The nascent RNA binding complex SFiNX licenses piRNA-guided heterochromatin formation

Abstract

The PIWI-interacting RNA (piRNA) pathway protects animal genome integrity in part through establishing repressive heterochromatin at transposon loci. Silencing requires piRNA-guided targeting of nuclear PIWI proteins to nascent transposon transcripts, yet the subsequent molecular events are not understood. Here, we identify SFiNX (Silencing Factor interacting Nuclear eXport variant), an interdependent protein complex required for Piwimediated co-transcriptional silencing in Drosophila. SFiNX consists of Nxf2-Nxt1, a gonadspecific variant of the heterodimeric mRNA export receptor Nxf1-Nxt1, and the Piwiassociated protein Panoramix. SFiNX mutant flies are sterile and exhibit transposon derepression because piRNA-loaded Piwi is unable to establish heterochromatin. Within SFiNX, Panoramix recruits the heterochromatin effectors, while the RNA binding Nxf2 protein licenses co-transcriptional silencing. Our data reveal how Nxf2 evolved from an RNA transport receptor into a co-transcriptional silencing factor. Thus, NXF-variants, which are abundant in metazoans, can have diverse molecular functions and might have been co-opted for host genome defense more broadly.Please note: In the first version of this bioRxiv preprint, we reported CLIP-seq data for Nxf2. In the meantime, we found an unexpected de-repression of gypsy (but not mdg1) transposons in the Cas9-engineered Nxf2-GFP line. We therefore do not trust the CLIP-seq data anymore and removed it from this updated submission. We apologize for any confusion that might have been caused by this.Batki, Schnabl, Wang et al. April 28, 2019 |The PIWI-interacting RNA (piRNA) pathway protects animal genome integrity in part through establishing repressive heterochromatin at transposon loci. Silencing requires piRNAguided targeting of nuclear PIWI proteins to nascent transposon transcripts, yet the subsequent molecular events are not understood. Here, we identify SFiNX (Silencing Factor interacting Nuclear eXport variant), an interdependent protein complex required for Piwi-mediated co-transcriptional silencing in Drosophila. SFiNX consists of Nxf2-Nxt1, a gonadspecific variant of the heterodimeric mRNA export receptor Nxf1-Nxt1, and the Piwi-associated protein Panoramix. SFiNX mutant flies are sterile and exhibit transposon de-repression because piRNA-loaded Piwi is unable to establish heterochromatin. Within SFiNX, Panoramix recruits the heterochromatin effectors, while the RNA binding Nxf2 protein licenses co-transcriptional silencing. Our data reveal how Nxf2 evolved from an RNA transport receptor into a cotranscriptional silencing factor. Thus, NXF-variants, which are abundant in metazoans, can have diverse molecular functions and might have been co-opted for host genome defense more broadly.

piRNA-guided co-transcriptional silencing coopts nuclear export factors

“…RIP-seq experiments from unperturbed cells found transposon RNAs enriched only with Panx, as reported (Sienski et al, 2015), but not with Nxf2 (Figure 4—figure supplement 1F), possibly due to low substrate availability combined with an insensitive assay. These results are consistent with another recent report that did not detect transposon enrichment in Nxf2 CLIP-seq from wild-type cells (Batki et al, 2019). However, two other studies identified transposon mRNA association with Nxf2 in CLIP-seq experiments upon depletion of the previously described TGS factor, Mael (Zhao et al, 2019), or by using a stable cell line and depletion of endogenous Nxf2 (Murano et al, 2019).…”

“…Both Nxf2 and Nxt1 were previously identified in screens for piRNA-guided silencing in somatic and germline cells, and their depletion resulted in female sterility (Czech et al, 2013; Handler et al, 2013; Muerdter et al, 2013). Contrary to expectations based upon previous findings (Sienski et al, 2015; Yu et al, 2015), we saw no enrichment for Piwi by mass spectrometry (Figure 1A), results that are consistent with another recent study (Batki et al, 2019). However, co-immunoprecipitation experiments detected weak but reproducible interactions between Piwi and Panx, Nxf2, and Nxt1, but not with a negative control (Figure 1—figure supplement 1D), suggesting that low amounts of transposon substrates in unperturbed cells and/or transient associations might push Piwi below the limit of detection by less sensitive approaches.…”

Specialization of theDrosophilanuclear export family protein, Nxf3, for piRNA precursor export

“…The work presented here, and other emerging realizations (Batki et al 2019;ElMaghraby et al 2019;Fabry et al 2019;Murano et al 2019;Zhao et al 2019) begin to paint a picture in which the evolutionary pressures exerted by the need to control mobile genetic elements have resulted in exaptation and dedication of nuclear export factor family members to the piRNA pathway. This specialization has taken different forms.…”

“…This specialization has taken different forms. The most easily understood is the adaptation of Nxf3 to export a specific class of non-canonically transcribed RNAs (ElMaghraby et al 2019), whereas the precise mechanism by which Nxf2 acts as a co-transcriptional silencing factor remains more mysterious (Batki et al 2019;Fabry et al 2019;Murano et al 2019;Zhao et al 2019). Ultimately, an understanding of how these proteins have assumed new roles through evolution will require a detailed understanding of how they are recruited to particular transcripts and how they mediate their downstream effects.…”

“…S1B). Ubiquitously expressed Nxf1 is responsible for bulk mRNA export (Herold et al 2001), while ovary-enriched Nxf2 functions in piRNA-guided transcriptional transposon silencing (Batki et al 2019;Fabry et al 2019;Murano et al 2019;Zhao et al 2019). Nxf3, and the testis-specific Nxf4, have not yet been ascribed functions.…”