RNA granules are sub-cellular compartments proposed to form by liquid-liquid phase separation (LLPS), a thermodynamic process that partitions molecules between dilute and condensed liquid phases. The mechanisms that localize liquid phases in cells, however, are not fully understood. P granules are RNA granules that form in the posterior of C. elegans embryos. Theoretical studies have suggested that spontaneous LLPS of the RNA-binding protein PGL-3 with RNA drives P granule assembly. We find that the PGL-3 phase is intrinsically labile and requires a second phase for stabilization in embryos. The second phase is formed by gel-like assemblies of the disordered protein MEG-3 that associate with liquid PGL-3 droplets in the embryo posterior. Co-assembly of gel and liquid phases confers local stability and long-range dynamics, both of which contribute to localized P granule assembly. Our findings suggest that condensation of RNA granules can be regulated spatially by gel-like polymers that stimulate LLPS locally in the cytoplasm.

SUMMARY The heavy occupancy of transposons in the genome implies that existing organisms have survived from multiple, independent rounds of transposon invasions. However, how and which host cell types survive the initial wave of transposon invasion remains unclear. We show that the germline stem cells can initiate a robust adaptive response that rapidly endogenizes invading P-element transposons by activating the DNA-damage checkpoint and piRNA production. We find that temperature modulates the P-element activity in germline stem cells, establishing a powerful tool to trigger transposon hyper-activation. Facing vigorous invasion, Drosophila first shut down oogenesis and induce selective apoptosis. Interestingly, a robust adaptive response occurs in ovarian stem cells through activation of the DNA-damage checkpoint. Within 4 days, the hosts amplify P-element-silencing piRNAs, repair DNA damage, subdue the transposon, and reinitiate oogenesis. We propose that this robust adaptive response can bestow upon organisms the ability to survive recurrent transposon invasions throughout evolution.

Puromycin is a tyrosyl-tRNA mimic that blocks translation by labeling and releasing elongating polypeptide chains from translating ribosomes. Puromycin has been used in molecular biology research for decades as a translation inhibitor. The development of puromycin antibodies and derivatized puromycin analogs has enabled the quantification of active translation in bulk and single-cell assays. More recently, in vivo puromycylation assays have become popular tools for localizing translating ribosomes in cells. These assays often use elongation inhibitors to purportedly inhibit the release of puromycin-labeled nascent peptides from ribosomes. Using in vitro and in vivo experiments in various eukaryotic systems, we demonstrate that, even in the presence of elongation inhibitors, puromycylated peptides are released and diffuse away from ribosomes. Puromycylation assays reveal subcellular sites, such as nuclei, where puromycylated peptides accumulate post-release and which do not necessarily coincide with sites of active translation. Our findings urge caution when interpreting puromycylation assays in vivo.