Interaction of RNA polymerase II and the small RNA machinery affects heterochromatic silencing in Drosophila

Abstract

BackgroundHeterochromatin is the tightly packaged dynamic region of the eukaryotic chromosome that plays a vital role in cellular processes such as mitosis and meiotic recombination. Recent experiments in Schizosaccharomyces pombe have revealed the structure of centromeric heterochromatin is affected in RNAi pathway mutants. It has also been shown in fission yeast that the heterochromatin barrier is traversed by RNA Pol II and that the passage of RNA Pol II through heterochromatin is important for heterochromatin structure. Thus, an intricate interaction between the RNAi machinery and RNA Pol II affects heterochromatin structure. However, the role of the RNAi machinery and RNA Pol II on the metazoan heterochromatin landscape is not known. This study analyses the interaction of the small RNA machinery and RNA Pol II on Drosophila heterochromatin structure.ResultsThe results in this paper show genetic and biochemical interaction between RNA Pol II (largest and second largest subunit) and small RNA silencing machinery components (dcr-2, ago1, ago2, piwi, Lip [D], aub and hls). Immunofluorescence analysis of polytene chromosomes from trans-heterozygotes of RNA Pol II and different mutations of the small RNA pathways show decreased H3K9me2 and mislocalization of Heterochromatin protein-1. A genetic analysis performed on these mutants showed a strong suppression of white-mottled4h position effect variegation. This was further corroborated by a western blot analysis and chromatin immunoprecipitation, which showed decreased H3K9me2 in trans-heterozygote mutants compared to wild type or single heterozygotes. Co-immunoprecipitation performed using Drosophila embryo extracts showed the RNA Pol II largest subunit interacting with Dcr-2 and dAGO1. Co-localization performed on polytene chromosomes showed RNA Pol II and dAGO1 overlapping at some sites.ConclusionOur experiments show a genetic and biochemical interaction between RNA Pol II (largest and second largest subunits) and the small RNA silencing machinery in Drosophila. The interaction has functional aspects in terms of determining H3K9me2 and HP-1 deposition at the chromocentric heterochromatin. Thus, RNA Pol II has an important role in establishing heterochromatin structure in Drosophila.

Separation of stem cell maintenance and transposon silencing functions of Piwi protein

“…Possibly, particular Piwiregulated transposons avoid the degradation by Aub/Ago-3 cytoplasmic piRNA machinery as a result of their masking by cytoplasmic proteins. Several reports have implicated Piwi in chromatin status maintenance (8,(39)(40)(41)(42)(43). We revealed that the loss of the nuclear Piwi in the piwi Nt ovaries enriches the chromatin context of telomeric transposons with H3K4me2 euchromatic marks and decreases the occupancy of heterochromatic marks such as H3K9me2/3 histones and HP1 protein (Fig.…”

The SelfishSegregation DistorterGene Complex ofDrosophila melanogaster

“…Some rasiRNAs are implicated in silencing heterochromatin in plants, fungi, and Drosophila, where ribonucleoprotein (RNP) complexes associated with rasiRNAs recruit silencing factors to the chromatin (Pal-Bhadra et al 2004;Verdel et al 2004;Buhler and Moazed 2007;Kavi and Birchler 2009;Wang and Elgin 2011). The most famous rasiRNA pathway in the germline is the Piwi pathway including P-elementinduced wimpy testis (Piwi), Aubergine (Aub), and Argonaute 3 (Ago3).…”

“…If SD/SD + heterozygotes suffer disrupted nuclear transport during spermatogenesis, then Rsp rasiRNAs, after associating with RNP complexes in the cytoplasm, may not be efficiently imported back into the nucleus for targeting to the Rsp satellite ( Figure 7C). Presumably the rasiRNAs direct chromatin remodeling complexes after meiosis in the testis, similar to RNAi-dependent heterochromatin formation in somatic tissues (Pal-Bhadra et al 2004;Kavi and Birchler 2009). Because Rsp s alleles have many (and Rsp i alleles have few) repeats, Rsp s -bearing spermatids might be disproportionately affected by the dearth of Rsp rasiRNAs in the nucleus and as a result fail to condense their pericentric heterochromatin (Ferree and Barbash 2007;Tao et al 2007).…”

“…The bending of satellite DNA (Radic et al 1987) and nucleosome spacing differences may have a role in facilitating the compaction of heterochromatin (Doshi et al 1991). Satellite sequences also generate rasiRNAs, whose role is not fully understood (Aravin et al 2003;Brennecke et al 2007); however, one likely role is in directing heterochromatin formation (Pal-Bhadra et al 2004;Kavi and Birchler 2009). …”

A Balance Between Euchromatic (JIL-1) and Heterochromatic [SU(VAR)2-5 and SU(VAR)3-9] Factors Regulates Position-Effect Variegation inDrosophila

“…Thus, to test whether the heterozygous JIL-1 z2 allele could counterbalance the suppression of the Su(var)3-9 06 or Su(var)2-5 05 loss-of-function alleles of the PEV of 118E-10, we compared the eye pigment levels of the various genotypes (Figures 1 and 2 and Table 1). Pigment assays were performed essentially as in Kavi and Birchler (2009) using three sets of 10 pooled fly heads from each genotype. Although both male and female flies were scored, due to sex differences only results from male flies are shown.…”