Both piRNA and siRNA Pathways Are Silencing Transcripts of the Suffix Element in the Drosophila melanogaster Germline and Somatic Cells

In the Drosophila melanogaster germline, the piRNA pathway silences retrotransposons as well as other transcribed repetitive elements. Suffix is an unusual short retroelement that was identified both as an actively transcribed repetitive element and also as an element at the 3′ ends of the Drosophila non-LTR F element. The copies of suffix that are F element-independent are far more actively transcribed than their counterparts on the F element. We studied the patterns of small RNAs targeting both strands of suffix in Drosophila ovaries using an RNase protection assay and the analysis of the corresponding RNA sequences from the libraries of total small RNAs. Our results indicate that suffix sense and antisense transcripts are targeted mainly by 23–29 nucleotides in length piRNAs and also by 21 nucleotides in length siRNAs. Suffix sense transcripts actively form longer RNA species, corresponding either to partial digestion products of the RNAi and Piwi pathways or to another RNA silencing mechanism. Both sense and antisense suffix transcripts accumulated in the ovaries of homozygous spn-E, piwi and aub mutants. These results provide evidence that suffix sense and antisense transcripts in the germ line and soma are targeted by both RNAi and Piwi pathways and that a Dicer-independent pathway of biogenesis of siRNAs could exist in Drosophila cells

Genome-wide profiling of forum domains in Drosophila melanogaster

Forum domains are stretches of chromosomal DNA that are excised from eukaryotic chromosomes during their spontaneous non-random fragmentation. Most forum domains are 50–200 kb in length. We mapped forum domain termini using FISH on polytene chromosomes and we performed genome-wide mapping using a Drosophila melanogaster genomic tiling microarray consisting of overlapping 3 kb fragments. We found that forum termini very often correspond to regions of intercalary heterochromatin and regions of late replication in polytene chromosomes. We found that forum domains contain clusters of several or many genes. The largest forum domains correspond to the main clusters of homeotic genes inside BX-C and ANTP-C, cluster of histone genes and clusters of piRNAs. PRE/TRE and transcription factor binding sites often reside inside domains and do not overlap with forum domain termini. We also found that about 20% of forum domain termini correspond to small chromosomal regions where Ago1, Ago2, small RNAs and repressive chromatin structures are detected. Our results indicate that forum domains correspond to big multi-gene chromosomal units, some of which could be coordinately expressed. The data on the global mapping of forum domains revealed a strong correlation between fragmentation sites in chromosomes, particular sets of mobile elements and regions of intercalary heterochromatin

Suffix-specific RNAi Leads to Silencing of F Element in Drosophila melanogaster

Separate conserved copies of suffix, a short interspersed Drosophila retroelement (SINE), and also divergent copies in the 3′ untranslated regions of the three genes, have already been described. Suffix has also been identified on the 3′ end of the Drosophila non-LTR F element, where it forms the last conserved domain of the reverse transcriptase (RT). In our current study, we show that the separate copies of suffix are far more actively transcribed than their counterparts on the F element. Transcripts from both strands of suffix are present in RNA preparations during all stages of Drosophila development, providing the potential for the formation of double-stranded RNA and the initiation of RNA interference (RNAi). Using in situ RNA hybridization analysis, we have detected the expression of both sense and antisense suffix transcripts in germinal cells. These sense and antisense transcripts are colocalized in the primary spermatocytes and in the cytoplasm of the nurse cells, suggesting that they form double-stranded RNA. We performed further analyses of suffix-specific small RNAs using northern blotting and SI nuclease protection assays. Among the total RNA preparations isolated from embryos, larvae, pupae and flies, suffix-specific small interfering RNAs (siRNAs) were detected only in pupae. In wild type ovaries, both the siRNAs and longer suffix-specific Piwi-interacting RNAs (piRNAs) were observed, whereas in ovaries of the Dicer-2 mutant, only piRNAs were detected. We further found by 3′ RACE that in pupae and ovaries, F element transcripts lacking the suffix sequence are also present. Our data provide direct evidence that suffix-specific RNAi leads to the silencing of the relative LINE (long interspersed element), F element, and suggests that SINE-specific RNA interference could potentially downregulate a set of genes possessing SINE stretches in their 5′ or 3′ non-coding regions. These data also suggest that double stranded RNAs possessing suffix are processed by both RNAi and an additional silencing mechanism

Binase cleaves cellular noncoding RNAs and affects coding mRNAs

Bacterial RNases are promising tools for the development of anticancer drugs. Neoplastic transformation leads to enhanced accumulation of rRNA and tRNA, and altered expression of regulatory noncoding RNAs. Cleavage of RNA in cancer cells is the main reason for the cytotoxic effects of exogenic RNases. We have shown that binase, a cytotoxic ribonuclease from Bacillus intermedius, affects the total amount of intracellular RNA and the expression of proapoptotic and antiapoptotic mRNAs. For four cell lines, we visualized cellular RNA by fluorescence microscopy, and determined RNA levels, viability and apoptosis by flow cytometry. We found that the level of cellular RNA was decreased in cells that were sensitive to the cytotoxic effects of binase. The RNA level was lowered by 44% in HEK cells transfected with the hSK4 gene of the Ca 2+-activated potassium channels (HEKhSK4) and by 20% in kit-transformed myeloid progenitor FDC-P1iR1171 cells. The most significant decrease in RNA levels was registered in the subpopulations of apoptotic cells. However, the binase-induced RNA decrease did not correlate with apoptosis. Kit-transformed cells with binase-induced RNA decrease retained viability if the interleukin-dependent proliferation pathway was activated. Using quantitative RT-PCR with RNA samples isolated from the binase-treated HEKhSK4 cells, we found that the amount of mRNA of the antiapoptotic bcl-2 gene in vivo was reduced about two-fold. In contrast, expression of the proapoptotic genes p53 and hSK4 was increased 1.5-fold and 4.3-fold, respectively. These results show that binase is a regulator of RNA-dependent processes of cell proliferation and apoptosis. © 2009 FEBS

Evidence for RNA synthesis in the intergenic region between enhancer and promoter and its inhibition by insulators in Drosophila melanogaster

Uncovering the nature of communication between enhancers, promoters and insulators is important for understanding the fundamental mechanisms that ensure appropriate gene expression levels. Here we describe an approach employing transient expression of genetic luciferase reporter gene constructs with quantitative RT–PCR analysis of transcription between an enhancer and Hsp70 promoter. We tested genetic constructs containing gypsy and/or Fab7 insulators in different orientations, and an enhancer from copia LTR-retroelement [(enh)copia]. A single gypsy or Fab7 insulator inserted between the promoter and enhancer in any polarity reduced enhancer action. A pair of insulators flanking the gene in any orientation exhibited increased insulation activity. We detected promoter-independent synthesis of non-coding RNA in the intergenic region of the constructs, which was induced by the enhancer in both directions and repressed by a single insulator or a pair of insulators. These results highlight the involvement of RNA-tracking mechanisms in the communications between enhancers and promoters, which are inhibited by insulators

Ribonuclease binase apoptotic signature in leukemic Kasumi-1 cells

Cytotoxic exogenous RNases triggering apoptotic response in malignant cells have potential as anticancer drugs; surprisingly, detailed characterization of the RNase-induced apoptosis has not been conducted so far. Here we show that a cytotoxic RNase from Bacillus intermedius (binase) induces extrinsic and intrinsic apoptotic pathways in leukemic Kasumi-1 cells. The experiments were performed using TaqMan Array Human Apoptosis 96-well Plate for gene expression analysis, and flow cytometry. Cytometric studies demonstrated dissipation of the mitochondrial membrane potential, opening of mitochondrial permeability transition pores, activation of caspases, increase of intracellular Ca 2+ and decrease of reactive oxygen species levels. We found that expression of 62 apoptotic genes is up-regulated, including 16 genes that are highly up-regulated, and only one gene was found to be down-regulated. The highest, 16 fold increase of the expression level was observed for TNF gene. Highly up-regulated genes also include the non-canonical NF-κB signaling pathway and inflammatory caspases 1,4. The obtained results suggest that binase induces evolutionary acquired cellular response to a microbial agent and triggers unusual apoptosis pathway. © 2013 Elsevier Masson SAS. All rights reserved

Oncogenic c-kit transcript is a target for binase

Mutational activation of c-Kit receptor tyrosine kinase is common in acute myelogenous leukemia. One such activating point mutation is the N822K replacement in the c-Kit protein. Here we investigate the selective cytotoxic effect of binase - RNase from Bacillus intermedius - on FDC-P1-N822K cells. These cells were derived from myeloid progenitor FDC-P1 cells, in which ectopic expression of N822K c-kit gene induces interleukin-3 independent growth. In order to determine whether the sensitivity of these cells to binase is caused by the expression of c-kit oncogene, the cytotoxicity of the RNase was studied in the presence of selective inhibitor of mutated c-Kit imatinib (Gleevec). Inhibition of mutated c-Kit protein leads to the loss of cell sensitivity to the apoptotic effect of binase, while the latter still decreases the amount of cellular RNA. Using green fluorescent protein as an expression marker for the c-Kit oncoprotein, we demonstrate that the elimination of c-Kit is the key factor in selective cytotoxicity of binase. Quantitative RT-PCR with RNA samples isolated from the binase-treated FDC-P1-N822K cells shows that binase treatment results in 41% reduction in the amount of c-kit mRNA. this indicates that the transcript of the activated mutant c-kit is the target for toxic action of binase. Thus, the combination of inhibition of oncogenic protein with the destruction of its mRNA is a promising approach to eliminating malignant cells. © 2010 Landes Bioscience

Paired termini stabilize antisense RNAs and enhance conditional gene silencing in Escherichia coli

Reliable methods for conditional gene silencing in bacteria have been elusive. To improve silencing by expressed antisense RNAs (asRNAs), we systematically altered several design parameters and targeted multiple reporter and essential genes in Escherichia coli. A paired termini (PT) design, where flanking inverted repeats create paired dsRNA termini, proved effective. PTasRNAs targeted against the ackA gene within the acetate kinase-phosphotransacetylase operon (ackA-pta) triggered target mRNA decay and a 78% reduction in AckA activity with high genetic penetrance. PTasRNAs are abundant and stable and function through an RNase III independent mechanism that requires a large stoichiometric excess of asRNA. Conditional ackA silencing reduced carbon flux to acetate and increased heterologous gene expression. The PT design also improved silencing of the essential fabI gene. Full anti-fabI PTasRNA induction prevented growth and partial induction sensitized cells to a FabI inhibitor. PTasRNAs have potential for functional genomics, antimicrobial discovery and metabolic flux control

Trans-natural antisense transcripts including noncoding RNAs in 10 species: implications for expression regulation

Natural antisense transcripts are at least partially complementary to their sense transcripts. Cis-Sense/Antisense pairs (cis-SAs) have been extensively characterized and known to play diverse regulatory roles, whereas trans-Sense/Antisense pairs (trans-SAs) in animals are poorly studied. We identified long trans-SAs in human and nine other animals, using ESTs to increase coverage significantly over previous studies. The percentage of transcriptional units (TUs) involved in trans-SAs among all TUs was as high as 4.13%. Particularly 2896 human TUs (or 2.89% of all human TUs) were involved in 3327 trans-SAs. Sequence complementarities over multiple segments with predicted RNA hybridization indicated that some trans-SAs might have sophisticated RNA–RNA pairing patterns. One-fourth of human trans-SAs involved noncoding TUs, suggesting that many noncoding RNAs may function by a trans-acting antisense mechanism. TUs in trans-SAs were statistically significantly enriched in nucleic acid binding, ion/protein binding and transport and signal transduction functions and pathways; a significant number of human trans-SAs showed concordant or reciprocal expression pattern; a significant number of human trans-SAs were conserved in mouse. This evidence suggests important regulatory functions of trans-SAs. In 30 cases, trans-SAs were related to cis-SAs through paralogues, suggesting a possible mechanism for the origin of trans-SAs. All trans-SAs are available at http://trans.cbi.pku.edu.cn/