Production of Small Noncoding RNAs from the flamenco Locus Is Regulated by the gypsy Retrotransposon of Drosophila melanogaster

Abstract

Protective mechanisms based on RNA silencing directed against the propagation of transposable elements are highly conserved in eukaryotes. The control of transposable elements is mediated by small noncoding RNAs, which derive from transposonrich heterochromatic regions that function as small RNA-generating loci. These clusters are transcribed and the precursor transcripts are processed to generate Piwi-interacting RNAs (piRNAs) and endogenous small interfering RNAs (endo-siRNAs), which silence transposable elements in gonads and somatic tissues. The flamenco locus is a Drosophila melanogaster small RNA cluster that controls gypsy and other transposable elements, and has played an important role in understanding how small noncoding RNAs repress transposable elements. In this study, we describe a cosuppression mechanism triggered by new euchromatic gypsy insertions in genetic backgrounds carrying flamenco alleles defective in gypsy suppression. We found that the silencing of gypsy is accompanied by the silencing of other transposons regulated by flamenco, and of specific flamenco sequences from which small RNAs against gypsy originate. This cosuppression mechanism seems to depend on a post-transcriptional regulation that involves both endo-siRNA and piRNA pathways and is associated with the occurrence of developmental defects. In conclusion, we propose that new gypsy euchromatic insertions trigger a post-transcriptional silencing of gypsy sense and antisense sequences, which modifies the flamenco activity. This cosuppression mechanism interferes with some developmental processes, presumably by influencing the expression of specific genes. KEYWORDS transposon; small RNA; RNA silencing; primary transcript; ecdysis E UKARYOTIC genomes consist in part of sequences derived from a wide variety of transposable elements (TEs), some of which can mobilize to new genomic locations (de Koning et al. 2011). A genomic consequence of their mobilization is the induction of new mutations and chromosomal rearrangements that may have deleterious effects on fitness. However, they may also provide a fundamental contribution to genetic variation and evolutionary changes (Fedoroff 2012;Warren et al. 2015;Elbarbary et al. 2016;Mita and Boeke 2016). TE activation is suppressed by specific silencing mechanisms that act both at the transcriptional level, through chromatin modifications, and at the post-transcriptional level (Buchon and Vaury 2006). Piwi-interacting RNAs (piRNAs), a distinct class of 24-to 30-nt-long RNAs produced by a Dicer-independent biogenesis pathway, are involved in the recognition and selective silencing of transposons during gametogenesis (Sarot et al. 2004;Kalmykova et al. 2005). In the Drosophila ovary germline, the coordinated action of aubergine (aub), Argonaute 3 (AGO3), and piwi suppresses activity of a broad group of TEs through the formation of piRNAs involving both a primary processing and a secondary "ping-pong" amplification loop (Brennecke et al. 2007;Gunawardane et al. 2007;Li et al. 2009;Malone...

Silencing of Euchromatic Transposable Elements as a Consequence of Nuclear Lamina Dysfunction

“…With this aim, we analyzed the mortality rate of Lam 4643/K2 mutant flies carrying a gypsy insertion in the cut locus (ct A allele). This gypsy insertion was isolated among flam A individuals, and the resulting mutant has the same genetic background as the original flam A strain [31]. We found that the ct A allele in heterozygous condition slightly but significantly increases mortality ( Figure 1, compare column 5 with 7), while in homozygous condition it greatly increases the mortality rate of the pharate (Figure 1, column 10).…”

“…However, we considered the possibility that this phenotype could depend on the genetic background. In a previous report, we showed that pharate mortality during the eclosion process can be induced by euchromatic insertions of the gypsy retrotransposon in genetic backgrounds that are permissive for gypsy transposition [31]. To test a possible involvement of gypsy, the X chromosomes of the original Lam 4643/K2 flies, derived from the wild type Canton S strain, were replaced with the X chromosome carrying flam A , a permissive allele of flamenco.…”

“…To test a possible involvement of gypsy, the X chromosomes of the original Lam 4643/K2 flies, derived from the wild type Canton S strain, were replaced with the X chromosome carrying flam A , a permissive allele of flamenco. The locus flamenco is a well characterized sncRNA cluster controlling gypsy and other retrotransposons [31]. In this genetic background, Lam inactivation induces a low but significant mortality rate during the pharate stage (Figure 1, columns 5 and 6).…”

“…We found that the ct A allele in heterozygous condition slightly but significantly increases mortality ( Figure 1, compare column 5 with 7), while in homozygous condition it greatly increases the mortality rate of the pharate (Figure 1, column 10). In addition, we tested a second gypsy insertion isolated in the flam A genetic background but in a different locus [31], obtaining a significant increase in pharate mortality when the mutation was in hemizygosis or homozygosis (Figure S1A, columns 4 and 5). These data suggest that mortality of pharate adults found in Lam mutant flies is linked to the presence of active gypsy elements in euchromatic loci.…”

Evolutionary landscape of mosquito viral integrations

“…piRNA clusters are dynamic components of the Drosophila melanogaster genome and their structural composition has been linked to differential regulatory abilities. For instance, the ability of the D. melanogaster master piRNA locus flamenco to control transposons such as gypsy , ZAM and /defix was shown to be dependent on frequent chromosomal rearrangements, loss or gain of fragmented TE sequences (Zanni et al 2013; Guida et al 2016). Additionally, variations in the composition of subtelomeric piRNA clusters were observed upon adaptation to laboratory conditions of D. melanogaster wild collected flies (Asif-Laidin et al 2017).…”