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

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

Expression patterns of <i>suffix</i> and F elements in <i>Drosophila menalogaster</i> ovaries.

<p><i>In situ</i> hybridizations with DIG-labeled, strand-specific RNA probes were performed (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000476#s4" target="_blank">Materials Methods</a>). (A, B) The patterns revealed by <i>suffix</i> sense and antisense probes, respectively. (C, D) The patterns revealed by F element sense and antisense probes, respectively. Black arrows indicate the transcripts detected in the cytoplasm of nurse cells. White arrows denote F element transcripts in follicle cells.</p

Northern blot analysis of F element-specific transcripts.

<p>Hybridizations were performed with F element-specific antisense or sense [³²P]-labeled RNA probes. The labeling is as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000476#pone-0000476-g002" target="_blank"><u>Figure 2</u></a>.</p

Hypothetical model for the concerted regulation of genes containing a SINE in their 5′ or 3′ non coding regions.

<p>Symmetrical transcription of a SINE sequence, co-transcribed as part of a number of larger RNA molecules, leads to the formation of dsRNA and thus to RNAi. As a result, SINE-specific siRNA molecules target the SINE-containing mRNAs, leading to the cleavage of the 5′ or 3′ non coding stretches that are important for gene expression and thus result in the concerted silencing of the SINE-containing genes.</p

Analysis of <i>suffix</i>-specific small RNAs by Northern blot analysis or SI nuclease protection assay.

<p>Numbers indicate the lengths of the RNA molecules in nt. M indicates RNA markers, containing a mixture of intact 25 and 14 nt RNAs and a fragmented 25 nt or 86 nt RNAs (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000476#s4" target="_blank">Materials and Methods</a>). (A) Blots containing fractionated total RNA samples isolated from embryos (E), larvae (L), pupae (P) and imago (I), were probed by <i>suffix</i>-specific antisense [³²P]-labeled RNA probes (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000476#pone-0000476-g001" target="_blank"><u>Figure 1</u></a>). The same results were obtained with an identical blot after hybridization with a <i>suffix</i>-specific sense [³²P]-labeled RNA probe (not shown). The total <i>Drosophila</i> RNA content on lanes is validated by hybridization with a 123 nt 5.8S RNA-specific probe (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000476#s4" target="_blank">Materials and Methods</a>). (B) SI nuclease protection assay of total <i>Drosophila</i> RNA isolated from <i>dcr²/dcr²</i> ovaries (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000476#s4" target="_blank">Materials and Methods</a>). [³²P]-labeled 86 nt antisense RNA probe corresponding to 5′ region of the <i>suffix</i> was hybridized overnight with 5 µg of total RNA (1) or with 5 µg of yeast tRNA (3). After SI nuclease digestion at 20°C the probes were separated on 12% denaturing polyacrylamide gel. 2–result obtained with 5 µg of total <i>Drosophila</i> RNA without hybridization (overnight incubation at 0°C). M–RNA marker. (C) SI nuclease protection assay of total <i>Drosophila</i> RNA isolated from the wild type ovaries. Indications are the same as in (B). Regions corresponding to siRNAs and piRNAs are shown in brackets.</p

Northern blot analysis of <i>suffix</i>-specific transcripts.

<p>Hybridizations were performed with <i>suffix</i>-specific antisense (as) or sense (s) [³²P]-labeled RNA probes. The lanes with poly(A)⁺ and poly(A)⁻ RNA samples, isolated from <i>Drosophila</i> embryos (E), larvae (L), pupae (P) and imago (I) are shown. Positions of the RNA markers in nt are shown on the right. The band of around 300 nt in length is shown on the left. Quantitation of the RNA content by hybridization with an rp49-specific probe is shown at the bottom.</p