embryo development

Conserved and highly expressed tRNA derived fragments in zebrafish

Background: Small non-coding RNAs (sncRNAs) are a class of transcripts implicated in several eukaryotic regulatory mechanisms, namely gene silencing and chromatin regulation. Despite significant progress in their identification by next generation sequencing (NGS) we are still far from understanding their full diversity and functional repertoire. Results: Here we report the identification of tRNA derived fragments (tRFs) by NGS of the sncRNA fraction of zebrafish. The tRFs identified are 18–30 nt long, are derived from specific 5′ and 3′ processing of mature tRNAs and are differentially expressed during development and in differentiated tissues, suggesting that they are likely produced by specific processing rather than random degradation of tRNAs. We further show that a highly expressed tRF (5′tRF-ProCGG) is cleaved in vitro by Dicer and has silencing ability, indicating that it can enter the RNAi pathway. A computational analysis of zebrafish tRFs shows that they are conserved among vertebrates and mining of publicly available datasets reveals that some 5′tRFs are differentially expressed in disease conditions, namely during infection and colorectal cancer. Conclusions: tRFs constitute a class of conserved regulatory RNAs in vertebrates and may be involved in mechanisms of genome regulation and in some diseases. Keywords: tRNA derived fragments, Zebrafish, Small non coding RNAs, tRNAspublishe

In Vivo Expression of MHC Class I Genes Depends on the Presence of a Downstream Barrier Element

Regulation of MHC class I gene expression is critical to achieve proper immune surveillance. In this work, we identify elements downstream of the MHC class I promoter that are necessary for appropriate in vivo regulation: a novel barrier element that protects the MHC class I gene from silencing and elements within the first two introns that contribute to tissue specific transcription. The barrier element is located in intergenic sequences 3′ to the polyA addition site. It is necessary for stable expression in vivo, but has no effect in transient transfection assays. Accordingly, in both transgenic mice and stably transfected cell lines, truncation of the barrier resulted in transcriptional gene silencing, increased nucleosomal density and decreased histone H3K9/K14 acetylation and H3K4 di-methylation across the gene. Significantly, distinct sequences within the barrier element govern anti-silencing and chromatin modifications. Thus, this novel barrier element functions to maintain transcriptionally permissive chromatin organization and prevent transcriptional silencing of the MHC class I gene, ensuring it is poised to respond to immune signaling

A Latent Pro-survival Function for the Mir-290-295 Cluster in Mouse Embryonic Stem Cells

MicroRNAs (miRNAs) post-transcriptionally regulate the expression of thousands of distinct mRNAs. While some regulatory interactions help to maintain basal cellular functions, others are likely relevant in more specific settings, such as response to stress. Here we describe such a role for the mir-290-295 cluster, the dominant miRNA cluster in mouse embryonic stem cells (mESCs). Examination of a target list generated from bioinformatic prediction, as well as expression data following miRNA loss, revealed strong enrichment for apoptotic regulators, two of which we validated directly: Caspase 2, the most highly conserved mammalian caspase, and Ei24, a p53 transcriptional target. Consistent with these predictions, mESCs lacking miRNAs were more likely to initiate apoptosis following genotoxic exposure to gamma irradiation or doxorubicin. Knockdown of either candidate partially rescued this pro-apoptotic phenotype, as did transfection of members of the mir-290-295 cluster. These findings were recapitulated in a specific mir-290-295 deletion line, confirming that they reflect miRNA functions at physiological levels. In contrast to the basal regulatory roles previously identified, the pro-survival phenotype shown here may be most relevant to stressful gestations, where pro-oxidant metabolic states induce DNA damage. Similarly, this cluster may mediate chemotherapeutic resistance in a neoplastic context, making it a useful clinical target.National Institutes of Health (U.S.) (NIH grant RO1-GM34277)National Cancer Institute (U.S.) (NCI grant PO1-CA42063)National Cancer Institute (U.S.) (NCI Cancer Center Support (core) grant P30-CA14051

Dicer Is Associated with Ribosomal DNA Chromatin in Mammalian Cells

Background: RNA silencing is a common term for pathways utilizing small RNAs as sequence-specific guides to repress gene expression. Components of the RNA silencing machinery are involved in different aspects of chromatin function in numerous organisms. However, association of RNA silencing with chromatin in mammalian cells remains unclear. Methodology/Principal Findings: Immunostaining of mitotic chromosomes with antibodies visualizing either endogenous or ectopically expressed Dicer in mammalian cells revealed association of the protein with ribosomal DNA (rDNA) repeats. Chromatin immunoprecipitations and bisulfite sequencing experiments indicated that Dicer is associated with transcribed regions of both active and silenced genes in rDNA arrays of interphase chromosomes. Metabolic labeling of the mouse embryonic stem (ES) cells lacking Dicer did not reveal apparent defect in rRNA biogenesis though pre-rRNA synthesis in these cells was decreased, likely as a consequence of their slower growth caused by the loss of miRNAs. We analyzed in detail chromatin structure of rDNA but did not find any epigenetic changes at rDNA loci in Dicer 2/2 ES cells. Instead, we found that rDNA methylation is rather low in primary tissues, contrasting with rDNA methylation patterns in transformed cell lines. Conclusion/Significance: We found that Dicer, a key component of RNA silencing pathways, can be detected in association with rDNA chromatin in mammalian cells. The role of this particular localization of Dicer is not readily apparent since th