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

Abstract

The piRNA pathway is a conserved, small RNA-based immune system that protects animal germ cell genomes from the harmful effects of transposon mobilisation. In Drosophila ovaries, most piRNAs originate from dual-strand clusters, which generate piRNAs from both genomic strands. Dual-strand clusters use non-canonical transcription mechanisms. Although transcribed by RNA polymerase II, cluster transcripts lack splicing signatures and poly(A) tails. mRNA processing is important for general mRNA export mediated by Nuclear export factor 1. Although UAP56, a component of the transcription and export complex, has been implicated in piRNA precursor export, it remains unknown how dual-strand cluster transcripts are specifically targeted for piRNA biogenesis by export from the nucleus to cytoplasmic processing centers. Here we report that dual-strand cluster transcript export requires CG13741/Bootlegger and the Drosophila Nuclear export factor family protein, Nxf3. Bootlegger is specifically recruited to piRNA clusters and in turn brings Nxf3. We find that Nxf3 specifically binds to piRNA precursors and is essential for their export to piRNA biogenesis sites, a process that is critical for germline transposon silencing. Our data shed light on how dual-strand clusters compensate for a lack of canonical features of mature mRNAs to be specifically exported via Nxf3, ensuring proper piRNA production.Kneuss et al., page 3 We thank Martin H. Fabry for help with computational analyses. We thank the CRUK Cambridge Institute Bioinformatics, Genomics, Microscopy, Research Instrumentation and Cell Services, and Proteomics Core Facilities for technical support. We thank the University of Cambridge Department of Genetics Fly Facility for microinjection services and fly stock generation. We thank the Vienna Drosophila Resource Center and the Bloomington Stock Center for fly stocks. We thank Julius Brennecke for anti-Piwi, anti-Aub and anti-Ago3 antibodies, William Theurkauf for anti-Rhi antibody and fly strains, Elisa Izaurralde for anti-Nxt1 antibody, and Paul Lasko for anti-UAP56 antibody.

A Heterochromatin-Specific RNA Export Pathway Facilitates piRNA Production

“…The Nxf3-Bootlegger pathway provides a glimpse into how biological information is sorted and spatially distributed in cells. We note that similar findings on the Nxf3-Bootlegger pathway have been published by Kneuss and colleagues during the proof stages of this manuscript (Kneuss et al, 2019).…”

piRNA-guided co-transcriptional silencing coopts nuclear export factors

“…Interestingly, while Nxf1, acting along with Nxt1, is crucial for canonical mRNA export (Braun et al, 2001; Fribourg et al, 2001; Herold et al, 2001; Wilkie et al, 2001), Nxf2 has been coopted by the piRNA pathway and functions in co-transcriptional gene silencing. Nxf3, which also is required for transposon repression in germ cells (Czech et al, 2013), is emerging as being critical for the export of piRNA precursors generated from dual-strand clusters in the germline (ElMaghraby et al, 2019; Kneuss et al, 2019. The role of Nxf4, whose expression is testis-specific, is yet to be established.…”

Functions of PIWI Proteins in Gene Regulation: New Arrows Added to the piRNA Quiver

“…First, piRNA cluster genes [74] are transcribed into long noncoding transcripts by RNA polymerase II via specific transcription factors or chromatin regulators [2,3] (Figure 1). The piRNA precursor transcript is then exported out of the nucleus in the nuage, with a specific export complex containing nuclear export factor 3 (Nxf3) in Drosophila [75,76]. There, Drosophila Ago3 associated with a maternally inherited piRNA recognizes and cleaves a complementary piRNA precursor transcript, generating a 5′ monophosphorylated RNA that interacts with Aub.…”