Full-length direct RNA sequencing uncovers stress-granule dependent RNA decay upon cellular stress.

Cells react to stress by triggering response pathways, leading to extensive alterations in the transcriptome to restore cellular homeostasis. The role of RNA metabolism in shaping the cellular response to stress is vital, yet the global changes in RNA stability under these conditions remain unclear. In this work, we employ direct RNA sequencing with nanopores, enhanced by 5' end adaptor ligation, to comprehensively interrogate the human transcriptome at single-molecule and nucleotide resolution. By developing a statistical framework to identify robust RNA length variations in nanopore data, we find that cellular stress induces prevalent 5' end RNA decay that is coupled to translation and ribosome occupancy. Unlike typical RNA decay models in normal conditions, we show that stress-induced RNA decay is dependent on XRN1 but does not depend on removal of the poly(A) tail. We observed that RNAs undergoing decay are predominantly enriched in the stress granule transcriptome. Inhibition of stress granule formation via genetic ablation of G3BP1 and G3BP2 fully rescues RNA length and suppresses stress-induced decay. Our findings reveal RNA decay as a key determinant of RNA metabolism upon cellular stress and dependent on stress-granule formation.

Deep learning and direct sequencing of labeled RNA captures transcriptome dynamics.

Quantification of the dynamics of RNA metabolism is essential for understanding gene regulation in health and disease. Existing methods rely on metabolic labeling of nascent RNAs and physical separation or inference of labeling through PCR-generated mutations, followed by short-read sequencing. However, these methods are limited in their ability to identify transient decay intermediates or co-analyze RNA decay with cis-regulatory elements of RNA stability such as poly(A) tail length and modification status, at single molecule resolution. Here we use 5-ethynyl uridine (5EU) to label nascent RNA followed by direct RNA sequencing with nanopores. We developed RNAkinet, a deep convolutional and recurrent neural network that processes the electrical signal produced by nanopore sequencing to identify 5EU-labeled nascent RNA molecules. RNAkinet demonstrates generalizability to distinct cell types and organisms and reproducibly quantifies RNA kinetic parameters allowing the combined interrogation of RNA metabolism and cis-acting RNA regulatory elements.

Senescence suppresses the integrated stress response and activates a stress-enhanced secretory phenotype.

Senescence is a state of indefinite cell cycle arrest associated with aging, cancer, and age-related diseases. Here, using label-based mass spectrometry, ribosome profiling and nanopore direct RNA sequencing, we explore the coordinated interaction of translational and transcriptional programs of human cellular senescence. We find that translational deregulation and a corresponding maladaptive integrated stress response (ISR) is a hallmark of senescence that desensitizes senescent cells to stress. We present evidence that senescent cells maintain high levels of eIF2α phosphorylation, typical of ISR activation, but translationally repress production of the stress response transcription factor 4 (ATF4) by ineffective bypass of the inhibitory upstream open reading frames. Surprisingly, ATF4 translation remains inhibited even after acute proteotoxic and amino acid starvation stressors, resulting in a highly diminished stress response. Furthermore, absent a response, stress augments the senescence secretory phenotype, thus intensifying a proinflammatory state that exacerbates disease. Our results reveal a novel mechanism that senescent cells exploit to evade an adaptive stress response and remain viable.

Ribonucleic Acid-Mediated Control of Protein Translation Under Stress.

Significance: The need of cells to constantly respond to endogenous and exogenous stress has necessitated the evolution of pathways to counter the deleterious effects of stress and to restore cellular homeostasis. The inability to activate a timely and adequate response can lead to disease and is a hallmark of aging. Besides protein-coding genes, cells contain a plethora of noncoding regulatory elements that allow cells to respond rapidly and efficiently to external stimuli by activating highly specific and tightly controlled mechanisms. Many of these programs converge on the regulation of translation, one of the most energy-consuming processes in cells. Recent Advances: The noncoding dimension of translational regulation includes short and long noncoding ribonucleic acids (ncRNAs), as well as messenger RNA features, such as the sequence and modification status of the 5' and 3' untranslated regions (UTRs), that do not change the amino acid sequence of the produced protein. Critical Issues: In this review, we discuss the regulatory role of the nonprotein-coding components of translation under stress, particularly oxidative stress. We conclude that the regulation of translation through ncRNAs, UTRs, and nucleotide modifications is emerging as a critical component of the stress response. Future Directions: Further areas of study using long-read sequencing technologies will be discussed. Antioxid. Redox Signal. 39, 374-389.

Biology of Stress Responses in Aging.

On April 28th, 2022, a group of scientific leaders gathered virtually to discuss molecular and cellular mechanisms of responses to stress. Conditions of acute, high-intensity stress are well documented to induce a series of adaptive responses that aim to promote survival until the stress has dissipated and then guide recovery. However, high-intensity or persistent stress that goes beyond the cell's compensatory capacity are countered with resilience strategies that are not completely understood. These adaptative strategies, which are an essential component of the study of aging biology, were the theme of the meeting. Specific topics discussed included mechanisms of proteostasis, such as the unfolded protein response (UPR) and the integrated stress response (ISR), as well as mitochondrial stress and lysosomal stress responses. Attention was also given to regulatory mechanisms and associated biological processes linked to age-related conditions, such as muscle loss and regeneration, cancer, senescence, sleep quality, and degenerative disease, with a general focus on the relevance of stress responses to frailty. We summarize the concepts and potential future directions that emerged from the discussion and highlight their relevance to the study of aging and age-related chronic diseases.

A BDNF-TrkB autocrine loop enhances senescent cell viability.

Cellular senescence is characterized by cell cycle arrest, resistance to apoptosis, and a senescence-associated secretory phenotype (SASP) whereby cells secrete pro-inflammatory and tissue-remodeling factors. Given that the SASP exacerbates age-associated pathologies, some aging interventions aim at selectively eliminating senescent cells. In this study, a drug library screen uncovered TrkB (NTRK2) inhibitors capable of triggering apoptosis of several senescent, but not proliferating, human cells. Senescent cells expressed high levels of TrkB, which supported senescent cell viability, and secreted the TrkB ligand BDNF. The reduced viability of senescent cells after ablating BDNF signaling suggested an autocrine function for TrkB and BDNF, which activated ERK5 and elevated BCL2L2 levels, favoring senescent cell survival. Treatment with TrkB inhibitors reduced the accumulation of senescent cells in aged mouse organs. We propose that the activation of TrkB by SASP factor BDNF promotes cell survival and could be exploited therapeutically to reduce the senescent-cell burden.

Characterizing and circumventing sequence restrictions for synthesis of circular RNA in vitro.

Just as eukaryotic circular RNA (circRNA) is a product of intracellular backsplicing, custom circRNA can be synthesized in vitro using a transcription template in which transposed halves of a split group I intron flank the sequence of the RNA to be circularized. Such permuted intron-exon (PIE) constructs have been used to produce circRNA versions of ribozymes, mimics of viral RNA motifs, a streptavidin aptamer, and protein expression vectors for genetic engineering and vaccine development. One limitation of this approach is the obligatory incorporation of small RNA segments (E1 and E2) into nascent circRNA at the site of end-joining. This restriction may preclude synthesis of small circRNA therapeutics and RNA nanoparticles that are sensitive to extraneous sequence, as well as larger circRNA mimics whose sequences must precisely match those of the native species on which they are modelled. In this work, we used serial mutagenesis and in vitro selection to determine how varying E1 and E2 sequences in a thymidylate synthase (td) group I intron PIE transcription template construct affects circRNA synthesis yield. Based on our collective findings, we present guidelines for the design of custom-tailored PIE transcription templates from which synthetic circRNAs of almost any sequence may be efficiently synthesized.

Translational Control during Cellular Senescence.

Senescence is a state of long-term cell cycle arrest that arises in cells that have incurred sublethal damage. While senescent cells no longer replicate, they remain metabolically active and further develop unique and stable phenotypes that are not present in proliferating cells. On one hand, senescent cells increase in size, maintain an active mTORC1 complex, and produce and secrete a substantial amount of inflammatory proteins as part of the senescence-associated secretory phenotype (SASP). On the other hand, these progrowth phenotypes contrast with the p53-mediated growth arrest typical of senescent cells that is associated with nucleolar stress and an inhibition of rRNA processing and ribosome biogenesis. In sum, translation in senescent cells paradoxically comprises both a global repression of translation triggered by DNA damage and a select increase in the translation of specific proteins, including SASP factors.

Mutations in the anticodon stem of tRNA cause accumulation and Met22-dependent decay of pre-tRNA in yeast.

During tRNA maturation in yeast, aberrant pre-tRNAs are targeted for 3'-5' degradation by the nuclear surveillance pathway, and aberrant mature tRNAs are targeted for 5'-3' degradation by the rapid tRNA decay (RTD) pathway. RTD is catalyzed by the 5'-3' exonucleases Xrn1 and Rat1, which act on tRNAs with an exposed 5' end due to the lack of certain body modifications or the presence of destabilizing mutations in the acceptor stem, T-stem, or tRNA fold. RTD is inhibited by mutation of MET22, likely due to accumulation of the Met22 substrate adenosine 3',5' bis-phosphate, which inhibits 5'-3' exonucleases. Here we provide evidence for a new tRNA quality control pathway in which intron-containing pre-tRNAs with destabilizing mutations in the anticodon stem are targeted for Met22-dependent pre-tRNA decay (MPD). Multiple SUP4οc anticodon stem variants that are subject to MPD each perturb the bulge-helix-bulge structure formed by the anticodon stem-loop and intron, which is important for splicing, resulting in substantial accumulation of end-matured unspliced pre-tRNA as well as pre-tRNA decay. Mutations that restore exon-intron structure commensurately reduce pre-tRNA accumulation and MPD. The MPD pathway can contribute substantially to decay of anticodon stem variants, since pre-tRNA decay is largely suppressed by removal of the intron or by restoration of exon-intron structure, each also resulting in increased tRNA levels. The MPD pathway is general as it extends to variants of tRNATyr(GUA) and tRNASer(CGA) These results demonstrate that the integrity of the anticodon stem-loop and the efficiency of tRNA splicing are monitored by a quality control pathway.

Widespread temperature sensitivity and tRNA decay due to mutations in a yeast tRNA.

Microorganisms have universally adapted their RNAs and proteins to survive at a broad range of temperatures and growth conditions. However, for RNAs, there is little quantitative understanding of the effects of mutations on function at high temperatures. To understand how variant tRNA function is affected by temperature change, we used the tRNA nonsense suppressor SUP4oc of the yeast Saccharomyces cerevisiae to perform a high-throughput quantitative screen of tRNA function at two different growth temperatures. This screen yielded comparative values for 9243 single and double variants. Surprisingly, despite the ability of S. cerevisiae to grow at temperatures as low as 15°C and as high as 39°C, the vast majority of variants that could be scored lost half or more of their function when evaluated at 37°C relative to 28°C. Moreover, temperature sensitivity of a tRNA variant was highly associated with its susceptibility to the rapid tRNA decay (RTD) pathway, implying that RTD is responsible for most of the loss of function of variants at higher temperature. Furthermore, RTD may also operate in a met22Δ strain, which was previously thought to fully inhibit RTD. Consistent with RTD acting to degrade destabilized tRNAs, the stability of a tRNA molecule can be used to predict temperature sensitivity with high confidence. These findings offer a new perspective on the stability of tRNA molecules and their quality control at high temperature.

Methodology for the High-Throughput Identification and Characterization of tRNA Variants That Are Substrates for a tRNA Decay Pathway.

The rapid tRNA decay (RTD) pathway is a tRNA quality control pathway known to degrade several specific hypomodified or destabilized tRNAs in the yeast Saccharomyces cerevisiae. In this chapter, we describe seven methods for identifying RTD substrates, with a focus on two new approaches: a high-throughput approach that utilizes a suppressor tRNA library, fluorescence-activated cell sorting, and deep sequencing, and has greatly expanded the known range of RTD substrates; and a poison primer extension assay that allows for the measurement of levels of suppressor tRNA variants, even in the presence of highly similar endogenous tRNAs. We also discuss different applications of the use of the high-throughput and poison primer extension methodologies for different problems in tRNA biology.

Identification of the determinants of tRNA function and susceptibility to rapid tRNA decay by high-throughput in vivo analysis.

Sequence variation in tRNA genes influences the structure, modification, and stability of tRNA; affects translation fidelity; impacts the activity of numerous isodecoders in metazoans; and leads to human diseases. To comprehensively define the effects of sequence variation on tRNA function, we developed a high-throughput in vivo screen to quantify the activity of a model tRNA, the nonsense suppressor SUP4oc of Saccharomyces cerevisiae. Using a highly sensitive fluorescent reporter gene with an ochre mutation, fluorescence-activated cell sorting of a library of SUP4oc mutant yeast strains, and deep sequencing, we scored 25,491 variants. Unexpectedly, SUP4oc tolerates numerous sequence variations, accommodates slippage in tertiary and secondary interactions, and exhibits genetic interactions that suggest an alternative functional tRNA conformation. Furthermore, we used this methodology to define tRNA variants subject to rapid tRNA decay (RTD). Even though RTD normally degrades tRNAs with exposed 5' ends, mutations that sensitize SUP4oc to RTD were found to be located throughout the sequence, including the anti-codon stem. Thus, the integrity of the entire tRNA molecule is under surveillance by cellular quality control machinery. This approach to assess activity at high throughput is widely applicable to many problems in tRNA biology.

Extracellular gluco-oligosaccharide degradation by Caulobacter crescentus.

The oligotrophic bacterium Caulobacter crescentus has the ability to metabolize various organic molecules, including plant structural carbohydrates, as a carbon source. The nature of ß-glucosidase (BGL)-mediated gluco-oligosaccharide degradation and nutrient transport across the outer membrane in C. crescentus was investigated. All gluco-oligosaccharides tested (up to celloheptose) supported growth in M2 minimal media but not cellulose or CM-cellulose. The periplasmic and outer membrane fractions showed highest BGL activity, but no significant BGL activity was observed in the cytosol or extracellular medium. Cells grown in cellobiose showed expression of specific BGLs and TonB-dependent receptors (TBDRs). Carbonyl cyanide 3-chlorophenylhydrazone lowered the rate of cell growth in cellobiose but not in glucose, indicating potential cellobiose transport into the cell by a proton motive force-dependent process, such as TBDR-dependent transport, and facilitated diffusion of glucose across the outer membrane via specific porins. These results suggest that C. crescentus acquires carbon from cellulose-derived gluco-oligosaccharides found in the environment by extracellular and periplasmic BGL activity and TBDR-mediated transport. This report on extracellular degradation of gluco-oligosaccharides and methods of nutrient acquisition by C. crescentus supports a broader suite of carbohydrate metabolic capabilities suggested by the C. crescentus genome sequence that until now have not been reported.