SUMMARY Stress granules are mRNA-protein assemblies formed from nontranslating mRNAs. Stress granules are important in the stress response and may contribute to some degenerative diseases. Here we describe the stress granule transcriptome of yeast and mammalian cells through RNA-Seq analysis of purified stress granule cores and smFISH validation. While essentially every mRNA, and some ncRNAs, can be targeted to stress granules, the targeting efficiency varies from <1% to >95%. mRNA accumulation in stress granules correlates with longer coding and UTR regions and poor translatability. Quantifying the RNA-Seq analysis by smFISH reveals only 10% of bulk mRNA molecules accumulate in mammalian stress granules, and only 185 genes have more than 50% of their mRNA molecules in stress granules. These results suggest stress granules may not represent a specific biological program of mRNP assembly, but instead form by condensation of nontranslating mRNPs in proportion to their length and lack of association with ribosomes.

Stress granules are non-membrane bound RNA-protein (RNP) assemblies that form when translation initiation is limited and contain a biphasic structure with stable core structures surrounded by a less concentrated shell. The order of assembly and disassembly of these two structures remains unknown. Time course analysis of granule assembly suggests that core formation is an early event in granule assembly. Stress granule disassembly is also a stepwise process with shell dissipation followed by core clearance. Perturbations that alter liquid-liquid phase separations (LLPS) driven by intrinsically disordered protein regions (IDR) of RNA binding proteins in vitro have the opposite effect on stress granule assembly in vivo. Taken together, these observations argue that stress granules assemble through a multistep process initiated by stable assembly of untranslated mRNPs into core structures, which could provide sufficient high local concentrations to allow for a localized LLPS driven by IDRs on RNA binding proteins.DOI: http://dx.doi.org/10.7554/eLife.18413.001

Stress granules are higher order assemblies of nontranslating mRNAs and proteins that form when translation initiation is inhibited. Stress granules are thought to form by protein-protein interactions of RNA-binding proteins. We demonstrate RNA homopolymers or purified cellular RNA forms assemblies in vitro analogous to stress granules. Remarkably, under conditions representative of an intracellular stress response, the mRNAs enriched in assemblies from total yeast RNA largely recapitulate the stress granule transcriptome. We suggest stress granules are formed by a summation of protein-protein and RNA-RNA interactions, with RNA self-assembly likely to contribute to other RNP assemblies wherever there is a high local concentration of RNA. RNA assembly in vitro is also increased by GR and PR dipeptide repeats, which are known to increase stress granule formation in cells. Since GR and PR dipeptides are involved in neurodegenerative diseases, this suggests that perturbations increasing RNA-RNA assembly in cells could lead to disease.

In response to foreign and endogenous doublestranded RNA (dsRNA), protein kinase R (PKR) and ribonuclease L (RNase L) reprogram translation in mammalian cells. PKR inhibits translation initiation through eIF2a phosphorylation, which triggers stress granule (SG) formation and promotes translation of stress responsive mRNAs. The mechanisms of RNase L-driven translation repression, its contribution to SG assembly, and its regulation of dsRNA stress-induced mRNAs are unknown. We demonstrate that RNase L drives translational shut-off in response to dsRNA by promoting widespread turnover of mRNAs. This alters stress granule assembly and reprograms translation by allowing translation of mRNAs resistant to RNase L degradation, including numerous antiviral mRNAs such as interferon (IFN)-b. Individual cells differentially activate dsRNA responses revealing variation that can affect cellular outcomes. This identifies bulk mRNA degradation and the resistance of antiviral mRNAs as the mechanism by which RNase L reprograms translation in response to dsRNA. (A) IF for SG-associated proteins G3BP1 and PABPC1 in WT and RL-KO U-2 OS cells. (B) G3BP1-positive foci from >30 WT and RL-KO U-2 OS cells binned by volume. (C) IF for G3BP1 and PABPC1 in parental RL-KO A549 cells stably expressing either RNase L (RL) or RNase L-R667A (RL-CM) 8 h post-poly(I:C). Images for G3BP1 and PABPC1 staining are shown in Figure S1E.

RNAs besides tRNA and rRNA contain chemical modifications, including the recently described 5′ nicotinamide-adenine dinucleotide (NAD + ) RNA in bacteria. Whether 5′ NAD-RNA exists in eukaryotes remains unknown. We demonstrate that 5′ NAD-RNA is found on subsets of nuclear and mitochondrial encoded mRNAs in Saccharomyces cerevisiae. NAD-mRNA appears to be produced cotranscriptionally because NAD-RNA is also found on pre-mRNAs, and only on mitochondrial transcripts that are not 5′ end processed. These results define an additional 5′ RNA cap structure in eukaryotes and raise the possibility that this 5′ NAD + cap could modulate RNA stability and translation on specific subclasses of mRNAs.T he number and prevalence of known chemical modifications on mRNAs have dramatically increased in the past several years (1). Quantification of these modification events suggests they occur in many RNAs (2, 3). Importantly, several of these modifications have functional consequences (4-6). For example, the presence of a single N 6 -methyladenosine within the 5′ UTR of an mRNA increases translation initiation (4). In addition, the methylation status of cytosine residues within the 3′ UTR of the p16(INK4) human mRNA affects mRNA stability (6). Due to the increasing sensitivity of RNA sequencing (RNA-Seq) and small-molecule mass spectrometry, it is reasonable to hypothesize that many novel chemical modifications within mRNAs remain to be discovered.One modification recently identified in bacteria is 5′ nicotinamide-adenine dinucleotide (NAD + )-linked RNA (7, 8). Because canonical bacterial RNAs contain a 5′ triphosphate terminus, addition of NAD + to the 5′ end of RNAs represents a rudimentary "capping" mechanism, perhaps designed to impart specific properties for these RNAs by granting them a more structurally complex 5′ end. Consistent with this idea, NAD + addition to RNAs appears to occur during transcription initiation (9), as opposed to the more complex eukaryote 5′ capping, which occurs after transcription has commenced (10). Because the NAD + modification defines the 5′ end of NAD-RNAs, this modification can affect RNA stability in Escherichia coli (7, 9).For decades, 5′ end classification and study of eukaryotic mRNAs have been restricted to canonical 7-methylguanosine (m 7 G) "caps" and their methylated variants (11). The m 7 G cap modulates numerous facets of mRNA metabolism, including stability (12, 13), translation (14, 15), and export (16). The importance of this modification is underscored by the substantial cellular machinery dedicated to its addition and removal (17,18). Thus, mRNAs containing noncanonical 5′ termini may have distinct properties and be subject to alternative metabolic events.Described herein is the identification of NAD-RNAs in the eukaryote Saccharomyces cerevisiae. Examples of NAD-RNAs in S. cerevisiae include nuclear encoded mRNAs for ribosomal proteins, as well as some mitochondrial encoded transcripts. Our data suggest that the NAD + moiety is added during initiation in both nuclear and mitoc...