Long non-coding RNAs (lncRNAs) have an undefined role in the pathobiology of glioblastoma multiforme (GBM). These tumors are genetically and phenotypically heterogeneous with transcriptome subtype-specific GBM stem-like cells (GSCs) that adapt to the brain tumor microenvironment, including hypoxic niches. We identified hypoxia-inducible factor 1 alpha-antisense RNA 2 (HIF1A-AS2) as a subtype-specific hypoxia-inducible lncRNA, upregulated in mesenchymal GSCs. Its deregulation affects GSC growth, self-renewal, and hypoxia-dependent molecular reprogramming. Among the HIF1A-AS2 interactome, IGF2BP2 and DHX9 were identified as direct partners. This association was needed for maintenance of expression of their target gene, HMGA1. Downregulation of HIF1A-AS2 led to delayed growth of mesenchymal GSC tumors, survival benefits, and impaired expression of HMGA1 in vivo. Our data demonstrate that HIF1A-AS2 contributes to GSCs' speciation and adaptation to hypoxia within the tumor microenvironment, acting directly through its interactome and targets and indirectly by modulating responses to hypoxic stress depending on the subtype-specific genetic context.

Stress-induced tRNA cleavage has been implicated in various cellular processes, where tRNA fragments play diverse regulatory roles. Angiogenin (ANG), a member of the RNase A superfamily, induces cleavage of tRNAs resulting in the formation of tRNA-derived stress-induced RNAs (tiRNAs) that contribute to translational reprogramming aiming at cell survival. In addition to cleaving tRNA anticodon loops, ANG has been shown to cleave 3'-CCA termini of tRNAs in vitro, although it is not known whether this process occurs in cells. It has also been suggested that tiRNAs can be generated independently of ANG, although the role of other stress-induced RNases in tRNA cleavage is poorly understood. Using gene editing and biochemical approaches, we examined the involvement of ANG in stress-induced tRNA cleavage by focusing on its cleavage of CCA-termini as well as anticodon loops. We show that ANG is not responsible for CCA-deactivation under sodium arsenite (SA) treatment in cellulo, and although ANG treatment significantly increases 3'-tiRNA levels in cells, the majority of 3'-tiRNAs retain their 3'-CCA termini. Instead, other RNases can cleave CCA-termini in cells, although with low efficiency. Moreover, in the absence of ANG, other RNases are able to promote the production of tiRNAs in cells. Depletion of RNH1 (an endogenous inhibitor of RNase A superfamily) promotes constitutively-produced tiRNAs and CCA-deactivated tRNAs in cells. Interestingly, SA treatment in RNH1-depleted cells did not increase the amount of tiRNAs or CCA-deactivated tRNAs, suggesting that RNase A superfamily enzymes are largely responsible for SA-induced tRNA cleavage. We show that interplay between stress-induced RNases cause targeting tRNAs in a stress-specific manner in cellulo.

Guanine (G)-rich nucleic acids can fold into G-quadruplex (G4) structures under permissive conditions. Although many RNAs contain sequences that fold into RNA G4s (rG4s) in vitro, their folding and functions in vivo are not well understood. In this report, we showed that the folding of putative rG4s in human cells into rG4 structures is dynamically regulated under stress. By using high-throughput dimethylsulfate (DMS) probing, we identified hundreds of endogenous stress-induced rG4s, and validated them by using an rG4 pull-down approach. Our results demonstrate that stress-induced rG4s are enriched in mRNA 3'-untranslated regions and enhance mRNA stability. Furthermore, stress-induced rG4 folding is readily reversible upon stress removal. In summary, our study revealed the dynamic regulation of rG4 folding in human cells and suggested that widespread rG4 motifs may have a global regulatory impact on mRNA stability and cellular stress response.

Fragile-X mental retardation autosomal homologue-1 (FXR1) is a muscle-enriched RNA-binding protein. FXR1 depletion is perinatally lethal in mice, Xenopus, and zebrafish; however, the mechanisms driving these phenotypes remain unclear. The FXR1 gene undergoes alternative splicing, producing multiple protein isoforms and mis-splicing has been implicated in disease. Furthermore, mutations that cause frameshifts in muscle-specific isoforms result in congenital multi-minicore myopathy. We observed that FXR1 alternative splicing is pronounced in the serine- and arginine-rich intrinsically disordered domain; these domains are known to promote biomolecular condensation. Here, we show that tissue-specific splicing of fxr1 is required for Xenopus development and alters the disordered domain of FXR1. FXR1 isoforms vary in the formation of RNA-dependent biomolecular condensates in cells and in vitro. This work shows that regulation of tissue-specific splicing can influence FXR1 condensates in muscle development and how mis-splicing promotes disease.

The production of ribosomes is an energy-intensive process owing to the intricacy of these massive macromolecular machines. Each human ribosome contains 80 ribosomal proteins and four non-coding RNAs. Accurate assembly requires precise regulation of protein and RNA subunits. In response to stress, the integrated stress response (ISR) rapidly inhibits global translation. How rRNA is coordinately regulated with the rapid inhibition of ribosomal protein synthesis is not known. Here, we show that stress specifically inhibits the first step of rRNA processing. Unprocessed rRNA is stored within the nucleolus, and when stress resolves, it re-enters the ribosome biogenesis pathway. Retention of unprocessed rRNA within the nucleolus aids in the maintenance of this organelle. This response is independent of the ISR or inhibition of cellular translation but is independently regulated. Failure to coordinately control ribosomal protein translation and rRNA production results in nucleolar fragmentation. Our study unveils how the rapid translational shut-off in response to stress coordinates with rRNA synthesis production to maintain nucleolar integrity.

Tourrière et al. (2013. J. Cell Biol. https://doi.org/10.1083/jcb.200212128) reported that G3BP1-S149 dephosphorylation promotes stress granule formation. We show that constructs used to establish this conclusion contain additional mutations causing these phenotypes, and that S149 phosphorylation status does not change upon stress.

Metazoan replication-dependent histone mRNAs are the only known eukaryotic mRNAs that lack a poly(A) tail, ending instead in a conserved stem-loop sequence, which is bound to the stem-loop binding protein (SLBP) on the histone mRNP. Histone mRNAs are rapidly degraded when DNA synthesis is inhibited in S phase in mammalian cells. Rapid degradation of histone mRNAs is initiated by oligouridylation of the 3' end of histone mRNAs and requires the cytoplasmic Lsm1-7 complex, which can bind to the oligo(U) tail. An exonuclease, 3'hExo, forms a ternary complex with SLBP and the stem-loop and is required for the initiation of histone mRNA degradation. The Lsm1-7 complex is also involved in degradation of polyadenylated mRNAs. It binds to the oligo(A) tail remaining after deadenylation, inhibiting translation and recruiting the enzymes required for decapping. Whether the Lsm1-7 complex interacts directly with other components of the mRNP is not known. We report here that the C-terminal extension of Lsm4 interacts directly with the histone mRNP, contacting both SLBP and 3'hExo. Mutants in the C-terminal tail of Lsm4 that prevent SLBP and 3'hExo binding reduce the rate of histone mRNA degradation when DNA synthesis is inhibited.

We show that 3-morpholinosydnonimine (SIN-1)-induced nitric oxide (NO) triggers the formation of SGs. Whereas the composition of NO-induced SGs is initially similar to sodium arsenite (SA)-induced type I (cytoprotective) SGs, the progressive loss of eIF3 over time converts them into pro-death (type II) SGs. NO-induced SG assembly requires the phosphorylation of eIF2α, but the transition to type II SGs is temporally linked to the mTOR-regulated displacement of eIF4F complexes from the m7 guanine cap. Whereas SA does not affect mitochondrial morphology or function, NO alters mitochondrial integrity and function, resulting in increased ROS production, decreased cytoplasmic ATP, and plasma membrane permeabilization, all of which are supported by type II SG assembly. Thus, cellular energy balance is linked to the composition and function of NO-induced SGs in ways that determine whether cells live or die.

In cells exposed to environmental stress, inhibition of translation initiation conserves energy for the repair of cellular damage. Untranslated mRNAs that accumulate in these cells move to discrete cytoplasmic foci known as stress granules (SGs). The assembly of SGs helps cells to survive under adverse environmental conditions. We have analyzed the mechanism by which hydrogen peroxide (H(2)O(2))-induced oxidative stress inhibits translation initiation and induces SG assembly in mammalian cells. Our data indicate that H(2)O(2) inhibits translation and induces the assembly of SGs. The assembly of H(2)O(2)-induced SGs is independent of the phosphorylation of eIF2α, a major trigger of SG assembly, but requires remodeling of the cap-binding eIF4F complex. Moreover, H(2)O(2)-induced SGs are compositionally distinct from canonical SGs, and targeted knockdown of eIF4E, a protein required for canonical translation initiation, inhibits H(2)O(2)-induced SG assembly. Our data reveal new aspects of translational regulation induced by oxidative insults.

We have transcriptionally profiled the genes differentially expressed in E. coli prey cells when predatorily attacked by Bdellovibrio bacteriovorus just prior to prey cell killing. This is a brief, approximately 20-25 min period when the prey cell is still alive but contains a Bdellovibrio cell in its periplasm or attached to and penetrating its outer membrane. Total RNA was harvested and labelled 15 min after initiating a semi-synchronous infection with an excess of Bdellovibrio preying upon E. coli and hybridised to a macroarray spotted with all predicted ORFs of E. coli. SAM analysis and t-tests were performed on the resulting data and 126 E. coli genes were found to be significantly differentially regulated by the prey upon attack by Bdellovibrio. The results were confirmed by QRT-PCR. Amongst the prey genes upregulated were a variety of general stress response genes, potentially "selfish" genes within or near prophages and transposable elements, and genes responding to damage in the periplasm and osmotic stress. Essentially, the presence of the invading Bdellovibrio and the resulting damage to the prey cell elicited a small "transcriptional scream", but seemingly no specific defensive mechanism with which to counter the Bdellovibrio attack. This supports other studies which do not find Bdellovibrio resistance responses in prey, and bodes well for its use as a "living antibiotic".

Histone proteins are synthesized in large amounts during S-phase to package the newly replicated DNA, and are among the most stable proteins in the cell. The replication-dependent (RD)-histone mRNAs expressed during S-phase end in a conserved stem-loop rather than a polyA tail. In addition, there are replication-independent (RI)-histone genes that encode histone variants as polyadenylated mRNAs. Most variants have specific functions in chromatin, but H3.3 also serves as a replacement histone for damaged histones in long-lived terminally differentiated cells. There are no reported replacement histone genes for histones H2A, H2B or H4. We report that a subset of RD-histone genes are expressed in terminally differentiated tissues as polyadenylated mRNAs, likely serving as replacement histone genes in long-lived non-dividing cells. Expression of two genes, HIST2H2AA3 and HIST1H2BC, is conserved in mammals. They are expressed as polyadenylated mRNAs in fibroblasts differentiated in vitro, but not in serum starved fibroblasts, suggesting that their expression is part of the terminal differentiation program. There are two histone H4 genes and an H3 gene that encode mRNAs that are polyadenylated and expressed at 5- to 10-fold lower levels than the mRNAs from H2A and H2B genes, which may be replacement genes for the H3.1 and H4 proteins.

Platinum-based antineoplastic drugs, such as cisplatin, are commonly used to induce tumor cell death. Cisplatin is believed to induce apoptosis as a result of cisplatin-DNA adducts that inhibit DNA and RNA synthesis. Although idea that DNA damage underlines anti-proliferative effects of cisplatin is dominant in cancer research, there is a poor correlation between the degree of the cell sensitivity to cisplatin and the extent of DNA platination. Here, we examined possible effects of cisplatin on post-transcriptional gene regulation that may contribute to cisplatin-mediated cytotoxicity. We show that cisplatin suppresses formation of stress granules (SGs), pro-survival RNA granules with multiple roles in cellular metabolism. Mechanistically, cisplatin inhibits cellular translation to promote disassembly of polysomes and aggregation of ribosomal subunits. As SGs are in equilibrium with polysomes, cisplatin-induced shift towards ribosomal aggregation suppresses SG formation. Our data uncover previously unknown effects of cisplatin on RNA metabolism.

Liquid-liquid phase separation (LLPS) mediates formation of membraneless condensates such as those associated with RNA processing, but the rules that dictate their assembly, substructure, and coexistence with other liquid-like compartments remain elusive. Here, we address the biophysical mechanism of this multiphase organization using quantitative reconstitution of cytoplasmic stress granules (SGs) with attached P-bodies in human cells. Protein-interaction networks can be viewed as interconnected complexes (nodes) of RNA-binding domains (RBDs), whose integrated RNA-binding capacity determines whether LLPS occurs upon RNA influx. Surprisingly, both RBD-RNA specificity and disordered segments of key proteins are non-essential, but modulate multiphase condensation. Instead, stoichiometry-dependent competition between protein networks for connecting nodes determines SG and P-body composition and miscibility, while competitive binding of unconnected proteins disengages networks and prevents LLPS. Inspired by patchy colloid theory, we propose a general framework by which competing networks give rise to compositionally specific and tunable condensates, while relative linkage between nodes underlies multiphase organization.

The animal replication-dependent (RD) histone mRNAs are coordinately regulated with chromosome replication. The RD-histone mRNAs are the only known cellular mRNAs that are not polyadenylated. Instead, the mature transcripts end in a conserved stem-loop (SL) structure. This SL structure interacts with the stem-loop binding protein (SLBP), which is involved in all aspects of RD-histone mRNA metabolism. We used several genomic methods, including high-throughput sequencing of cross-linked immunoprecipitate (HITS-CLIP) to analyze the RNA-binding landscape of SLBP. SLBP was not bound to any RNAs other than histone mRNAs. We performed bioinformatic analyses of the HITS-CLIP data that included (i) clustering genes by sequencing read coverage using CVCA, (ii) mapping the bound RNA fragment termini, and (iii) mapping cross-linking induced mutation sites (CIMS) using CLIP-PyL software. These analyses allowed us to identify specific sites of molecular contact between SLBP and its RD-histone mRNA ligands. We performed in vitro crosslinking assays to refine the CIMS mapping and found that uracils one and three in the loop of the histone mRNA SL preferentially crosslink to SLBP, whereas uracil two in the loop preferentially crosslinks to a separate component, likely the 3'hExo. We also performed a secondary analysis of an iCLIP data set to map UPF1 occupancy across the RD-histone mRNAs and found that UPF1 is bound adjacent to the SLBP-binding site. Multiple proteins likely bind the 3' end of RD-histone mRNAs together with SLBP.

Resistance to chemotherapy drugs is a serious therapeutic problem and its underlying molecular mechanisms are complex. Stress granules (SGs), cytoplasmic ribonucleoprotein complexes assembled in cells exposed to stress, are implicated in various aspects of cancer cell metabolism and survival. SGs promote the survival of stressed cells by reprogramming gene expression and inhibiting pro-apoptotic signaling cascades. We show that the vinca alkaloid (VA) class of anti-neoplastic agents potently activates a SG-mediated stress response program. VAs inhibit translation initiation by simultaneous activation of eIF4E-BP1 and phosphorylation of eIF2α, causing polysome disassembly and SG assembly. VA-induced SGs contain canonical SG components but lack specific signaling molecules. Blocking VA-induced SG assembly by inactivating eIF4EBP1 or inhibiting eIF2α phosphorylation decreases cancer cell viability and promotes apoptosis. Our data describe previously unappreciated effects of VAs on cellular RNA metabolism and illuminate the roles of SGs in cancer cell survival.

Autoantibody profiles represent important patient stratification markers in systemic sclerosis (SSc). Here, we performed serum-immunoprecipitations with patient antibodies followed by mass spectrometry (LC-MS/MS) to obtain an unbiased view of all possible autoantibody targets and their associated molecular complexes recognized by SSc.

Nuclear bodies (NBs) are structures that concentrate proteins, RNAs, and ribonucleoproteins that perform functions essential to gene expression. How NBs assemble is not well understood. We studied the Drosophila histone locus body (HLB), a NB that concentrates factors required for histone mRNA biosynthesis at the replication-dependent histone gene locus. We coupled biochemical analysis with confocal imaging of both fixed and live tissues to demonstrate that the Drosophila Multi Sex Combs (Mxc) protein contains multiple domains necessary for HLB assembly. An important feature of this assembly process is the self-interaction of Mxc via two conserved N-terminal domains: a LisH domain and a novel self-interaction facilitator (SIF) domain immediately downstream of the LisH domain. Molecular modeling suggests that the LisH and SIF domains directly interact, and mutation of either the LisH or the SIF domain severely impairs Mxc function in vivo, resulting in reduced histone mRNA accumulation. A region of Mxc between amino acids 721 and 1481 is also necessary for HLB assembly independent of the LisH and SIF domains. Finally, the C-terminal 195 amino acids of Mxc are required for recruiting FLASH, an essential histone mRNA-processing factor, to the HLB. We conclude that multiple domains of the Mxc protein promote HLB assembly in order to concentrate factors required for histone mRNA biosynthesis.

In response to severe environmental stresses eukaryotic cells shut down translation and accumulate components of the translational machinery in stress granules (SGs). Since they contain mainly mRNA, translation initiation factors and 40S ribosomal subunits, they have been referred to as dominant accumulations of stalled translation preinitiation complexes. Here we present evidence that the robust heat shock-induced SGs of S. cerevisiae also contain translation elongation factors eEF3 (Yef3p) and eEF1Bγ2 (Tef4p) as well as translation termination factors eRF1 (Sup45p) and eRF3 (Sup35p). Despite the presence of the yeast prion protein Sup35 in heat shock-induced SGs, we found out that its prion-like domain is not involved in the SGs assembly. Factors eEF3, eEF1Bγ2 and eRF1 were accumulated and co-localized with Dcp2 foci even upon a milder heat shock at 42°C independently of P-bodies scaffolding proteins. We also show that eEF3 accumulations at 42°C determine sites of the genuine SGs assembly at 46°C. We suggest that identification of translation elongation and termination factors in SGs might help to understand the mechanism of the eIF2α factor phosphorylation-independent repression of translation and SGs assembly.

Membraneless RNA granules originate via phase separation events driven by multivalent interactions. As RNA is the defining component of such granules, we examined how RNA contributes to granule assembly. Expansion of hexanucleotide GGGGCC (G4C2) repeats in the first intron of C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia (C9-ALS/FTD). We describe a biophysical phenomenon whereby G4C2 RNA (rG4C2) promotes the phase separation of RNA granule proteins in vitro and in cells. The ability of rG4C2 to promote phase separation is dependent on repeat length and RNA structure because rG4C2 must assume a G-quadruplex conformation to promote granule assembly. We demonstrate a central role for RNA in promoting phase separations and implicate rG4C2 G-quadruplex structures in the pathogenesis of C9-ALS/FTD.

Stress-induced phosphorylation of eIF2alpha inhibits global protein synthesis to conserve energy for repair of stress-induced damage. Stress-induced translational arrest is observed in cells expressing a nonphosphorylatable eIF2alpha mutant (S51A), which indicates the existence of an alternative pathway of translational control. In this paper, we show that arsenite, heat shock, or ultraviolet irradiation promotes transfer RNA (tRNA) cleavage and accumulation of tRNA-derived, stress-induced small RNAs (tiRNAs). We show that angiogenin, a secreted ribonuclease, is required for stress-induced production of tiRNAs. Knockdown of angiogenin, but not related ribonucleases, inhibits arsenite-induced tiRNA production and translational arrest. In contrast, knockdown of the angiogenin inhibitor RNH1 enhances tiRNA production and promotes arsenite-induced translational arrest. Moreover, recombinant angiogenin, but not RNase 4 or RNase A, induces tiRNA production and inhibits protein synthesis in the absence of exogenous stress. Finally, transfection of angiogenin-induced tiRNAs promotes phospho-eIF2alpha-independent translational arrest. Our results introduce angiogenin and tiRNAs as components of a phospho-eIF2alpha-independent stress response program.