Non-coding small RNAs as versatile regulators of germline gene expression programs

The RNA-guided targeting of nucleic acids is an ancient and conserved mechanism of cellular immunity that has been evolutionary adapted and diversified to regulate eukaryotic gene expression. In animal germ cells, PIWI-interacting small RNAs (piRNAs) have been extensively characterized as a defense mechanism targeting transposable elements (TEs) to promote fertility and genome integrity. In a nutshell: loaded into PIWI effector proteins, piRNA sequences provide mRNA targeting specificity by antisense-complementarity, promoting gene silencing through a variety of mechanisms. Yet, piRNA sequences do not necessarily match TEs, pointing to extended possibilities in gene regulation.

Studying piRNA pathway functions in the context of the developing C. elegans germline we show that spermatogenic genes are susceptible to piRNA-mediated transcriptional silencing, and this function is required to ensure proper germline gene expression patterning and germ cell differentiation. In addition, our work revealed an intriguing aspect of small RNA biology: piRNA pathway components localize into diverse and distinct phase separated condensates present in the nuclear periphery. These condensates, also known as germ granules, are enriched in RNA-binding proteins and RNAs and are suspected to regulate post-transcriptional processes important for germ cell fate specification and function. Our results show that the organization of germ granules changes dynamically during development, and only a particular configuration enable nuclear piRNA silencing at a specific time and location in the germline tissue. This suggests that changes in germ granule composition directly influence nuclear processes through the modulation of small RNA related activities.

Overall the results of this work show that the function of piRNAs can be repurposed to regulate endogenous transcriptional programs during development and might contribute to expand the notion that piRNAs do not only function as a cellular immune system but also act as extremely versatile regulators of gene expression in animals.

Bacterial retrons encode phage-sensing toxin/antitoxin systems

The first prokaryotic reverse transcriptases (RTs) were discovered thirty years ago in genetic elements called retrons. Retron RTs produce short single-stranded DNAs, known as msDNA (multicopy single-stranded DNA). Despite extensive efforts, the function of retrons and msDNA has remained elusive until recently. We found that Retron-Sen2, present throughout Salmonella Typhimurium strains and member of the most prevalent retron family across bacteria, encodes a novel tripartite toxin/antitoxin (TA) system. The RT and msDNA form an antitoxin complex, which counteracts a toxin, encoded by the accessory retron gene. When msDNA biosynthesis is perturbed, the retron toxin inhibits bacterial growth in specific conditions. To further illuminate the physiological role of this novel TA system, we devised a tandem high-throughput genetics screen (TIC/TAC; Toxin Inhibition/Activation Conjugation). TIC/TAC enabled us to identify numerous proteins that trigger or block the Retron-Sen2 TA with the strongest of them being of phage origin. We show that the phage triggers activate the retron toxin, by directly interacting and altering the msDNA, and that bacteria use retrons of this family to defend against different phages through abortive infection. Overall, the Sen2 family of retrons encode for a new type of tripartite TA systems, which are used to defend against phages. Our TIC/TAC methodology opens new paths to identify triggers and blockers for any TA system.