In Drosophila melanogaster the COM Locus Directs the Somatic Silencing of Two Retrotransposons through both Piwi-Dependent and -Independent Pathways

BACKGROUND: In the Drosophila germ line, repeat-associated small interfering RNAs (rasiRNAs) ensure genomic stability by silencing endogenous transposable elements. This RNA silencing involves small RNAs of 26-30 nucleotides that are mainly produced from the antisense strand and function through the Piwi protein. Piwi belongs to the subclass of the Argonaute family of RNA interference effector proteins, which are expressed in the germline and in surrounding somatic tissues of the reproductive apparatus. In addition to this germ-line expression, Piwi has also been implicated in diverse functions in somatic cells. PRINCIPAL FINDINGS: Here, we show that two LTR retrotransposons from Drosophila melanogaster, ZAM and Idefix, are silenced by an RNA silencing pathway that has characteristics of the rasiRNA pathway and that specifically recognizes and destroys the sense-strand RNAs of the retrotransposons. This silencing depends on Piwi in the follicle cells surrounding the oocyte. Interestingly, this silencing is active in all the somatic tissues examined from embryos to adult flies. In these somatic cells, while the silencing still involves the strict recognition of sense-strand transcripts, it displays the marked difference of being independent of the Piwi protein. Finally, we present evidence that in all the tissues examined, the repression is controlled by the heterochromatic COM locus. CONCLUSION: Our data shed further light on the silencing mechanism that acts to target Drosophila LTR retrotransposons in somatic cells throughout fly development. They demonstrate that different RNA silencing pathways are involved in ovarian versus other somatic tissues, since Piwi is necessary for silencing in the former tissues but is dispensable in the latter. They further demonstrate that these pathways are controlled by the heterochromatic COM locus which ensures the overall protection of Drosophila against the detrimental effects of random retrotransposon mobilization

In C. elegans, High Levels of dsRNA Allow RNAi in the Absence of RDE-4

C. elegans Dicer requires an accessory double-stranded RNA binding protein, RDE-4, to enact the first step of RNA interference, the cleavage of dsRNA to produce siRNA. While RDE-4 is typically essential for RNAi, we report that in the presence of high concentrations of trigger dsRNA, rde-4 deficient animals are capable of silencing a transgene. By multiple criteria the silencing occurs by the canonical RNAi pathway. For example, silencing is RDE-1 dependent and exhibits a decrease in the targeted mRNA in response to an increase in siRNA. We also find that high concentrations of dsRNA trigger lead to increased accumulation of primary siRNAs, consistent with the existence of a rate-limiting step during the conversion of primary to secondary siRNAs. Our studies also revealed that transgene silencing occurs at low levels in the soma, even in the presence of ADARs, and that at least some siRNAs accumulate in a temperature-dependent manner. We conclude that an RNAi response varies with different conditions, and this may allow an organism to tailor a response to specific environmental signals

PIWI Associated siRNAs and piRNAs Specifically Require the Caenorhabditis elegans HEN1 Ortholog henn-1

Small RNAs—including piRNAs, miRNAs, and endogenous siRNAs—bind Argonaute proteins to form RNA silencing complexes that target coding genes, transposons, and aberrant RNAs. To assess the requirements for endogenous siRNA formation and activity in Caenorhabditis elegans, we developed a GFP-based sensor for the endogenous siRNA 22G siR-1, one of a set of abundant siRNAs processed from a precursor RNA mapping to the X chromosome, the X-cluster. Silencing of the sensor is also dependent on the partially complementary, unlinked 26G siR-O7 siRNA. We show that 26G siR-O7 acts in trans to initiate 22G siRNA formation from the X-cluster. The presence of several mispairs between 26G siR-O7 and the X-cluster mRNA, as well as mutagenesis of the siRNA sensor, indicates that siRNA target recognition is permissive to a degree of mispairing. From a candidate reverse genetic screen, we identified several factors required for 22G siR-1 activity, including the chromatin factors mes-4 and gfl-1, the Argonaute ergo-1, and the 3′ methyltransferase henn-1. Quantitative RT–PCR of small RNAs in a henn-1 mutant and deep sequencing of methylated small RNAs indicate that siRNAs and piRNAs that associate with PIWI clade Argonautes are methylated by HENN-1, while siRNAs and miRNAs that associate with non-PIWI clade Argonautes are not. Thus, PIWI-class Argonaute proteins are specifically adapted to associate with methylated small RNAs in C. elegans

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

Analysis of MicroRNA Expression in Embryonic Developmental Toxicity Induced by MC-RR

As cynobacterial blooms frequently occur in fresh waters throughout the world, microcystins (MCs) have caused serious damage to both wildlife and human health. MCs are known to have developmental toxicity, however, the possible molecular mechanism is largely unknown. This is the first toxicological study to integrate post-transcriptomic, proteomic and bioinformatics analysis to explore molecular mechanisms for developmental toxicity of MCs in zebrafish. After being microinjected directly into embryos, MC-RR dose-dependently decreased survival rates and increased malformation rates of embryos, causing various embryo abnormalities including loss of vascular integrity and hemorrhage. Expressions of 31 microRNAs (miRNAs) and 78 proteins were significantly affected at 72 hours post-fertilisation (hpf). Expressions of miR-430 and miR-125 families were also significantly changed. The altered expressions of miR-31 and miR-126 were likely responsible for the loss of vascular integrity. MC-RR significantly reduced the expressions of a number of proteins involved in energy metabolism, cell division, protein synthesis, cytoskeleton maintenance, response to stress and DNA replication. Bioinformatics analysis shows that several aberrantly expressed miRNAs and proteins (involved in various molecular pathways) were predicted to be potential MC-responsive miRNA-target pairs, and that their aberrant expressions should be the possible molecular mechanisms for the various developmental defects caused by MC-RR

Hsmar1 transposition is sensitive to the topology of the transposon donor and the target

Hsmar1 is a member of the Tc1-mariner superfamily of DNA transposons. These elements mobilize within the genome of their host by a cut-and-paste mechanism. We have exploited the in vitro reaction provided by Hsmar1 to investigate the effect of DNA supercoiling on transposon integration. We found that the topology of both the transposon and the target affect integration. Relaxed transposons have an integration defect that can be partially restored in the presence of elevated levels of negatively supercoiled target DNA. Negatively supercoiled DNA is a better target than nicked or positively supercoiled DNA, suggesting that underwinding of the DNA helix promotes target interactions. Like other Tc1-mariner elements, Hsmar1 integrates into 5′-TA dinucleotides. The direct vicinity of the target TA provides little sequence specificity for target interactions. However, transposition within a plasmid substrate was not random and some TA dinucleotides were targeted preferentially. The distribution of intramolecular target sites was not affected by DNA topology

RNA deep sequencing reveals differential MicroRNA expression during development of sea urchin and sea star

microRNAs (miRNAs) are small (20-23 nt), non-coding single stranded RNA molecules that act as post-transcriptional regulators of mRNA gene expression. They have been implicated in regulation of developmental processes in diverse organisms. The echinoderms, Strongylocentrotus purpuratus (sea urchin) and Patiria miniata (sea star) are excellent model organisms for studying development with well-characterized transcriptional networks. However, to date, nothing is known about the role of miRNAs during development in these organisms, except that the genes that are involved in the miRNA biogenesis pathway are expressed during their developmental stages. In this paper, we used Illumina Genome Analyzer (Illumina, Inc.) to sequence small RNA libraries in mixed stage population of embryos from one to three days after fertilization of sea urchin and sea star (total of 22,670,000 reads). Analysis of these data revealed the miRNA populations in these two species. We found that 47 and 38 known miRNAs are expressed in sea urchin and sea star, respectively, during early development (32 in common). We also found 13 potentially novel miRNAs in the sea urchin embryonic library. miRNA expression is generally conserved between the two species during development, but 7 miRNAs are highly expressed in only one species. We expect that our two datasets will be a valuable resource for everyone working in the field of developmental biology and the regulatory networks that affect it. The computational pipeline to analyze Illumina reads is available at http://www.benoslab.pitt.edu/services.html. © 2011 Kadri et al

Toxicity in mice expressing short hairpin RNAs gives new insight into RNAi

Short hairpin RNAs can provide stable gene silencing via RNA interference. Recent studies have shown toxicity in vivo that appears to be related to saturation of the endogenous microRNA pathway. Will these findings limit the therapeutic use of such hairpins

Alternative initiation and splicing in dicer gene expression in human breast cells

INTRODUCTION: Dicer is a ribonuclease that mediates RNA interference both at the transcriptional and the post-transcriptional levels. Human dicer gene expression is regulated in different tissues. Dicer is responsible for the synthesis of microRNAs and short temporal (st)RNAs that regulate the expression of many genes. Thus, understanding the control of the expression of the dicer gene is essential for the appreciation of double-stranded (ds)RNA-mediated pathways of gene expression. Human dicer mRNA has many upstream open reading frames (uORFs) at the 5'-leader sequences (the nucleotide sequence between the 5'-end and the start codon of the major ORF), and we studied whether these elements at the 5'-leader sequences regulate the expression of the dicer gene. METHOD: We determined the 5'-leader sequences of the dicer mRNAs in human breast cells by 5'-RACE and S1-nuclease protection analysis. We have analyzed the functions of the 5'-leader variants by reporter gene expression in vitro and in vivo. RESULTS: We found that the dicer transcripts in human breast cells vary in the sequence of their 5'-leader sequences, and that alternative promoter selection along with alternative splicing of the 5'-terminal exons apparently generate these variations. The breast cell has at least two predominant forms of dicer mRNAs, one of which has an additional 110 nucleotides at the 5'-end. Sequence comparison revealed that the first 80 nucleotides of these mRNA isoforms are encoded by a new exon located approximately 16 kb upstream of the reported start site. There are 30 extra nucleotides added to the previously reported exon 1. The human breast cells studied predominantly express two 5'-leader variants of dicer mRNAs, one with the exons 2 and 3 (long form) and the other without them (short form). By reporter gene expression analysis we found that the exon 2 and 3 sequences at the 5'-leader sequences are greatly inhibitory for the translation of the mRNA into protein. CONCLUSION: Dicer gene expression in human breast cells is regulated by alternative promoter selection to alter the length and composition of the 5'-leader sequence of its mRNA. Furthermore, alternative splicing of its exon 2 and 3 sequences of their pre-mRNA creates a more translationally competent mRNA in these cells