Ribosomal modification protein rimK-like family member A activates betaine-homocysteine S-methyltransferase 1 to ameliorate hepatic steatosis.

Nonalcoholic fatty liver disease (NAFLD) is a serious threat to public health, but its underlying mechanism remains poorly understood. In screening important genes using Gene Importance Calculator (GIC) we developed previously, ribosomal modification protein rimK-like family member A (RIMKLA) was predicted as one essential gene but its functions remained largely unknown. The current study determined the roles of RIMKLA in regulating glucose and lipid metabolism. RIMKLA expression was reduced in livers of human and mouse with NAFLD. Hepatic RIMKLA overexpression ameliorated steatosis and hyperglycemia in obese mice. Hepatocyte-specific RIMKLA knockout aggravated high-fat diet (HFD)-induced dysregulated glucose/lipid metabolism in mice. Mechanistically, RIMKLA is a new protein kinase that phosphorylates betaine-homocysteine S-methyltransferase 1 (BHMT1) at threonine 45 (Thr45) site. Upon phosphorylation at Thr45 and activation, BHMT1 eliminated homocysteine (Hcy) to inhibit the activity of transcription factor activator protein 1 (AP1) and its induction on fatty acid synthase (FASn) and cluster of differentiation 36 (CD36) gene transcriptions, concurrently repressing lipid synthesis and uptake in hepatocytes. Thr45 to alanine (T45A) mutation inactivated BHMT1 to abolish RIMKLA's repression on Hcy level, AP1 activity, FASn/CD36 expressions, and lipid deposition. BHMT1 overexpression rescued the dysregulated lipid metabolism in RIMKLA-deficient hepatocytes. In summary, RIMKLA is a novel protein kinase that phosphorylates BHMT1 at Thr45 to repress lipid synthesis and uptake. Under obese condition, inhibition of RIMKLA impairs BHMT1 activity to promote hepatic lipid deposition.

A Novel tsRNA, m7G-3' tiRNA LysTTT, Promotes Bladder Cancer Malignancy Via Regulating ANXA2 Phosphorylation.

Emerging evidence indicates that transfer RNA (tRNA)-derived small RNAs (tsRNAs), originated from tRNA with high abundance RNA modifications, play an important role in many complex physiological and pathological processes. However, the biological functions and regulatory mechanisms of modified tsRNAs in cancer remain poorly understood. Here, it is screened for and confirmed the presence of a novel m7G-modified tsRNA, m7G-3'-tiRNA LysTTT (mtiRL), in a variety of chemical carcinogenesis models by combining small RNA sequencing with an m7G small RNA-modified chip. Moreover, it is found that mtiRL, catalyzed by the tRNA m7G-modifying enzyme mettl1, promotes bladder cancer (BC) malignancy in vitro and in vivo. Mechanistically, mtiRL is found to specifically bind the oncoprotein Annexin A2 (ANXA2) to promote its Tyr24 phosphorylation by enhancing the interactions between ANXA2 and Yes proto-oncogene 1 (Yes1), leading to ANXA2 activation and increased p-ANXA2-Y24 nuclear localization in BC cells. Together, these findings define a critical role for mtiRL and suggest that targeting this novel m7G-modified tsRNA can be an efficient way for to treat BC.

A LATS2 and ALKBH5 positive feedback loop supports their oncogenic roles.

N(6)-methyladenosine (m6A) critically regulates RNA dynamics in various biological processes. The m6A demethylase ALKBH5 promotes tumorigenesis of glioblastoma, while the intricate web that orchestrates its regulation remains enigmatic. Here, we discover that cell density affects ALKBH5 subcellular localization and m6A dynamics. Mechanistically, ALKBH5 is phosphorylated by the large tumor suppressor kinase 2 (LATS2), preventing its nuclear export and enhancing protein stability. Furthermore, phosphorylated ALKBH5 reciprocally erases m6A from LATS2 mRNA, thereby stabilizing this transcript. Unexpectedly, LATS2 depletion suppresses glioblastoma stem cell self-renewal independent of yes-associated protein activation. Additionally, deficiency in either LATS2 or ALKBH5 phosphorylation impedes tumor progression in mouse xenograft models. Moreover, high levels of LATS2 expression and ALKBH5 phosphorylation are associated with tumor malignancy in patients with gliomas. Collectively, our study unveils an oncogenic positive feedback loop between LATS2 and ALKBH5, revealing a non-canonical branch of the Hippo pathway for RNA processing and suggesting potential anti-cancer interventions.

Phosphorylated SHMT2 Regulates Oncogenesis Through m6A Modification in Lung Adenocarcinoma.

Targeting cancer-specific metabolic processes is a promising therapeutic strategy. Here, this work uses a compound library that directly inhibits metabolic enzymes to screen the potential metabolic targets in lung adenocarcinoma (LUAD). SHIN1, the specific inhibitor of serine hydroxymethyltransferase 1/2 (SHMT1/2), has a highly specific inhibitory effect on LUAD cells, and this effect depends mainly on the overexpression of SHMT2. This work clarifies that mitogen-activated protein kinase 1 (MAPK1)-mediated phosphorylation at Ser90 is the key mechanism underlying SHMT2 upregulation in LUAD and that this phosphorylation stabilizes SHMT2 by reducing STIP1 homology and U-box containing protein 1 (STUB1)-mediated ubiquitination and degradation. SHMT2-Ser90 dephosphorylation decreases S-adenosylmethionine levels in LUAD cells, resulting in reduced N6-methyladenosine (m6A) levels in global RNAs without affecting total protein or DNA methylation. Methylated RNA immunoprecipitation sequencing (MeRIP-Seq) and RNA sequencing (RNA-Seq) analyses further demonstrate that SHMT2-Ser90 dephosphorylation accelerates the RNA degradation of oncogenic genes by reducing m6A modification, leading to the inhibition of tumorigenesis. Overall, this study elucidates a new regulatory mechanism of SHMT2 during oncogenesis and provides a theoretical basis for targeting SHMT2 as a therapeutic target in LUAD.

Identification of plants' functional counterpart of the metazoan mediator of DNA Damage checkpoint 1.

Induction of DNA damage triggers rapid phosphorylation of the histone H2A.X (γH2A.X). In animals, mediator of DNA damage checkpoint 1 (MDC1) binds γH2A.X through a tandem BRCA1 carboxyl-terminal (tBRCT) domain and mediates recruitment of downstream effectors of DNA damage response (DDR). However, readers of this modification in plants have remained elusive. We show that from the Arabidopsis BRCT domain proteome, BCP1-4 proteins with tBRCT domains are involved in DDR. Through its tBRCT domain BCP4 binds γH2A.X in vitro and localizes to DNA damage-induced foci in an H2A.X-dependent manner. BCP4 also contains a domain that interacts directly with NBS1 and thus acts as a functional counterpart of MDC1. We also show that BCP1, that contains two tBRCT domains, co-localizes with γH2A.X but it does not bind γH2A.X suggesting functional similarity with human PAXIP1. A phylogenetic analysis supports that PAXIP1 and MDC1 in metazoa and their plant counterparts evolved independently from common ancestors with tBRCT domains. Collectively, our study reveals missing components and provides mechanistic and evolutionary insights into plant DDR.

The BHLHE40‒PPM1F‒AMPK pathway regulates energy metabolism and is associated with the aggressiveness of endometrial cancer.

BHLHE40 is a basic helix-loop-helix transcription factor that is involved in multiple cell activities including differentiation, cell cycle, and epithelial-to-mesenchymal transition. While there is growing evidence to support the functions of BHLHE40 in energy metabolism, little is known about the mechanism. In this study, we found that BHLHE40 expression was downregulated in cases of endometrial cancer of higher grade and advanced disease. Knockdown of BHLHE40 in endometrial cancer cells resulted in suppressed oxygen consumption and enhanced extracellular acidification. Suppressed pyruvate dehydrogenase (PDH) activity and enhanced lactated dehydrogenase (LDH) activity were observed in the knockdown cells. Knockdown of BHLHE40 also led to dephosphorylation of AMPKα Thr172 and enhanced phosphorylation of pyruvate dehydrogenase E1 subunit alpha 1 (PDHA1) Ser293 and lactate dehydrogenase A (LDHA) Tyr10. These results suggested that BHLHE40 modulates PDH and LDH activity by regulating the phosphorylation status of PDHA1 and LDHA. We found that BHLHE40 enhanced AMPKα phosphorylation by directly suppressing the transcription of an AMPKα-specific phosphatase, PPM1F. Our immunohistochemical study showed that the expression of BHLHE40, PPM1F, and phosphorylated AMPKα correlated with the prognosis of endometrial cancer patients. Because AMPK is a central regulator of energy metabolism in cancer cells, targeting the BHLHE40‒PPM1F‒AMPK axis may represent a strategy to control cancer development.

Resection of DNA double-strand breaks activates Mre11-Rad50-Nbs1- and Rad9-Hus1-Rad1-dependent mechanisms that redundantly promote ATR checkpoint activation and end processing in Xenopus egg extracts.

Sensing and processing of DNA double-strand breaks (DSBs) are vital to genome stability. DSBs are primarily detected by the ATM checkpoint pathway, where the Mre11-Rad50-Nbs1 (MRN) complex serves as the DSB sensor. Subsequent DSB end resection activates the ATR checkpoint pathway, where replication protein A, MRN, and the Rad9-Hus1-Rad1 (9-1-1) clamp serve as the DNA structure sensors. ATR activation depends also on Topbp1, which is loaded onto DNA through multiple mechanisms. While different DNA structures elicit specific ATR-activation subpathways, the regulation and mechanisms of the ATR-activation subpathways are not fully understood. Using DNA substrates that mimic extensively resected DSBs, we show here that MRN and 9-1-1 redundantly stimulate Dna2-dependent long-range end resection and ATR activation in Xenopus egg extracts. MRN serves as the loading platform for ATM, which, in turn, stimulates Dna2- and Topbp1-loading. Nevertheless, MRN promotes Dna2-mediated end processing largely independently of ATM. 9-1-1 is dispensable for bulk Dna2 loading, and Topbp1 loading is interdependent with 9-1-1. ATR facilitates Mre11 phosphorylation and ATM dissociation. These data uncover that long-range end resection activates two redundant pathways that facilitate ATR checkpoint signaling and DNA processing in a vertebrate system.

LncNFYB promotes the proliferation of rheumatoid arthritis fibroblast-like synoviocytes via LncNFYB/ANXA2/ERK1/2 axis.

Long noncoding RNAs (lncRNAs) are specifically expressed in different diseases and regulate disease progression. To explore the functions of rheumatoid arthritis (RA)-specific lncRNA, we determined the lncRNA expression profile of fibroblast-like synoviocytes (FLS) obtained from patients with RA and osteoarthritis (OA) using a LncRNA microarray and identified up-regulated LncNFYB in RA as a potential therapeutic target. Using gain- and loss-of-function studies, LncNFYB was proven to promote FLS proliferation and cell cycle progress but not affect their invasion, migration, and apoptotic abilities. Further investigation discovered that LncRNA could combine with annexin A2 (ANXA2) and enhance the level of phospho-ANXA2 (Tyr24) in the plasma membrane area, which induced the activation of ERK1/2 to promote proliferation. These findings provide new insights into the biological functions of LncNFYB on modification of FLS, which may be exploited for the therapy of RA.

LINC00969 inhibits proliferation with metastasis of breast cancer by regulating phosphorylation of PI3K/AKT and ILP2 expression through HOXD8.

Background: Breast cancer (BC) is a malignancy that is inadequately treated and poses a significant global health threat to females. The aberrant expression of long noncoding RNAs (lncRNAs) acts as a complex with a precise regulatory role in BC progression. LINC00969 has been linked to pyroptotic cell death and resistance to gefitinib in lung cancer cells. However, the precise function and regulatory mechanisms of LINC00969 in BC remain largely unexplored. Methods: Cell proliferation, migration, and invasion of BC cells were evaluated using CCK-8 and Transwell assays. Western blotting was employed to analyze the protein expression levels of HOXD8, ILP2, PI3K, t-AKT, and p-AKT. Results: LINC00969 was drastically reduced in BC tissues LINC00969 overexpression markedly suppressed proliferation, migration, and invasion, and blocked PI3K and p-AKT protein expression in MCF-7 cells. Activation of the PI3K/AKT pathway reversed the suppressive effect of LINC0096 overexpression on the proliferation, migration, and invasion of MCF-7 cells. Moreover, LINC00969 overexpression enhanced HOXD8 and blocked ILP2 protein expression in MCF-7 cells. In contrast, activating the PI3K/AKT pathway had no effect on HOXD8 and blocked ILP2 protein expression in MCF-7 cells overexpressing LINC00969. HOXD8 knockdown enhanced ILP2, PI3K, and p-AKT protein expression, and the proliferation, migration, and invasion of MCF-7 cells co-transfected with si-HOXD8 and ov-LINC00969. LINC00969 regulated HOXD8 via binding to miR-425-5p. Conclusion: LINC00969 inhibits the proliferation and metastasis of BC cells by regulating PI3K/AKT phosphorylation through HOXD8/ILP2.

An siRNA library screen identifies CYLD and USP34 as deubiquitinases that regulate GPCR-p38 MAPK signaling and distinct inflammatory responses.

G protein-coupled receptors (GPCRs) are highly druggable and implicated in numerous diseases, including vascular inflammation. GPCR signals are transduced from the plasma membrane as well as from endosomes and controlled by posttranslational modifications. The thrombin-activated GPCR protease-activated receptor-1 is modified by ubiquitin. Ubiquitination of protease-activated receptor-1 drives recruitment of transforming growth factor-ß-activated kinase-1-binding protein 2 (TAB2) and coassociation of TAB1 on endosomes, which triggers p38 mitogen-activated protein kinase-dependent inflammatory responses in endothelial cells. Other endothelial GPCRs also induce p38 activation via a noncanonical TAB1-TAB2-dependent pathway. However, the regulatory processes that control GPCR ubiquitin-driven p38 inflammatory signaling remains poorly understood. We discovered mechanisms that turn on GPCR ubiquitin-dependent p38 signaling, however, the mechanisms that turn off the pathway are not known. We hypothesize that deubiquitination is an important step in regulating ubiquitin-driven p38 signaling. To identify specific deubiquitinating enzymes (DUBs) that control GPCR-p38 mitogen-activated protein kinase signaling, we conducted a siRNA library screen targeting 96 DUBs in endothelial cells and HeLa cells. We identified nine DUBs and validated the function two DUBs including cylindromatosis and ubiquitin-specific protease-34 that specifically regulate thrombin-induced p38 phosphorylation. Depletion of cylindromatosis expression by siRNA enhanced thrombin-stimulated p38 signaling, endothelial barrier permeability, and increased interleukin-6 cytokine expression. Conversely, siRNA knockdown of ubiquitin-specific protease-34 expression decreased thrombin-promoted interleukin-6 expression and had no effect on thrombin-induced endothelial barrier permeability. These studies suggest that specific DUBs distinctly regulate GPCR-induced p38-mediated inflammatory responses.

A Role of PI3K/Akt Signaling in Oocyte Maturation and Early Embryo Development.

A serine/threonine-specific protein kinase B (PKB), also known as Akt, is a key factor in the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway that regulates cell survival, metabolism and proliferation. Akt phosphorylates many downstream specific substrates, which subsequently control the nuclear envelope breakdown (NEBD), centrosome maturation, spindle assembly, chromosome segregation, and cytokinesis. In vertebrates, Akt is also an important player during oogenesis and preimplantation development. In the signaling pathways regulating mRNA translation, Akt is involved in the control of mammalian target of rapamycin complex 1 (mTORC1) and thereby regulates the activity of a translational repressor, the eukaryotic initiation factor 4E (eIF4E) binding protein 1 (4E-BP1). In this review, we summarize the functions of Akt in mitosis, meiosis and early embryonic development. Additionally, the role of Akt in the regulation of mRNA translation is addressed with respect to the significance of this process during early development.

Multi-functional regulation of cGAS by the nuclear localization signal2 (NLS2) motif: Nuclear localization, enzyme activity and protein degradation.

Cyclic GMP-AMP synthase (cGAS), which recognizes double-stranded DNA (dsDNA) and activates the innate immune system, is mainly localized in the cytosol, but also shows nuclear localization. Here, we sought to determine the role of nuclear cGAS by mutating known nuclear localization signal (NLS) motifs in cGAS and assessing its functionality by monitoring phosphorylation of the downstream target, interferon regulatory factor-3 (IRF3). Interestingly, NLS2-mutated cGAS failed to promote phosphorylation of IRF3, reflecting the loss of its ability to produce cyclic GMP-AMP (cGAMP). We further found that insertion of an NLS from SV40 large T antigen could not restore this loss of activity, indicating that this loss was attributable to the mutation of NLS2 itself, but not dependent on the inability of cGAS to enter the nucleus. NLS2-mutant cGAS protein also showed decreased stability dependent on polyubiquitination, an effect that was independent of both its loss of catalytic function and its inability to enter into the nucleus. Collectively, these findings indicate that the NLS2 motif of cGAS is not only involved in regulating the subcellular localization of cGAS protein but also influences its stability and enzymatic activity through independent mechanisms, highlighting the novel roles of NLS2 in regulating the intracellular functions of cGAS.

Disease mutations and phosphorylation alter the allosteric pathways involved in autoinhibition of protein phosphatase 2A.

Mutations in protein phosphatase 2A (PP2A) are connected to intellectual disability and cancer. It has been hypothesized that these mutations might disrupt the autoinhibition and phosphorylation-induced activation of PP2A. Since they are located far from both the active and substrate binding sites, it is unclear how they exert their effect. We performed allosteric pathway analysis based on molecular dynamics simulations and combined it with biochemical experiments to investigate the autoinhibition of PP2A. In the wild type (WT), the C-arm of the regulatory subunit B56δ obstructs the active and substrate binding sites exerting a dual autoinhibition effect. We find that the disease mutant, E198K, severely weakens the allosteric pathways that stabilize the C-arm in the WT. Instead, the strongest allosteric pathways in E198K take a different route that promotes exposure of the substrate binding site. To facilitate the allosteric pathway analysis, we introduce a path clustering algorithm for lumping pathways into channels. We reveal remarkable similarities between the allosteric channels of E198K and those in phosphorylation-activated WT, suggesting that the autoinhibition can be alleviated through a conserved mechanism. In contrast, we find that another disease mutant, E200K, which is in spatial proximity of E198, does not repartition the allosteric pathways leading to the substrate binding site; however, it may still induce exposure of the active site. This finding agrees with our biochemical data, allowing us to predict the activity of PP2A with the phosphorylated B56δ and provide insight into how disease mutations in spatial proximity alter the enzymatic activity in surprisingly different mechanisms.

CDK12 and Integrator-PP2A complex modulates LEO1 phosphorylation for processive transcription elongation.

Cyclin-dependent kinase 12 (CDK12) interacts with cyclin K to form a functional nuclear kinase that promotes processive transcription elongation through phosphorylation of the C-terminal domain of RNA polymerase II (Pol II). To gain a comprehensive understanding of CDK12's cellular function, we used chemical genetic and phosphoproteomic screening to identify a landscape of nuclear human CDK12 substrates, including regulators of transcription, chromatin organization, and RNA splicing. We further validated LEO1, a subunit of the polymerase-associated factor 1 complex (PAF1C), as a bona fide cellular substrate of CDK12. Acute depletion of LEO1, or substituting LEO1 phosphorylation sites with alanine, attenuated PAF1C association with elongating Pol II and impaired processive transcription elongation. Moreover, we discovered that LEO1 interacts with and is dephosphorylated by the Integrator-PP2A complex (INTAC) and that INTAC depletion promotes the association of PAF1C with Pol II. Together, this study reveals an uncharacterized role for CDK12 and INTAC in regulating LEO1 phosphorylation, providing important insights into gene transcription and its regulation.

Autophosphorylation of αCaMKII regulates alcohol consumption by controlling sedative effects of alcohol and alcohol-induced loss of excitatory synapses.

Calcium/calmodulin-dependent kinase II (CaMKII) is a key enzyme at the glutamatergic synapses. CAMK2A gene variants have been linked with alcohol use disorder (AUD) by an unknown mechanism. Here, we looked for the link between αCaMKII autophosphorylation and the AUD aetiology. Autophosphorylation-deficient heterozygous αCaMKII mutant mice (T286A+/- ) were trained in the IntelliCages to test the role of αCaMKII activity in AUD-related behaviours. The glutamatergic synapses morphology in CeA was studied in the animals drinking alcohol using 3D electron microscopy. We found that T286A+/- mutants consumed less alcohol and were more sensitive to sedating effects of alcohol, as compared to wild-type littermates (WT). After voluntary alcohol drinking, T286A+/- mice had less excitatory synapses in the CeA, as compared to alcohol-naive animals. This change correlated with alcohol consumption was not reversed after alcohol withdrawal and not observed in WT mice. Our study suggests that αCaMKII autophosphorylation affects alcohol consumption by controlling sedative effects of alcohol and preventing synaptic loss in the individuals drinking alcohol. This finding advances our understanding of the molecular processes that regulate alcohol dependence.

FMRP phosphorylation and interactions with Cdh1 regulate association with dendritic RNA granules and MEF2-triggered synapse elimination.

Fragile X Messenger Ribonucleoprotein (FMRP) is necessary for experience-dependent, developmental synapse elimination and the loss of this process may underlie the excess dendritic spines and hyperconnectivity of cortical neurons in Fragile X Syndrome, a common inherited form of intellectual disability and autism. Little is known of the signaling pathways that regulate synapse elimination and if or how FMRP is regulated during this process. We have characterized a model of synapse elimination in CA1 neurons of organotypic hippocampal slice cultures that is induced by expression of the active transcription factor Myocyte Enhancer Factor 2 (MEF2) and relies on postsynaptic FMRP. MEF2-induced synapse elimination is deficient in Fmr1 KO CA1 neurons, and is rescued by acute (24 h), postsynaptic and cell autonomous reexpression of FMRP in CA1 neurons. FMRP is an RNA binding protein that suppresses mRNA translation. Derepression is induced by posttranslational mechanisms downstream of metabotropic glutamate receptor signaling. Dephosphorylation of FMRP at S499 triggers ubiquitination and degradation of FMRP which then relieves translation suppression and promotes synthesis of proteins encoded by target mRNAs. Whether this mechanism functions in synapse elimination is not known. Here we demonstrate that phosphorylation and dephosphorylation of FMRP at S499 are both necessary for synapse elimination as well as interaction of FMRP with its E3 ligase for FMRP, APC/Cdh1. Using a bimolecular ubiquitin-mediated fluorescence complementation (UbFC) assay, we demonstrate that MEF2 promotes ubiquitination of FMRP in CA1 neurons that relies on activity and interaction with APC/Cdh1. Our results suggest a model where MEF2 regulates posttranslational modifications of FMRP via APC/Cdh1 to regulate translation of proteins necessary for synapse elimination.

B-lymphoid tyrosine kinase-mediated FAM83A phosphorylation elevates pancreatic tumorigenesis through interacting with ß-catenin.

Abnormal activation of Wnt/ß-catenin-mediated transcription is closely associated with the malignancy of pancreatic cancer. Family with sequence similarity 83 member A (FAM83A) was shown recently to have oncogenic effects in a variety of cancer types, but the biological roles and molecular mechanisms of FAM83A in pancreatic cancer need further investigation. Here, we newly discovered that FAM83A binds directly to ß-catenin and inhibits the assembly of the cytoplasmic destruction complex thus inhibiting the subsequent phosphorylation and degradation. FAM83A is mainly phosphorylated by the SRC non-receptor kinase family member BLK (B-lymphoid tyrosine kinase) at tyrosine 138 residue within the DUF1669 domain that mediates the FAM83A-ß-catenin interaction. Moreover, FAM83A tyrosine 138 phosphorylation enhances oncogenic Wnt/ß-catenin-mediated transcription through promoting ß-catenin-TCF4 interaction and showed an elevated nucleus translocation, which inhibits the recruitment of histone deacetylases by TCF4. We also showed that FAM83A is a direct downstream target of Wnt/ß-catenin signaling and correlates with the levels of Wnt target genes in human clinical pancreatic cancer tissues. Notably, the inhibitory peptides that target the FAM83A-ß-catenin interaction significantly suppressed pancreatic cancer growth and metastasis in vitro and in vivo. Our results revealed that blocking the FAM83A cascade signaling defines a therapeutic target in human pancreatic cancer.

Phosphorylation mutation impairs the promoting effect of spastin on neurite outgrowth without affecting its microtubule severing ability.

Spastin, a microtubule-severing enzyme, is known to be important for neurite outgrowth. However, the role of spastin post-translational modification, particularly its phosphorylation regulation in neuronal outgrowth, remains unclear. This study aimed to investigate the effects of eliminating spastin phosphorylation on the neurite outgrowth of rat hippocampal neurons. To accomplish this, we constructed a spastin mutant with eleven potential phosphorylation sites mutated to alanine. The phosphorylation levels of the wildtype spastin (WT) and the mutant (11A) were then detected using Phos-tag SDS-PAGE. The spastin constructs were transfected into COS7 cells for the observation of microtubule severing, and into rat hippocampal neurons for the detection of neuronal outgrowth. The results showed that compared to the spastin WT, the phosphorylation levels were significantly reduced in the spastin 11A mutant. The spastin mutant 11A impaired its ability to promote neurite length, branching, and complexity in hippocampal neurons, but did not affect its ability to sever microtubules in COS7 cells. In conclusion, the data suggest that mutations at multiple phosphorylation sites of spastin do not impair its microtubule cleavage ability in COS7 cells, but reduce its ability to promote neurite outgrowth in rat hippocampal neurons.

Critical role of Wat1/Pop3 in regulating the TORC1 signalling pathway in fission yeast S. pombe.

The target of rapamycin (TOR), a major pathway for the regulation of cell growth and proliferation is conserved from yeast to humans. Fission yeast contains two tor complexes, TORC1 is crucial for cell growth while TORC2 gets activated under stress conditions. Pop3/Wat1, a mammalian Lst8 ortholog is an important component of both TOR complexes and has been implicated in the oxidative stress response pathway. Here in this study, the genetic interaction analysis revealed a synthetic lethal interaction of wat1 with tor2-287 mutant cells. Co-immunoprecipitation analysis revealed Wat1 interacts with TORC1 components Tor2, Mip1, and Tco89 while wat1-17 mutant protein fails to interact with these proteins. In the absence of Wat1, the cells arrest at G1 phase with reduced cell size at non-permissive temperature reminiscent of tor2-287 mutant phenotype. Similarly, inactivation of Wat1 results in the failure of TORC1 mediated phosphorylation of Psk1 and Rps602, leading to dysregulation of amino acid permeases and delocalization of Gaf1, a DNA binding transcription factor. Overall, we have hypothesized that Wat1/Pop3 is required to execute the function of TORC1.

RNA methyltransferase NSun2 deficiency promotes neurodegeneration through epitranscriptomic regulation of tau phosphorylation.

Epitranscriptomic regulation adds a layer of post-transcriptional control to brain function during development and adulthood. The identification of RNA-modifying enzymes has opened the possibility of investigating the role epitranscriptomic changes play in the disease process. NOP2/Sun RNA methyltransferase 2 (NSun2) is one of the few known brain-enriched methyltransferases able to methylate mammalian non-coding RNAs. NSun2 loss of function due to autosomal-recessive mutations has been associated with neurological abnormalities in humans. Here, we show NSun2 is expressed in adult human neurons in the hippocampal formation and prefrontal cortex. Strikingly, we unravel decreased NSun2 protein expression and an increased ratio of pTau/NSun2 in the brains of patients with Alzheimer's disease (AD) as demonstrated by Western blotting and immunostaining, respectively. In a well-established Drosophila melanogaster model of tau-induced toxicity, reduction of NSun2 exacerbated tau toxicity, while overexpression of NSun2 partially abrogated the toxic effects. Conditional ablation of NSun2 in the mouse brain promoted a decrease in the miR-125b m6A levels and tau hyperphosphorylation. Utilizing human induced pluripotent stem cell (iPSC)-derived neuronal cultures, we confirmed NSun2 deficiency results in tau hyperphosphorylation. We also found that neuronal NSun2 levels decrease in response to amyloid-beta oligomers (AßO). Notably, AßO-induced tau phosphorylation and cell toxicity in human neurons could be rescued by overexpression of NSun2. Altogether, these results indicate that neuronal NSun2 deficiency promotes dysregulation of miR-125b and tau phosphorylation in AD and highlights a novel avenue for therapeutic targeting.