The PIWI-interacting RNA (piRNA) pathway protects 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 Piwi-mediated co-transcriptional silencing in Drosophila. SFiNX consists of Nxf2-Nxt1, a gonad-specific 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 heterochromatin effectors, Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:
Accurate segregation of chromosomes during meiosis—critical for genome stability across sexual cycles—relies on homologous recombination initiated by DNA double-strand breaks (DSBs) made by the Spo11 protein
1
,
2
. DSB formation is regulated and tied to the elaboration of large-scale chromosome structures
3
–
5
, but the protein assemblies that execute and control DNA breakage are poorly understood. We address this through molecular characterization of
Saccharomyces cerevisiae
RMM proteins (Rec114, Mei4 and Mer2)—essential, conserved components of the DSB machinery
2
. Each subcomplex of Rec114–Mei4 (a 2:1 heterotrimer) or Mer2 (a coiled-coil-containing homotetramer) is monodisperse in solution, but they independently condense with DNA into reversible nucleoprotein clusters that share properties with phase-separated systems. Multivalent interactions drive condensation. Mutations that weaken protein–DNA interactions strongly disrupt both condensate formation and DSBs
in vivo
, strongly correlating these processes.
In vitro
, condensates fuse into mixed RMM clusters that further recruit Spo11 complexes. Our data show how the DSB machinery self-assembles on chromosome axes to create centers of DSB activity. We propose that multilayered control of Spo11 arises from the recruitment of regulatory components and modulation of biophysical properties of the condensates.
Highlights d REC114 directly interacts with ANKRD31, a novel factor required for normal fertility d ANKRD31 influences the distribution of double-strand breaks genome-wide d ANKRD31 is essential for the recombination between X and Y chromosomes d A crystal structure reveals a PH domain in REC114 and its contacts with ANKRD31
Small self-cleaving ribozymes catalyze site-specific cleavage of their own phosphodiester backbone with implications for viral genome replication, pre-mRNA processing, and alternative splicing. We report on the 2.1-Å crystal structure of the hatchet ribozyme product, which adopts a compact pseudosymmetric dimeric scaffold, with each monomer stabilized by long-range interactions involving highly conserved nucleotides brought into close proximity of the scissile phosphate. Strikingly, the catalytic pocket contains a cavity capable of accommodating both the modeled scissile phosphate and its flanking 5′ nucleoside. The resulting modeled precatalytic conformation incorporates a splayed-apart alignment at the scissile phosphate, thereby providing structure-based insights into the in-line cleavage mechanism. We identify a guanine lining the catalytic pocket positioned to contribute to cleavage chemistry. The functional relevance of structure-based insights into hatchet ribozyme catalysis is strongly supported by cleavage assays monitoring the impact of selected nucleobase and atom-specific mutations on ribozyme activity.
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