Molecular and Cellular Biology

Growth recovery from serum starvation requires the activation of PI3 kinase (PI3K)-PDK1-Akt-mTOR pathways. Claspin plays multiple important roles in regulation of DNA replication as a mediator for the cellular response to replication stress, an integral replication fork factor that facilitates replication fork progression and a factor that promotes initiation by recruiting Cdc7 kinase. Here, we report a novel role of Claspin in growth recovery from serum starvation. In the absence of Claspin, cells do not proceed into S phase and eventually die. Claspin interacts with PI3K and mTOR, and is required for activation of PI3K-PDK1-mTOR and for that of mTOR downstream factors, p70S6K and 4E-BP1, but not for p38 MAPK cascade during the recovery from serum starvation. PDK1 interacts with Claspin, notably with CKBD, in a manner dependent on phosphorylation of the latter protein, and is required for interaction of mTOR with Claspin. p53 and ROS (Reactive Oxygen Species) inhibitors increased survival of Claspin-deficient cells released from serum starvation. Thus, Claspin plays a novel role as a mediator/protein platform for nutrition-induced proliferation/survival signaling by activating the mTOR pathway.

Telomeres perform multiple functions to maintain genome stability, including telomere length regulation, chromosome end protection, and meiotic chromosome dynamics. These functions are governed by shelterin, a telomere-binding protein complex. Here, we show that efficient pre-mRNA splicing of the Schizosaccharomyces pombe shelterin components Rap1 and Poz1 ensures sufficient protein levels, which are critical for telomere maintenance. Our analyses revealed that Tls1 and Cay1 act at distinct steps in splicing, specifically affecting rap1 and poz1 transcripts: Tls1 strongly interacts with Brr2 (a splicing factor), whereas Cay1 preferentially associates with introns. Accordingly, deletion of tls1 and cay1 synergistically impaired splicing of rap1 and poz1 transcripts and reduced their protein levels, leading to abnormal telomere elongation. Removal of introns from the rap1 and poz1 genes restored normal protein levels and telomere length, confirming that defective splicing underlies these defects. Analyses of the phenotypes of single and double tls1{Delta} and cay1{Delta} mutants revealed that different telomere functions vary in their dependence on Rap1 levels: telomere length regulation and, to a lesser extent, meiosis require higher protein abundance, whereas chromosome end protection can be sustained with minimal amounts. These findings reveal a hierarchical requirement for Rap1 across telomere functions and establish a framework for understanding how splicing-dependent regulation of shelterin components coordinates multiple aspects of telomere biology.

The fine balance between cellular homeostasis and stress response is crucial for cell survival. In this study, we identify translation regulation of specific autophagy (ATGs) genes by translation repressor Sbp1 upon hydroxyurea-mediated genotoxic stress. Sbp1 localizes to reversible, mRNA-containing granules specifically upon HU stress in RNA recognition motif 1 (RRM1) and RGG motif-dependent manner. Granule localization is independent of eIF4G1 binding despite increased arginine methylation. Deletion of SBP1 increased the tolerance to HU. RNA sequencing of polysome fractions identified ATG1, 2, 9 mRNAs being translationally upregulated in{Delta} sbp1 upon HU stress. Translation of TEL1 and MEC1, the upstream activators of autophagy, also increased in{Delta} sbp1. Concomitantly, increased autophagy and decreased NHEJ repair in{Delta} sbp1 cells was observed. Together, these findings identify Sbp1 as a regulator of the autophagy pathway in response to genotoxic stress by modulating autophagy at two levels, possibly mediated by its ability to localize to granules.

The Fanconi anemia (FA) protein FANCD2, and MCM8/9 heterohexameric helicase complex are critical for maintaining genomic integrity in response to replication stress. However, the nature of their relationship remains unclear. Here, we show that MCM8/9 physically interacts and functionally cooperates with FANCD2 during the repair of DNA interstrand crosslinks (ICLs). Using immunofluorescence and co-immunoprecipitation studies, we show that MCM8/9 interacts with FANCD2 through its core domain, independently of DNA. FANCD2 is essential for the recruitment of MCM9 to ICL damage induced nuclear foci and acts downstream the FANCD2I monoubiquitination. Although MCM8/9 foci formation requires its intact ATPase activity, BRCv motif and HROB, these are not required for FANCD2 binding, highlighting a distinction between physical interaction and functional activation. Interestingly, FANCD2 foci formation increase in MCM8 or MCM9 knockout cells, suggesting that MCM8/9 functions to mitigate replication associate stress. {gamma}H2AX DNA damage assays and cell survival assays show that combined loss of MCM9 and FANCD2 do not cause any additive DNA damage beyond individual knockouts, indicating an epistatic relationship and suggests they function in the same DNA repair pathway. Together, our findings identify MCM8/9 as a downstream effector of the FA pathway critical for resolving ICL induced DNA damage. HighlightsO_LIMCM8/9 interacts and colocalize with FANCD2 upon ICL DNA damage. C_LIO_LIFANCD2 is essential for recruitment of MCM9 to DNA damage site. C_LIO_LIMCM8/9 is epistatic to FANCD2 and within the same DNA damage response pathway. C_LI

The canonical function of the Mdm2 oncoprotein is widely recognized as the negative regulation of the p53 tumor suppressor. However, growing evidence indicates that its physiological activities extend far beyond p53. Here, we show that Mdm2 promotes cell cycle progression at the G2/M phase through the ubiquitin-mediated degradation of the substrate recognition adaptor Cdt2 of the CRL4Cdt2 E3 ubiquitin ligase complex, independently of p53. The attenuation of CRL4Cdt2 activity by Mdm2 stabilizes its cell cycle-specific substrates including p21, Set8, and Cdt1, at the G2/M phase following their proteasomal degradation in the S phase. Furthermore, the delay in cell cycle progression at the G2/M phase and the decreased cell proliferation observed in the absence of Mdm2 are largely caused by an increase in Cdt2 and a subsequent decrease in p21. Collectively, our data illustrate a previously unexplored mechanism by which Mdm2 regulates the cell cycle and promotes cellular proliferation by neutralising the CR4Cdt2 E3 ubiquitin ligase activity and subsequently stabilizing p21.

Stress granules (SGs) are dynamic, non-membranous structures composed of non-translating mRNAs and various proteins and play critical roles in cell survival under stressed conditions. Extensive proteomics analyses have been performed to identify proteins in SGs; however, the molecular functions of these components in SG formation remain unclear. In this report, we show that ubiquitin-associated protein 2-like (UBAP2L) is a novel component of SGs. UBAP2L localized to SGs in response to various stresses, and its depletion significantly suppressed SG organization. Proteomics and RNA sequencing analyses found that UBAP2L formed a protein-RNA complex with Ras-GTP-activating protein SH3 domain binding protein 1 (G3BP1) and small nucleolar RNAs (snoRNAs). In vitro binding analysis demonstrated that snoRNAs were required for UBAP2L association with G3BP1. In addition, decreased expression of snoRNAs reduced the interaction between UBAP2L and G3BP1 and suppressed SG formation. Our results reveal a critical role of a novel SG component, the UBAP2L/snoRNA/G3BP1 protein-RNA complex, and provide new insights into the regulation of SG assembly.

The telomere damage response is a critical mechanism regulating human aging and disease progression. Deprotected telomeres activate the cytosolic DNA-sensing cGAS-STING pathway; however, the underlying mechanism remains unclear. Here, we discover that histone H3.3 is required for cGAS-STING pathway activation in cells with deprotected telomeres. Expression of the TRF2 dominant-negative mutant, TRF2{Delta}B{Delta}M, induces telomere dysfunction in fibroblast cells, triggering cGAS-STING pathway activation and growth inhibition. Histone H3.3 depletion significantly reduces this activation, highlighting its critical role in linking telomere deprotection to the cGAS-STING-mediated innate immune signaling. Furthermore, we assess the role of histone H3.3 in telomere fusion. Our finding reveal that histone H3.3 regulates cGAS-STING signaling by controlling telomere fusion. Additionally, depletion of histone H3.3 chaperones, including ATRX, DAXX, and HIRA, inhibits telomere fusion and cGAS-STING pathway activation, underscoring the role of histone H3.3 in telomere maintenance and the DNA damage response. Collectively, our study establishes histone H3.3 as a key regulator of telomere fusion and telomere dysfunction-induced cGAS-STING pathway activation, revealing the correlation between histones and telomeric DNA damage response.

The Ku heterodimer (Ku70/Ku80) binds DNA ends with high affinity but without sequence specificity and, upon binding ends created by double-stranded breaks (DSBs), initiates canonical nonhomologous end-joining (c-NHEJ). Ku also localizes to functional telomeres where its c-NHEJ activity is inhibited. Interestingly, Ku has been co-opted at telomeres across species, where it performs varied telomeric functions. In humans, Ku is essential for its role in telomere maintenance, but how it associates with human telomeres is not known. Analysis of Kus telomere association in different populations of cen3tel cells, which had a wide range of average telomere lengths, supported Kus localization at human telomeres primarily via protein-protein interaction. We also found that the Ku70 and Ku80 5 helices, which are on opposing sides of the heterodimer and were previously implicated in Saccharomyces cerevisiae Kus NHEJ and telomeric functions, respectively, participated in Kus telomere association in human cells. While the Ku70 5 mutant showed increased interaction with TRF2, the Ku80 5 mutant was not impacted for TRF2 association. Interestingly, residues altered to impair Kus DNA end-binding function were also involved in TRF2 interaction and telomere association. Overall, our results suggest protein-protein interactions as the primary mode by which Ku associates with human telomeres.

Protein degradation by ubiquitin proteasome system (UPS) is the major selective proteolytic pathway responsible for the degradation of short lived proteins ranging from regulatory proteins to abnormal proteins. Many diseases are associated with abnormal protein degradation; occasionally such dysregulated protein degradation is compensated by various transcriptional and translational control mechanisms in the cell. Among those pathways CCR4-NOT protein complex is responsible for transcriptional and transitional control of various gene expressions. Furthermore, CCR4-NOT complex also has a RING type ubiquitin ligase (E3) which is required for the degradation of several proteins. Here we report a novel function that the CCR4-NOT complex 3-5 exonuclease Caf1 is involved in ubiquitindependent degradation of short lived proteins by the 26S proteasome in yeast Saccharomyces cerevisiae. caf1 deletion results in stabilization of R-Ura3 (N-end rule) and Ub-V76-Ura3 (Ubiquitin fusion degradation) substrates from proteasomal degradation. Additionally, caf1 deletion accumulates ubiquitin-modified Ub-V76-Ura3 proteins and Caf1 binds to poly-ubiquitin conjugates and linear tetra ubiquitin chains. Surprisingly, Caf1 interacts with 19S regulatory particle complex of the 26S proteasome. Therefore, we conclude that Caf1 has an exciting novel function as an ubiquitin shuttle factor in which Caf1 targets ubiquitin-modified proteins to 26S proteasome for efficient degradation.

Induction of unfolded protein response (UPR) involves activation of transcription factor Hac1p that facilitates the transactivation of genes encoding ER-chaperones. Hac1p is encoded by HAC1 pre-mRNA harboring an intron and a bipartite element (BE) at its 3'-UTR. This precursor RNA undergoes a reversible and differential intra-nuclear mRNA decay by the nuclear exosome/CTEXT at various phases of UPR. In this investigation, using a combination of genetic, and biochemical approach, the Rab-GTPase Ypt1p is demonstrated to control UPR signaling dynamics. Regulation of UPR by Ypt1p relies on its characteristic nuclear localization in absence of ER-stress resulting in its strong association with pre-HAC1 mRNA at its 3'-UTR that promotes sequential recruitments of Nrd1-Nab3p-Sen1p (NNS) complex [->] CTEXT [->] the nuclear exosome onto the pre-HAC1 mRNA that is accompanied by its rapid and selective nuclear decay. This accelerated 3'[->]5' mRNA decay produces a pre-HAC1 mRNA pool lacking the functional BE thus causing its inefficient targeting to Ire1p foci leading to their diminished splicing and translation. ER stress triggers a rapid relocalization of Ypt1p to the cytoplasm with its consequent dissociation from pre-HAC1 mRNA thereby causing a decreased recruitment of NNS/exosome/CTEXT to precursor HAC1 RNA leading to its diminished 3'[->]5' degradation by the exosome. This diminished decay produces an increased abundance of pre-HAC1 mRNA population with intact functional BE leading to its enhanced recruitment to Ire1p foci that is followed by its increased splicing and translation. This enhanced translation produces a huge burst of Hac1p that rapidly transactivates the genes encoding ER-chaperones.

Shwachman-Diamond syndrome (SDS) is a ribosomopathy characterized by neutropenia, pancreatic insufficiency, skeletal defects, and predisposition to leukemia. Most cases result from biallelic SBDS mutations that impairing 80S ribosome and polysome assembly. In yeast lacking SDO1 (the SBDS ortholog), growth slows dramatically and the p38 ortholog Hog1 signaling is elevated by multiple types of stress. SBDS-deficient HeLa cells exhibited reduced proliferation and slowed cell cycling. The p38 kinase was constitutively activated in SBDS mutants and SDS patient-derived blood cells. Because ZAK detects ribosome dysfunction, its activation links ribosomal defects to stress kinase pathways in SDS. Suppressing p38 or its upstream activator ZAK restored cell growth and reduced stress signaling. These findings reveal an evolutionarily conserved-independent mechanism via p38 drives SDS pathophysiology and identifies stress kinases as potential therapeutic targets for ribosomal dysfunction.

Unfolded protein response (UPR) is a cellular strategy to increase the protein folding capacity of cells in response to stress within the endoplasmic reticulum (ER). In metazoan cells, three major UPR sensors Ire1, PERK and ATF6 work in concert by simultaneously activating intracellular signaling pathways and modulating a series of physiological processes such as attenuation of the general protein synthesis and expression of protein chaperones. In yeast Saccharomyces cerevisiae, Ire1 is known to be the only UPR sensor, which mediates splicing of HAC1 mRNA in the cytoplasm and derepresses its translation. Hac1 is a transcription factor that increases the expression of protein folding enzymes and chaperones, thus enhancing the protein folding capacity of cells. In this study, we provide compelling evidence that kinase Slt2 plays a significant role in facilitating both the splicing and translation of HAC1 mRNA, while also serving as a key mediator in the activation of UPR genes through an alternative route. We also provide evidence that human extracellular signal-regulated kinase 1 (ERK1) or ERK2 served as a functional substitute for yeast Slt2 in the context of UPR. Furthermore, ERK1 exhibits an enhanced activation in human primary cells when grown in the presence of ER stressor. These findings collectively suggest that Slt2 responds to ER stress by activating the Ire1 pathway as well as initiating a parallel signaling pathway.

Ribosome biogenesis is a crucial process requiring enormous transcriptional output. In budding yeast, the expression of 138 ribosomal protein (RP) genes and over 200 ribosome biogenesis (RiBi) genes is regulated by an intricate network of factors, including the nutrient-sensitive transcription activator Sfp1 and the NuA4 coactivator/acetyltransferase complex. Nutrient starvation or inhibition of TORC1 by rapamycin leads to repression of RP and RiBi genes, in part through blocking Sfp1 nuclear localization and NuA4-dependent chromatin acetylation. Here, we demonstrate that Sfp1 physically interacts with NuA4 in a TORC1-dependent manner. Our results indicate that Sfp1, along with NuA4, regulate the transcription of RiBi and RP genes via distinct mechanisms depending on promoter architectures. Sfp1 promotes histone acetylation at the promoters without affecting NuA4 recruitment. In contrast, NuA4 does impact Sfp1 binding but specifically at two classes of RP genes. Importantly, NuA4 acetylates Sfp1 at lysines 655 and 657, regulating its function. Cells expressing Sfp1 with acetyl-mimicking mutations exhibit increased expression of RiBi genes while RP genes remain stable. However, the same mutants lead to the loss of Sfp1 binding/activity at RiBi genes when cells are under non-optimal growth conditions. Mimicking constitutive acetylation of Sfp1 also limits the transcriptional burst of RP genes upon addition of glucose. Altogether, these results draw an intricate functional relationship between Sfp1 and NuA4 to control ribosome biogenesis, fine-tuning transcription output in different growth conditions.

PDCD4 is a tumor suppressor, known to affect protein translation by binding to a component of the eIF4F complex, eIF4A, and reducing its helicase activity, which is necessary for the 48S preinitiation complex formation and scanning of the 5 untranslated region of mRNA. PDCD4 has also been shown to interact with the ribosome and with translation initiation factors eIF4G, eIF4G2, eIF3, and PABP, all of which participate both in initiation and the closed-loop structure that couples initiation and termination. To investigate whether PDCD4 modulates initiation and termination through these interactions, we used a reconstituted mammalian translation system and pre-termination complexes purified from rabbit reticulocyte lysate. We found that PDCD4 suppresses early initiation events prior to eIF4F complex binding to the cap structure on mRNA. Moreover, inhibition of the helicase activity of eIF4A by PDCD4 is lost when the 40S subunit is present. Inhibition of 48S complex formation was also observed in the presence of the truncated eIF4G fragment p50 or the eIF4G2 isoform, both of which interact with eIF4A but lack the eIF4E-binding domain. PDCD4-mediated inhibition of initiation persisted regardless of the presence of PABP. During translation termination, PDCD4 did not affect eIF4A activity, indicating that its regulatory function toward eIF4A is stage-specific and restricted to initiation. Finally, we discovered that PDCD4 additively stimulates peptide release together with eIF3, eIF4G2, and PABP, but competes with eIF4F. Thus, PDCD4 employs a complex molecular mechanism targeting multiple translation factors to regulate different stages of protein synthesis.

DBC1 plays a critical role in various cellular functions notably cell proliferation, transcription, histone modification and adipogenesis. Current reports about the role of DBC1 in tumorigenesis are paradoxical and designate DBC1 both as a tumor suppressor or an oncogene. Here, using small T antigen of polyoma virus (PyST) as a tool, we have delineated a signaling mechanism that connects LKB1 to AKT1 via DBC1. We report that PyST associates with DBC1 and leads to its down-regulation. Our results also show that PyST expression promotes LKB1 activation which in turn leads to in the downregulation of DBC1 protein. Absence of DBC1 results in transcriptional upregulation and consequently enhanced protein levels of TRB3. TRB3 sequesters AKT1, and consequently the phosphorylation and activity of AKT1 is compromised. This ultimately results in inactivation of pro-survival pathways triggered via AKT1 signaling. Our studies thus provide an insight into a signaling pathway that connects LKB1, DBC1, TRB3 and AKT1.

Yeast flocculation relies on cell surface flocculin proteins encoded by the FLO1 gene. The expression of FLO1 is antagonistically regulated by the Tup1-Cyc8 and the Swi-Snf complexes. The Post translational modifications of core histones regulate the transcription of Tup1-Cyc8-regulated genes. However, the mechanisms by which the physical presence of tail residues regulate FLO1 transcription process and flocculation is yet to be completely understood. Through screening we have identified a new region within the N-terminal tail of histone H3 regulating the transcription of FLO1 and FLO5. One of the histone H3 N-terminal truncation mutants H3{Delta}(17-24) showed higher FLO1 expression compared to wild-type H3. Results revealed that in absence of 17-24 stretch the occupancy of Cyc8 decreases from the upstream regions of FLO1. Additionally, analysis suggests that Hda1 is required for the Cyc8-mediated repression of FLO1. Altogether we demonstrate that 17-24 stretch is essential for the Tup1 independent binding of Cyc8 at the promoters assisted by Hda1, leading to the strong repression of FLO1 transcription. In the absence of the 17-24 stretch, Cyc8 cannot bind, resulting in uncontrolled transcription of FLO1.

Interstrand crosslinks are cytotoxic lesions that inhibit essential processes including replication and transcription. Replication fork reversal occurs in response to interstrand crosslink inducing drug, MMC, but how replication fork reversal promotes repair of interstrand crosslinks is poorly understood. Here, we investigated the role of the RAD54L translocase in interstrand crosslink repair. We found RAD54L is required to promote nascent DNA degradation in FANCD2 and FANCA-depleted cells consistent with a previous study indicating RAD54L promotes replication fork reversal. We further show RAD54L activity is required for formation of radial chromosomes in FANCD2-deficient cells suggesting fork reversal may be required to generate the intermediate undergoing aberrant fusion in FANC-deficient cells. Finally, we demonstrate FANCD2 foci accumulate and DSBs persist in RAD54L-deficient cells indicating RAD54L is required for efficient repair of DSBs. Together, our results indicate RAD54L plays multiple roles in efficient processing and repair of interstrand crosslinks.

Loss-of-function mutations in Cdk5rap2 is associated with the developmental disorders, primary microcephaly and primordial dwarfism, but the underlying molecular link remains obscure. Here, we show that Cdk5rap2 loss in BJ-5ta human fibroblasts triggers senescence that is associated with proliferation defect, which is manifested as small body size in Cdk5rap2an/an mice. In fibroblasts, Cdk5rap2 loss induces p53 Ser15 phosphorylation that correlates with decreased level of the p53 phosphatase, WIP1. Ectopic WIP1 expression reverses senescence in Cdk5rap2-depleted cells, linking senescence to WIP1 downregulation. Cdk5rap2 interacts with GSK3{beta}, increasing inhibitory Ser9 phosphorylation in GSK3{beta}, which phosphorylates and tags {beta}-catenin for degradation. Thus, Cdk5rap2 loss decreases GSK3{beta} Ser9 phosphorylation and increases GSK3{beta} activity, reducing {beta}-catenin that affects expression of NF-{kappa}B target genes, including WIP1. Consequently, Cdk5rap2 or {beta}-catenin depletion downregulates WIP1. GSK3{beta} Inhibition in Cdk5rap2-depleted cells restores {beta}-catenin and WIP1 levels, reducing p53 Ser15 phosphorylation and preventing senescence. Conversely, WIP1 inhibition increases p53 Ser15 phosphorylation and senescence in Cdk5rap2-depleted cells lacking GSK3{beta} activity. Senescence through GSK3{beta}/{beta}-catenin downregulation of WIP1 may contribute to the developmental disorders associated with Cdk5rap2 loss-of-function.

Bud27 is a prefoldin-like protein that participates in transcriptional regulation mediated by the three RNA polymerases in Saccharomyces cerevisiae. Lack of Bud27 significantly affects RNA pol III transcription, although the involved mechanisms have not been characterized. Here we show that Bud27 regulates the phosphorylation state of the RNA pol III transcriptional repressor, Maf1, influences its nuclear localization, and likely its activity. We demonstrate that Bud27 is associated with the Maf1 main phosphatase PP4 in vivo, and that this interaction is required for proper Maf1 dephosphorylation. Lack of Bud27 decreases the interaction among PP4 and Maf1, Maf1 dephosphorylation, and its nuclear entry. Our data uncover a new nuclear function of Bud27, identify PP4 as a novel Bud27 interactor and demonstrate the effect of this prefoldin-like on the posttranslational regulation of Maf1. Finally, our data reveal a broader effect of Bud27 on PP4 activity by influencing, at least, the phosphorylation of Rad53.

Potassium is an essential element for all living organisms. As the most abundant intracellular cation, it is vital for osmoregulation, maintenance of membrane potential, macromolecule transport and enzyme function. In this study, we identify potassium homeostasis as a previously unknown regulator of autophagy through the phosphatase activities of Ppz1 and Ppz2. We find that overexpression of either Ppz1 or Ppz2 triggers autophagy under nutrient-replete conditions, whereas the loss of both causes severe autophagy defects. Strikingly, deletion of the potassium transporters Trk1 and Trk2, which are substrates of Ppz1 and Ppz2-mediated dephosphorylation, restores autophagic activity in ppz1{Delta} ppz2{Delta} cells. Furthermore, intracellular potassium concentrations declined during autophagy induction in wild-type cells but remained stable in ppz1{Delta} ppz2{Delta} mutants. Collectively, these findings establish Ppz1 and Ppz2 as pivotal regulators of autophagy and underscore intracellular potassium reduction as a primary determinant of this process. Summary statementThis study demonstrates that potassium regulation by the phosphatases, Ppz1 and Ppz2, which suppress the major potassium transporters Trk1 and Trk2, is indispensable for autophagy induction in yeast.