Arrayed CRISPRi library to suppress genes required for Schizosaccharomyces pombe viability.

The fission yeast, Schizosaccharomyces pombe, is an excellent eukaryote model organism for studying essential biological processes. Its genome contains ∼1,200 genes essential for cell viability, most of which are evolutionarily conserved. To study these essential genes, resources enabling conditional perturbation of target genes are required. Here, we constructed comprehensive arrayed libraries of plasmids and strains to knock down essential genes in S. pombe using dCas9-mediated CRISPRi. These libraries cover ∼98% of all essential genes in fission yeast. We estimate that in ∼60% of these strains, transcription of a target gene was repressed so efficiently that cell proliferation was significantly inhibited. To demonstrate the usefulness of these libraries, we performed metabolic analyses with knockdown strains and revealed flexible interaction among metabolic pathways. Libraries established in this study enable comprehensive functional analyses of essential genes in S. pombe and will facilitate the understanding of essential biological processes in eukaryotes.

A meiotic driver hijacks an epigenetic reader to disrupt mitosis in noncarrier offspring.

Killer meiotic drivers (KMDs) are selfish genetic elements that distort Mendelian inheritance by selectively killing meiotic products lacking the KMD element, thereby promoting their own propagation. Although KMDs have been found in diverse eukaryotes, only a limited number of them have been characterized at the molecular level, and their killing mechanisms remain largely unknown. In this study, we identify that a gene previously deemed essential for cell survival in the fission yeast Schizosaccharomyces pombe is a single-gene KMD. This gene, tdk1, kills nearly all tdk1Δ progeny in a tdk1+ × tdk1Δ cross. By analyzing polymorphisms of tdk1 among natural strains, we identify a resistant haplotype, HT3. This haplotype lacks killing ability yet confers resistance to killing by the wild-type tdk1. Proximity labeling experiments reveal an interaction between Tdk1, the protein product of tdk1, and the epigenetic reader Bdf1. Interestingly, the nonkilling Tdk1-HT3 variant does not interact with Bdf1. Cryoelectron microscopy further elucidated the binding interface between Tdk1 and Bdf1, pinpointing mutations within Tdk1-HT3 that disrupt this interface. During sexual reproduction, Tdk1 forms stable Bdf1-binding nuclear foci in all spores after meiosis. These foci persist in germinated tdk1Δ progeny and impede chromosome segregation during mitosis by generating aberrant chromosomal adhesions. This study identifies a KMD that masquerades as an essential gene and reveals the molecular mechanism by which this KMD hijacks cellular machinery to execute killing. Additionally, we unveil that losing the hijacking ability is an evolutionary path for this single-gene KMD to evolve into a nonkilling resistant haplotype.

Structural duality enables a single protein to act as a toxin-antidote pair for meiotic drive.

In sexual reproduction, selfish genetic elements known as killer meiotic drivers (KMDs) bias inheritance by eliminating gametes that do not carry them. The selective killing behavior of most KMDs can be explained by a toxin-antidote model, where a toxin harms all gametes while an antidote provides resistance to the toxin in carriers. This study investigates whether and how the KMD element tdk1 in the fission yeast Schizosaccharomyces pombe deploys this strategy. Intriguingly, tdk1 relies on a single protein product, Tdk1, for both killing and resistance. We show that Tdk1 exists in a nontoxic tetrameric form during vegetative growth and meiosis but transforms into a distinct toxic form in spores. This toxic form acquires the ability to interact with the histone reader Bdf1 and assembles into supramolecular foci that disrupt mitosis in noncarriers after spore germination. In contrast, Tdk1 synthesized during germination of carrier spores is nontoxic and acts as an antidote, dismantling the preformed toxic Tdk1 assemblies. Replacement of the N-terminal region of Tdk1 with a tetramer-forming peptide reveals its dual roles in imposing an autoinhibited tetrameric conformation and facilitating the assembly of supramolecular foci when autoinhibition is released. Moreover, we successfully reconstituted a functional KMD element by combining a construct that exclusively expresses Tdk1 during meiosis ("toxin-only") with another construct that expresses Tdk1 specifically during germination ("antidote-only"). This work uncovers a remarkable example of a single protein employing structural duality to form a toxin-antidote pair, expanding our understanding of the mechanisms underlying toxin-antidote systems.

Quantitative proteomics and phosphoproteomics profiling of meiotic divisions in the fission yeast Schizosaccharomyces pombe.

In eukaryotes, chromosomal DNA is equally distributed to daughter cells during mitosis, whereas the number of chromosomes is halved during meiosis. Despite considerable progress in understanding the molecular mechanisms that regulate mitosis, there is currently a lack of complete understanding of the molecular mechanisms regulating meiosis. Here, we took advantage of the fission yeast Schizosaccharomyces pombe, for which highly synchronous meiosis can be induced, and performed quantitative proteomics and phosphoproteomics analyses to track changes in protein expression and phosphorylation during meiotic divisions. We compared the proteomes and phosphoproteomes of exponentially growing mitotic cells with cells harvested around meiosis I, or meiosis II in strains bearing either the temperature-sensitive pat1-114 allele or conditional ATP analog-sensitive pat1-as2 allele of the Pat1 kinase. Comparing pat1-114 with pat1-as2 also allowed us to investigate the impact of elevated temperature (25 °C versus 34 °C) on meiosis, an issue that sexually reproducing organisms face due to climate change. Using TMTpro 18plex labeling and phosphopeptide enrichment strategies, we performed quantification of a total of 4673 proteins and 7172 phosphosites in S. pombe. We found that the protein level of 2680 proteins and the rate of phosphorylation of 4005 phosphosites significantly changed during progression of S. pombe cells through meiosis. The proteins exhibiting changes in expression and phosphorylation during meiotic divisions were represented mainly by those involved in the meiotic cell cycle, meiotic recombination, meiotic nuclear division, meiosis I, centromere clustering, microtubule cytoskeleton organization, ascospore formation, organonitrogen compound biosynthetic process, carboxylic acid metabolic process, gene expression, and ncRNA processing, among others. In summary, our findings provide global overview of changes in the levels and phosphorylation of proteins during progression of S. pombe cells through meiosis at normal and elevated temperatures, laying the groundwork for further elucidation of the functions and importance of specific proteins and their phosphorylation in regulating meiotic divisions in this yeast.

Evolutionary Modes of wtf Meiotic Driver Genes in Schizosaccharomyces pombe.

Killer meiotic drivers are a class of selfish genetic elements that bias inheritance in their favor by destroying meiotic progeny that do not carry them. How killer meiotic drivers evolve is not well understood. In the fission yeast, Schizosaccharomyces pombe, the largest gene family, known as the wtf genes, is a killer meiotic driver family that causes intraspecific hybrid sterility. Here, we investigate how wtf genes evolve using long-read-based genome assemblies of 31 distinct S. pombe natural isolates, which encompass the known genetic diversity of S. pombe. Our analysis, involving nearly 1,000 wtf genes in these isolates, yields a comprehensive portrayal of the intraspecific diversity of wtf genes. Leveraging single-nucleotide polymorphisms in adjacent unique sequences, we pinpoint wtf gene-containing loci that have recently undergone gene conversion events and infer their ancestral state. These events include the revival of wtf pseudogenes, lending support to the notion that gene conversion plays a role in preserving this gene family from extinction. Moreover, our investigation reveals that solo long terminal repeats of retrotransposons, frequently found near wtf genes, can act as recombination arms, influencing the upstream regulatory sequences of wtf genes. Additionally, our exploration of the outer boundaries of wtf genes uncovers a previously unrecognized type of directly oriented repeats flanking wtf genes. These repeats may have facilitated the early expansion of the wtf gene family in S. pombe. Our findings enhance the understanding of the mechanisms influencing the evolution of this killer meiotic driver gene family.

Ribosomes hibernate on mitochondria during cellular stress.

Cell survival under nutrient-deprived conditions relies on cells' ability to adapt their organelles and rewire their metabolic pathways. In yeast, glucose depletion induces a stress response mediated by mitochondrial fragmentation and sequestration of cytosolic ribosomes on mitochondria. This cellular adaptation promotes survival under harsh environmental conditions; however, the underlying mechanism of this response remains unknown. Here, we demonstrate that upon glucose depletion protein synthesis is halted. Cryo-electron microscopy structure of the ribosomes show that they are devoid of both tRNA and mRNA, and a subset of the particles depicted a conformational change in rRNA H69 that could prevent tRNA binding. Our in situ structural analyses reveal that the hibernating ribosomes tether to fragmented mitochondria and establish eukaryotic-specific, higher-order storage structures by assembling into oligomeric arrays on the mitochondrial surface. Notably, we show that hibernating ribosomes exclusively bind to the outer mitochondrial membrane via the small ribosomal subunit during cellular stress. We identify the ribosomal protein Cpc2/RACK1 as the molecule mediating ribosomal tethering to mitochondria. This study unveils the molecular mechanism connecting mitochondrial stress with the shutdown of protein synthesis and broadens our understanding of cellular responses to nutrient scarcity and cell quiescence.

Clr4SUV39H1 ubiquitination and non-coding RNA mediate transcriptional silencing of heterochromatin via Swi6 phase separation.

Transcriptional silencing by RNAi paradoxically relies on transcription, but how the transition from transcription to silencing is achieved has remained unclear. The Cryptic Loci Regulator complex (CLRC) in Schizosaccharomyces pombe is a cullin-ring E3 ligase required for silencing that is recruited by RNAi. We found that the E2 ubiquitin conjugating enzyme Ubc4 interacts with CLRC and mono-ubiquitinates the histone H3K9 methyltransferase Clr4SUV39H1, promoting the transition from co-transcriptional gene silencing (H3K9me2) to transcriptional gene silencing (H3K9me3). Ubiquitination of Clr4 occurs in an intrinsically disordered region (Clr4IDR), which undergoes liquid droplet formation in vitro, along with Swi6HP1 the effector of transcriptional gene silencing. Our data suggests that phase separation is exquisitely sensitive to non-coding RNA (ncRNA) which promotes self-association of Clr4, chromatin association, and di-, but not tri- methylation instead. Ubc4-CLRC also targets the transcriptional co-activator Bdf2BRD4, down-regulating centromeric transcription and small RNA (sRNA) production. The deubiquitinase Ubp3 counteracts both activities.

Kinesin-5/Cut7 C-terminal tail phosphorylation is essential for microtubule sliding force and bipolar mitotic spindle assembly.

Kinesin-5 motors play an essential role during mitotic spindle assembly in many organisms1,2,3,4,5,6,7,8,9,10,11: they crosslink antiparallel spindle microtubules, step toward plus ends, and slide the microtubules apart.12,13,14,15,16,17 This activity separates the spindle poles and chromosomes. Kinesin-5s are not only plus-end-directed but can walk or be carried toward MT minus ends,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34 where they show enhanced localization.3,5,7,27,29,32 The kinesin-5 C-terminal tail interacts with and regulates the motor, affecting structure, motility, and sliding force of purified kinesin-535,36,37 along with motility and spindle assembly in cells.27,38,39 The tail contains phosphorylation sites, particularly in the conserved BimC box.6,7,40,41,42,43,44 Nine mitotic tail phosphorylation sites were identified in the kinesin-5 motor of the fission yeast Schizosaccharomyces pombe,45,46,47,48 suggesting that multi-site phosphorylation may regulate kinesin-5s. Here, we show that mutating all nine sites to either alanine or glutamate causes temperature-sensitive lethality due to a failure of bipolar spindle assembly. We characterize kinesin-5 localization and sliding force in the spindle based on Cut7-dependent microtubule minus-end protrusions in cells lacking kinesin-14 motors.39,49,50,51,52 Imaging and computational modeling show that Cut7p simultaneously moves toward the minus ends of protrusion MTs and the plus ends of spindle midzone MTs. Phosphorylation mutants show dramatic decreases in protrusions and sliding force. Comparison to a model of force to create protrusions suggests that tail truncation and phosphorylation mutants decrease Cut7p sliding force similarly to tail-truncated human Eg5.36 Our results show that C-terminal tail phosphorylation is required for kinesin-5/Cut7 sliding force and bipolar spindle assembly in fission yeast.

Actomyosin clusters as active units shaping living matter.

Stress generation by the actin cytoskeleton shapes cells and tissues. Despite impressive progress in live imaging and quantitative physical descriptions of cytoskeletal network dynamics, the connection between processes at molecular scales and spatiotemporal patterns at the cellular scale is still unclear. Here, we review studies reporting actomyosin clusters of micrometre size and with lifetimes of several minutes in a large number of organisms, ranging from fission yeast to humans. Such structures have also been found in reconstituted systems in vitro and in theoretical analyses of cytoskeletal dynamics. We propose that tracking these clusters could provide a simple readout for characterising living matter. Spatiotemporal patterns of clusters could serve as determinants of morphogenetic processes that have similar roles in diverse organisms.

Negative regulation of APC/C activation by MAPK-mediated attenuation of Cdc20Slp1 under stress.

Mitotic anaphase onset is a key cellular process tightly regulated by multiple kinases. The involvement of mitogen-activated protein kinases (MAPKs) in this process has been established in Xenopus egg extracts. However, the detailed regulatory cascade remains elusive, and it is also unknown whether the MAPK-dependent mitotic regulation is evolutionarily conserved in the single-cell eukaryotic organisms such as fission yeast (Schizosaccharomyces pombe). Here, we show that two MAPKs in S. pombe indeed act in concert to restrain anaphase-promoting complex/cyclosome (APC/C) activity upon activation of the spindle assembly checkpoint (SAC). One MAPK, Pmk1, binds to and phosphorylates Slp1Cdc20, the co-activator of APC/C. Phosphorylation of Slp1Cdc20 by Pmk1, but not by Cdk1, promotes its subsequent ubiquitylation and degradation. Intriguingly, Pmk1-mediated phosphorylation event is also required to sustain SAC under environmental stress. Thus, our study establishes a new underlying molecular mechanism of negative regulation of APC/C by MAPK upon stress stimuli, and provides a previously unappreciated framework for regulation of anaphase entry in eukaryotic cells.

Cdc42 mobility and membrane flows regulate fission yeast cell shape and survival.

Polarized exocytosis induced by local Cdc42 GTPase activity results in membrane flows that deplete low-mobility membrane-associated proteins. A reaction-diffusion particle model comprising Cdc42 positive feedback activation, hydrolysis by GTPase-activating proteins (GAPs), and flow-induced displacement by exo/endocytosis shows that flow-induced depletion of low mobility GAPs promotes polarization. We modified Cdc42 mobility in Schizosaccharomyces pombe by replacing its prenylation site with 1, 2 or 3 repeats of the Rit C-terminal membrane-binding domain (ritC), yielding alleles with progressively lower mobility and increased flow-coupling. While Cdc42-1ritC cells are viable and polarized, Cdc42-2ritC polarize poorly and Cdc42-3ritC are inviable, in agreement with model's predictions. Deletion of Cdc42 GAPs restores viability to Cdc42-3ritC cells, verifying the model's prediction that GAP deletion increases Cdc42 activity at the expense of polarization. Our work demonstrates how membrane flows are an integral part of Cdc42-driven pattern formation and require Cdc42-GTP to turn over faster than the surface on which it forms.

Mild Heat Stress Alters the Physical State and Structure of Membranes in Triacylglycerol-Deficient Fission Yeast, Schizosaccharomyces pombe.

We investigated whether the elimination of two major enzymes responsible for triacylglycerol synthesis altered the structure and physical state of organelle membranes under mild heat shock conditions in the fission yeast, Schizosaccharomyces pombe. Our study revealed that key intracellular membrane structures, lipid droplets, vacuoles, the mitochondrial network, and the cortical endoplasmic reticulum were all affected in mutant fission yeast cells under mild heat shock but not under normal growth conditions. We also obtained direct evidence that triacylglycerol-deficient cells were less capable than wild-type cells of adjusting their membrane physical properties during thermal stress. The production of thermoprotective molecules, such as HSP16 and trehalose, was reduced in the mutant strain. These findings suggest that an intact system of triacylglycerol metabolism significantly contributes to membrane protection during heat stress.

Cross-regulations of two connected domains form a mechanical circuit for steady force transmission during clathrin-mediated endocytosis.

Mechanical forces are transmitted from the actin cytoskeleton to the membrane during clathrin-mediated endocytosis (CME) in the fission yeast Schizosaccharomyces pombe. End4p directly transmits force in CME by binding to both the membrane (through the AP180 N-terminal homology [ANTH] domain) and F-actin (through the talin-HIP1/R/Sla2p actin-tethering C-terminal homology [THATCH] domain). We show that 7 pN force is required for stable binding between THATCH and F-actin. We also characterized a domain in End4p, Rend (rod domain in End4p), that resembles R12 of talin. Membrane localization of Rend primes the binding of THATCH to F-actin, and force-induced unfolding of Rend at 15 pN terminates the transmission of force. We show that the mechanical properties (mechanical stability, unfolding extension, hysteresis) of Rend and THATCH are tuned to form a circuit for the initiation, transmission, and termination of force between the actin cytoskeleton and membrane. The mechanical circuit by Rend and THATCH may be conserved and coopted evolutionarily in cell adhesion complexes.

Nitrogen signaling factor triggers a respiration-like gene expression program in fission yeast.

Microbes have evolved intricate communication systems that enable individual cells of a population to send and receive signals in response to changes in their immediate environment. In the fission yeast Schizosaccharomyces pombe, the oxylipin nitrogen signaling factor (NSF) is part of such communication system, which functions to regulate the usage of different nitrogen sources. Yet, the pathways and mechanisms by which NSF acts are poorly understood. Here, we show that NSF physically interacts with the mitochondrial sulfide:quinone oxidoreductase Hmt2 and that it prompts a change from a fermentation- to a respiration-like gene expression program without any change in the carbon source. Our results suggest that NSF activity is not restricted to nitrogen metabolism alone and that it could function as a rheostat to prepare a population of S. pombe cells for an imminent shortage of their preferred nutrients.

Fission yeast Duc1 links to ER-PM contact sites and influences PM lipid composition and cytokinetic ring anchoring.

Cytokinesis is the final stage of the cell cycle that results in the physical separation of daughter cells. To accomplish cytokinesis, many organisms build an actin- and myosin-based cytokinetic ring (CR) that is anchored to the plasma membrane (PM). Defects in CR-PM anchoring can arise when the PM lipid phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2] is depleted. In Schizosaccharomyces pombe, reduced PM PI(4,5)P2 results in a CR that cannot maintain a medial position and slides toward one cell end, resulting in two differently sized daughter cells. S. pombe PM PI(4,5)P2 is synthesized by the phosphatidylinositol 4-phosphate 5-kinase (PI5-kinase) Its3, but what regulates this enzyme to maintain appropriate PM PI(4,5)P2 levels in S. pombe is not known. To identify Its3 regulators, we used proximity-based biotinylation, and the uncharacterized protein Duc1 was specifically detected. We discovered that Duc1 decorates the PM except at the cell division site and that its unique localization pattern is dictated by binding to the endoplasmic reticulum (ER)-PM contact site proteins Scs2 and Scs22. Our evidence suggests that Duc1 also binds PI(4,5)P2 and helps enrich Its3 at the lateral PM, thereby promoting PM PI(4,5)P2 synthesis and robust CR-PM anchoring.

An improved tetracycline-inducible expression system for fission yeast.

The ability to manipulate gene expression is valuable for elucidating gene function. In the fission yeast Schizosaccharomyces pombe, the most widely used regulatable expression system is the nmt1 promoter and its two attenuated variants. However, these promoters have limitations, including a long lag, incompatibility with rich media and unsuitability for non-dividing cells. Here, we present a tetracycline-inducible system free of these shortcomings. Our system features the enotetS promoter, which achieves a similar induced level and a higher induction ratio compared to the nmt1 promoter, without exhibiting a lag. Additionally, our system includes four weakened enotetS variants, offering an expression range similar to that of the nmt1 series promoters but with more intermediate levels. To enhance usability, each promoter is combined with a Tet-repressor-expressing cassette in an integration plasmid. Importantly, our system can be used in non-dividing cells, enabling the development of a synchronous meiosis induction method with high spore viability. Moreover, our system allows for the shutdown of gene expression and the generation of conditional loss-of-function mutants. This system provides a versatile and powerful tool for manipulating gene expression in fission yeast.

3,3'-Diindolylmethane disrupts the endoplasmic reticulum and nuclear envelope in Schizosaccharomyces pombe.

3,3'-Diindolylmethane is recognized for its anti-cancer activities in various pathways, though its mechanism remains to be fully elucidated. Previous studies have shown that 3,3'-Diindolylmethane disturbed the localization of Cut11, a nuclear pore complex subunit in Schizosaccharomyces pombe. This study further reveals that in Schizosaccharomyces pombe, 3,3'-Diindolylmethane also disrupts other components of nuclear envelope, causing GFP-NLS leakage, making it evident that 3,3'-Diindolylmethane disrupts the nuclear envelope. 3,3'-Diindolylmethane also disturbs the localization of GFP-ADEL and Ost4, which are endoplasmic reticulum lumen proteins and membrane proteins respectively, suggesting the function of 3,3'-Diindolylmethane on endoplasmic reticulum disturbance. The nuclear envelope repairment, normal nuclear envelope physical properties, and lipid metabolism homeostasis are crucial for cell survival in the presence of 3,3'-Diindolylmethane. These findings provide new insights into the understanding and development of 3,3'-Diindolylmethane as an anti-cancer agent.

Characterization of Shy1, the Schizosaccharomyces pombe homolog of human SURF1.

Cytochrome c oxidase (complex IV) is the terminal enzyme in the mitochondrial respiratory chain. As a rare neurometabolic disorder caused by mutations in the human complex IV assembly factor SURF1, Leigh Syndrome (LS) is associated with complex IV deficiency. In this study, we comprehensively characterized Schizosaccharomyces pombe Shy1, the homolog of human SURF1. Bioinformatics analysis revealed that Shy1 contains a conserved SURF1 domain that links to the biogenesis of complex IV and shares high structural similarity with its homologs in Saccharomyces cerevisiae and humans. Our study showed that Shy1 is required for the expression of mtDNA-encoded genes and physically interacts with structural subunits and assembly factors of complex IV. Interestingly, Rip1, the subunit of ubiquinone-cytochrome c oxidoreductase or cytochrome bc1 complex (complex III), can also co-immunoprecipitate with Shy1, suggesting Shy1 may be involved in the assembly of the mitochondrial respiratory chain supercomplexes. This conclusion is further corroborated by our BN-PAGE analysis. Unlike its homologs, deletion of shy1 does not critically disrupt respiratory chain assembly, indicating the presence of the compensatory mechanism(s) within S. pombe that ensure mitochondrial functionality. Collectively, our investigation elucidates that Shy1 plays a pivotal role in the sustainability of the regular function of mitochondria by participating in the assembly of complex IV in S. pombe.

Differential Cytoophidium Assembly between Saccharomyces cerevisiae and Schizosaccharomyces pombe.

The de novo synthesis of cytidine 5'-triphosphate (CTP) is catalyzed by the enzyme CTP synthase (CTPS), which is known to form cytoophidia across all three domains of life. In this study, we use the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe as model organisms to compare cytoophidium assembly under external environmental and intracellular CTPS alterations. We observe that under low and high temperature conditions, cytoophidia in fission yeast gradually disassemble, while cytoophidia in budding yeast remain unaffected. The effect of pH changes on cytoophidia maintenance in the two yeast species is different. When cultured in the yeast-saturated cultured medium, cytoophidia in fission yeast disassemble, while cytoophidia in budding yeast gradually form. Overexpression of CTPS results in the presence and maintenance of cytoophidia in both yeast species from the log phase to the stationary phase. In summary, our results demonstrate differential cytoophidium assembly between Saccharomyces cerevisiae and Schizosaccharomyces pombe, the two most studied yeast species.

Uridylation regulates mRNA decay directionality in fission yeast.

Cytoplasmic mRNA decay is effected by exonucleolytic degradation in either the 5' to 3' or 3' to 5' direction. Pervasive terminal uridylation is implicated in mRNA degradation, however, its functional relevance for bulk mRNA turnover remains poorly understood. In this study, we employ genome-wide 3'-RACE (gw3'-RACE) in the model system fission yeast to elucidate the role of uridylation in mRNA turnover. We observe widespread uridylation of shortened poly(A) tails, promoting efficient 5' to 3' mRNA decay and ensuring timely and controlled mRNA degradation. Inhibition of this uridylation process leads to excessive deadenylation and enhanced 3' to 5' mRNA decay accompanied by oligouridylation. Strikingly we found that uridylation of poly(A) tails and oligouridylation of non-polyadenylated substrates are catalysed by different terminal uridyltransferases Cid1 and Cid16 respectively. Our study sheds new light on the intricate regulatory mechanisms underlying bulk mRNA turnover, demonstrating the role of uridylation in modulating mRNA decay pathways.