During cellular stress, protein synthesis is severely reduced and bulk mRNA is recruited to stress granules (SGs). Previously, we showed that the SG-recruited IGF2 mRNA-binding protein 1 (IGF2BP1) interferes with target mRNA degradation during cellular stress. Whether this requires the formation of SGs remained elusive. Here, we demonstrate that the sustained inhibition of visible SGs requires the concomitant knockdown of TIA1, TIAR and G3BP1. FRAP and photo-conversion studies, however, indicate that these proteins only transiently associate with SGs. This suggests that instead of forming a rigid scaffold for mRNP recruitment, TIA proteins and G3BP1 promote SG-formation by constantly replenishing mRNPs. In contrast, RNA-binding proteins like IGF2BP1 or HUR, which are dispensable for SG-assembly, are stably associated with SGs and the IGF2BP1/HUR-G3BP1 association is increased during stress. The depletion of IGF2BP1 enhances the degradation of target mRNAs irrespective of inhibiting SG-formation, whereas the turnover of bulk mRNA remains unaffected when SG-formation is impaired. Together these findings indicate that the stabilization of mRNAs during cellular stress is facilitated by the formation of stable mRNPs, which are recruited to SGs by TIA proteins and/or G3BP1. Importantly, however, the aggregation of mRNPs to visible SGs is dispensable for preventing mRNA degradation.

Retrons are genetic retroelements, commonly found in bacterial genomes and recently repurposed as genome editing tools. Their encoded reverse transcriptase (RT) produces a multi-copy single-stranded DNA (msDNA). Despite our understanding of their complex biosynthesis, the function of msDNAs and therefore, the physiological role of retrons has remained elusive. We establish that the retron-Sen2 in Salmonella Typhimurium encodes a toxin, which we have renamed as RcaT (Retron cold-anaerobic Toxin). RcaT is activated when msDNA biosynthesis is perturbed and its toxicity is higher at ambient temperatures or during anaerobiosis. The RT and msDNA form together the antitoxin unit, with the RT binding RcaT, and the msDNA enabling the antitoxin activity. Using another E. coli retron, we establish that this toxin/antitoxin function is conserved, and that RT-toxin interactions are cognate.Altogether, retrons constitute a novel family of tripartite toxin/antitoxin systems..

19Retrons are genetic retroelements, commonly found in bacterial genomes and recently 20 repurposed as genome editing tools. Their encoded reverse transcriptase (RT) produces a 21 multi-copy single-stranded DNA (msDNA). Despite our understanding of their complex 22 biosynthesis, the function of msDNAs and therefore, the physiological role of retrons has 23 remained elusive. We establish that the retron-Sen2 in Salmonella Typhimurium encodes a 24 toxin, which we have renamed as RcaT (Retron cold-anaerobic Toxin). RcaT is activated when 25 msDNA biosynthesis is perturbed and its toxicity is higher at ambient temperatures or during 26 anaerobiosis. The RT and msDNA form together the antitoxin unit, with the RT binding RcaT, 27 and the msDNA enabling the antitoxin activity. Using another E. coli retron, we establish that 28 this toxin/antitoxin function is conserved, and that RT-toxin interactions are cognate. 29Altogether, retrons constitute a novel family of tripartite toxin/antitoxin systems. 30 31 1A), do not affect msDNA biosynthesis 13,18 , and neither their sequence nor the position in 54 retron elements are conserved. 55 56The bottleneck to understand the natural function of retrons has been the absence of 57 phenotypes associated with retron deletions. The first retron-deletion phenotype was reported 58 for retron-Sen2 of Salmonella enterica subsp. enterica ser. Typhimurium str. 14028s (STm), 59wherein the RT-Sen2 was found to be essential for STm survival in calves 18 . This was because 60 it allows STm to grow in anaerobic conditions, present in calf intestines 19 . Here, we report that 61 retron-Sen2 deletion mutants are also unable to grow at lower temperatures. By exploiting the 62 retron cold-sensitivity phenotype, we show that the retron-Sen2 accessory gene STM14_4640 63 (rcaT) encodes a bona fide toxin. Perturbing msDNA biosynthesis at any stage results in toxin 64 activation, and thereby, growth inhibition in anaerobic conditions and cold. Although 65 reminiscent of Toxin/Antitoxin (TA) systems, which are composed of a protein or RNA antitoxin 66 and cognate toxin 20 , retron-Sen2 forms a novel tripartite TA system: RcaT is the toxin, and 67 the RT-msDNA complex is the antitoxin. Using another retron encoded by E. coli NILS-16 21 , 68 3 retron-Eco9, we demonstrate that this TA function is conserved and that the RT provides 69 specificity to the TA system. We propose that bacterial retrons function as TA systems, where 70 the RT-msDNA antitoxins directly inhibit retron-encoded toxins, by forming inactive msDNA-71 RT-toxin complexes. 72 73 RESULTS 74Perturbations in msDNA-Sen2 biosynthesis inhibit STm growth in cold 75As part of a larger chemical-genetics effort, we profiled the fitness of a single-gene deletion 76STm library 22 across hundreds of conditions (unpublished data). The two gene deletions that 77 led to the highest growth sensitivity at room temperature (cold-sensitivity) were ΔrrtT (ΔRT-78 Sen2) and ΔxseA (Fig. 1C). Both RT-Sen2 and Exo VII are involved in msDNA-Sen2 126Retron-Sen2 i...