Stress-specific differences in assembly and composition of stress granules and related foci.

Cells have developed different mechanisms to respond to stress, including the formation of cytoplasmic foci known as stress granules (SGs). SGs are dynamic and formed as a result of stress-induced inhibition of translation. Despite enormous interest in SGs due to their contribution to the pathogenesis of several human diseases, many aspects of SG formation are poorly understood. SGs induced by different stresses are generally assumed to be uniform, although some studies suggest that different SG subtypes and SG-like cytoplasmic foci exist. Here, we investigated the molecular mechanisms of SG assembly and characterized their composition when induced by various stresses. Our data revealed stress-specific differences in composition, assembly and dynamics of SGs and SG-like cytoplasmic foci. Using a set of genetically modified haploid human cells, we determined the molecular circuitry of stress-specific translation inhibition upstream of SG formation and its relation to cell survival. Finally, our studies characterize cytoplasmic stress-induced foci related to, but distinct from, canonical SGs, and also introduce haploid cells as a valuable resource to study RNA granules and translation control mechanisms.

RNA biology of angiogenin: Current state and perspectives.

Angiogenin (ANG) is a secreted ribonuclease best known for its ability to promote formation of blood vessels. Extensive research over many years has elucidated its structure and biophysical properties, although our knowledge of molecular mechanisms underlying ANG-associated biologic processes remains limited. Intriguingly, many of processes require the ribonuclease activity of ANG, thus highlighting the importance of identifying and characterizing RNA targets and intermediates of ANG-mediated endonucleolytic cleavage. While ANG demonstrates ribonuclease activity toward many RNA substrates in vitro, specific target of ANG, namely mature tRNA, was only recently identified in vivo. ANG-mediated tRNA cleavage is an unorthodox manner of generating non-coding RNAs with diverse biologic activities. In addition, the ribonuclease activity of ANG has been reported to be crucial for rRNA transcription. Here we critically discuss various aspects of ANG biology related to its RNase activity and discuss areas in need of further investigation.

Enhanced arginine methylation of programmed cell death 4 protein during nutrient deprivation promotes tumor cell viability.

The role of programmed cell death 4 (PDCD4) in tumor biology is context-dependent. PDCD4 is described as a tumor suppressor, but its coexpression with protein arginine methyltransferase 5 (PRMT5) promotes accelerated tumor growth. Here, we report that PDCD4 is methylated during nutrient deprivation. Methylation occurs because of increased stability of PDCD4 protein as well as increased activity of PRMT5 toward PDCD4. During nutrient deprivation, levels of methylated PDCD4 promote cell viability, which is dependent on an enhanced interaction with eIF4A. Upon recovery from nutrient deprivation, levels of methylated PDCD4 are regulated by phosphorylation, which controls both the localization and stability of methylated PDCD4. This study reveals that, in response to particular environmental cues, the role of PDCD4 is up-regulated and is advantageous for cell viability. These findings suggest that the methylated form of PDCD4 promotes tumor viability during nutrient deprivation, ultimately allowing the tumor to grow more aggressively.

High-resolution genome-wide mapping of chromosome-arm-scale truncations induced by CRISPR-Cas9 editing.

Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) is a powerful tool for introducing targeted mutations in DNA, but recent studies have shown that it can have unintended effects such as structural changes. However, these studies have not yet looked genome wide or across data types. Here we performed a phenotypic CRISPR-Cas9 scan targeting 17,065 genes in primary human cells, revealing a 'proximity bias' in which CRISPR knockouts show unexpected similarities to unrelated genes on the same chromosome arm. This bias was found to be consistent across cell types, laboratories, Cas9 delivery methods and assay modalities, and the data suggest that it is caused by telomeric truncations of chromosome arms, with cell cycle and apoptotic pathways playing a mediating role. Additionally, a simple correction is demonstrated to mitigate this pervasive bias while preserving biological relationships. This previously uncharacterized effect has implications for functional genomic studies using CRISPR-Cas9, with applications in discovery biology, drug-target identification, cell therapies and genetic therapeutics.

Genome sequences of 31 mycobacteriophages isolated on Mycobacterium smegmatis mc2155 at room temperature.

We report the genome sequences of 31 mycobacteriophages isolated on Mycobacterium smegmatis mc2155 at room temperature. The genomes add to the diversity of Clusters A, B, C, G, and K. Collectively, the genomes include 70 novel protein-coding genes that have no close relatives among the actinobacteriophages.

Selective Cleavage at CCA Ends and Anticodon Loops of tRNAs by Stress-Induced RNases.

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.

Translation inhibition and suppression of stress granules formation by cisplatin.

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.

Bisphenol A promotes stress granule assembly and modulates the integrated stress response.

Bisphenol-A (BPA) is a ubiquitous precursor of polycarbonate plastics that is found in the blood and serum of >92% of Americans. While BPA has been well documented to act as a weak estrogen receptor (ER) agonist, its effects on cellular stress are unclear. Here, we demonstrate that high-dose BPA causes stress granules (SGs) in human cells. A common estrogen derivative, ß-estradiol, does not trigger SGs, indicating the mechanism of SG induction is not via the ER pathway. We also tested other structurally related environmental contaminants including the common BPA substitutes BPS and BPF, the industrial chemical 4-nonylphenol (4-NP) and structurally related compounds 4-EP and 4-VP, as well as the pesticide 2,4-dichlorophenoxyacetic acid (2,4-D). The variable results from these related compounds suggest that structural homology is not a reliable predictor of the capacity of a compound to cause SGs. Also, we demonstrate that BPA acts primarily through the PERK pathway to generate canonical SGs. Finally, we show that chronic exposure to a low physiologically relevant dose of BPA suppresses SG assembly upon subsequent acute stress. Interestingly, this SG inhibition does not affect phosphorylation of eIF2α or translation inhibition, thus uncoupling the physical assembly of SGs from translational control. Our work identifies additional effects of BPA beyond endocrine disruption that may have consequences for human health.

The Role of RNA in Biological Phase Separations.

Phase transitions that alter the physical state of ribonucleoprotein particles contribute to the spacial and temporal organization of the densely packed intracellular environment. This allows cells to organize biologically coupled processes as well as respond to environmental stimuli. RNA plays a key role in phase separation events that modulate various aspects of RNA metabolism. Here, we review the role that RNA plays in ribonucleoprotein phase separations.

The role of RNA modifications in the regulation of tRNA cleavage.

Transfer RNA (tRNA) have been harbingers of many paradigms in RNA biology. They are among the first recognized noncoding RNA (ncRNA) playing fundamental roles in RNA metabolism. Although mainly recognized for their role in decoding mRNA and delivering amino acids to the growing polypeptide chain, tRNA also serve as an abundant source of small ncRNA named tRNA fragments. The functional significance of these fragments is only beginning to be uncovered. Early on, tRNA were recognized as heavily post-transcriptionally modified, which aids in proper folding and modulates the tRNA:mRNA anticodon-codon interactions. Emerging data suggest that these modifications play critical roles in the generation and activity of tRNA fragments. Modifications can both protect tRNA from cleavage or promote their cleavage. Modifications to individual fragments may be required for their activity. Recent work has shown that some modifications are critical for stem cell development and that failure to deposit certain modifications has profound effects on disease. This review will discuss how tRNA modifications regulate the generation and activity of tRNA fragments.

Nitric oxide triggers the assembly of "type II" stress granules linked to decreased cell viability.

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.

RNA G-Quadruplexes in Biology: Principles and Molecular Mechanisms.

G-quadruplexes (G4s) are extremely stable DNA or RNA secondary structures formed by sequences rich in guanine. These structures are implicated in many essential cellular processes, and the number of biological functions attributed to them continues to grow. While DNA G4s are well understood on structural and, to some extent, functional levels, RNA G4s and their functions have received less attention. The presence of bona fide RNA G4s in cells has long been a matter of debate. The development of G4-specific antibodies and ligands hinted on their presence in vivo, but recent advances in RNA sequencing coupled with chemical footprinting suggested the opposite. In this review, we will critically discuss the biology of RNA G4s focusing on the molecular mechanisms underlying their proposed functions.

ALS/FTD-Associated C9ORF72 Repeat RNA Promotes Phase Transitions In Vitro and in Cells.

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.

Methods to Classify Cytoplasmic Foci as Mammalian Stress Granules.

Cells are often challenged by sudden environmental changes. Stress Granules (SGs), cytoplasmic ribonucleoprotein complexes that form in cells exposed to stress conditions, are implicated in various aspects of cell metabolism and survival. SGs modulate cellular signaling pathways, post-transcriptional gene expression, and stress response programs. The formation of these mRNA-containing granules is directly connected to cellular translation. SG assembly is triggered by inhibited translation initiation, and SG disassembly is promoted by translation activation or by inhibited translation elongation. This relationship is further highlighted by SG composition. Core SG components are stalled translation pre-initiation complexes, mRNA, and selected RNA-binding Proteins (RBPs). The purpose of SG assembly is to conserve cellular energy by sequestering translationally stalled housekeeping mRNAs, allowing for the enhanced translation of stress-responsive proteins. In addition to the core constituents, such as stalled translation preinitiation complexes, SGs contain a plethora of other proteins and signaling molecules. Defects in SG formation can impair cellular adaptation to stress and can thus promote cell death. SGs and similar RNA-containing granules have been linked to a number of human diseases, including neurodegenerative disorders and cancer, leading to the recent interest in classifying and defining RNA granule subtypes. This protocol describes assays to characterize and quantify mammalian SGs.

Vinca alkaloid drugs promote stress-induced translational repression and stress granule formation.

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.

Protein arginine methyltransferase 5 accelerates tumor growth by arginine methylation of the tumor suppressor programmed cell death 4.

Programmed cell death 4 (PDCD4) has been described as a tumor suppressor, with high expression correlating with better outcomes in a number of cancer types. Yet a substantial number of cancer patients with high PDCD4 in tumors have poor survival, suggesting that oncogenic pathways may inhibit or change PDCD4 function. Here, we explore the significance of PDCD4 in breast cancer and identify protein arginine methyltransferase 5 (PRMT5) as a cofactor that radically alters PDCD4 function. Specifically, we find that coexpression of PDCD4 and PRMT5 in an orthotopic model of breast cancer causes accelerated tumor growth and that this growth phenotype is dependent on both the catalytic activity of PRMT5 and a site of methylation within the N-terminal region of PDCD4. In agreement with the xenograft model, elevated PDCD4 expression was found to correlate with worse outcome within the cohort of breast cancer patients whose tumors contain higher levels of PRMT5. These results reveal a new cofactor for PDCD4 that alters its tumor suppressor functions and point to the utility of PDCD4/PRMT5 status as both a prognostic biomarker and a potential target for chemotherapy.