Interface Passivation of a Pyridine-Based Bifunctional Molecule for Inverted Perovskite Solar Cells.

Organic-inorganic hybrid perovskite solar cells (PSCs) have recently been demonstrated to be promising renewable harvesters because of their prominent photovoltaic power conversion efficiency (PCE), although their stability and efficiency still have not reached commercial criteria. Trouble-oriented analyses showcase that defect reduction among the grain boundaries and interfaces in the prepared perovskite polycrystalline films is a practical strategy, which has prompted researchers to develop functional molecules for interface passivation. Herein, the pyridine-based bifunctional molecule dimethylpyridine-3,5-dicarboxylate (DPDC) was employed as the interface between the electron-transport layer and perovskite layer, which achieved a champion PCE of 21.37% for an inverted MAPbI3-based PSC, which was greater than 18.64% for the control device. The mechanistic studies indicated that the significantly improved performance was mainly attributed to the remarkably enhanced fill factor with a value greater than 83%, which was primarily due to the nonradiative recombination suppression offered by the passivation effect of DPDC. Moreover, the promoted carrier mobility together with the enlarged crystal size contributed to a higher short-circuit current density. In addition, an increase in the open-circuit voltage was also observed in the DPDC-treated PSC, which benefited from the improved work function for reducing the energy loss during carrier transport. Furthermore, the DPDC-treated PSC showed substantially enhanced stability, with an over 80% retention rate of its initial PCE value over 300 h even at a 60% relative humidity level, which was attributed to the hydrophobic nature of the DPDC molecule and effective defect passivation. This work is expected not only to serve as an effective strategy for using a pyridine-based bifunctional molecule to passivate perovskite interfaces to enhance photovoltaic performance but also to shed light on the interface passivation mechanism.

Single-cell RNA-seq analysis decodes the kidney microenvironment induced by polystyrene microplastics in mice receiving a high-fat diet.

In recent years, the environmental health issue of microplastics has aroused an increasingly significant concern. Some studies suggested that exposure to polystyrene microplastics (PS-MPs) may lead to renal inflammation and oxidative stress in animals. However, little is known about the essential effects of PS-MPs with high-fat diet (HFD) on renal development and microenvironment. In this study, we provided the single-cell transcriptomic landscape of the kidney microenvironment induced by PS-MPs and HFD in mouse models by unbiased single-cell RNA sequencing (scRNA-seq). The kidney injury cell atlases in mice were evaluated after continued PS-MPs exposure, or HFD treated for 35 days. Results showed that PS-MPs plus HFD treatment aggravated the kidney injury and profibrotic microenvironment, reshaping mouse kidney cellular components. First, we found that PS-MPs plus HFD treatment acted on extracellular matrix organization of renal epithelial cells, specifically the proximal and distal convoluted tubule cells, to inhibit renal development and induce ROS-driven carcinogenesis. Second, PS-MPs plus HFD treatment induced activated PI3K-Akt, MAPK, and IL-17 signaling pathways in endothelial cells. Besides, PS-MPs plus HFD treatment markedly increased the proportions of CD8+ effector T cells and proliferating T cells. Notably, mononuclear phagocytes exhibited substantial remodeling and enriched in oxidative phosphorylation and chemical carcinogenesis pathways after PS-MPs plus HFD treatment, typified by alterations tissue-resident M2-like PF4+ macrophages. Multispectral immunofluorescence and immunohistochemistry identified PF4+ macrophages in clear cell renal cell carcinoma (ccRCC) and adjacent normal tissues, indicating that activate PF4+ macrophages might regulate the profibrotic and pro-tumorigenic microenvironment after renal injury. In conclusion, this study first systematically revealed molecular variation of renal cells and immune cells in mice kidney microenvironment induced by PS-MPs and HFD with the scRNA-seq approach, which provided a molecular basis for decoding the effects of PS-MPs on genitourinary injury and understanding their potential profibrotic and carcinogenesis in mammals.

Unveiling the impact of tertiary lymphoid structures on immunotherapeutic responses of clear cell renal cell carcinoma.

Tertiary lymphoid structures (TLS) are organized aggregates of immune cells that form under pathological conditions. However, the predictive value of TLS in clear cell renal cell carcinoma (ccRCC) for immunotherapies remains unclear. We comprehensively assessed the implications for prognosis and immunological responses of the TLS spatial and maturation heterogeneity in 655 ccRCC patients. A higher proportion of early-TLS was found in peritumoral TLS, while intratumoral TLS mainly comprised secondary follicle-like TLS (SFL-TLS), indicating markedly better survival. Notably, presence of TLS, especially intratumoral TLS and SFL-TLS, significantly correlated with better survival and objective reflection rate for ccRCC patients receiving anti-Programmed Cell Death Protein-1 (PD-1)/Programmed Cell Death-Ligand-1 (PD-L1) immunotherapies. In peritumoral TLS cluster, primary follicle-like TLS, the proportion of tumor-associated macrophages, and Treg infiltration in the peritumoral regions increased prominently, suggesting an immunosuppressive tumor microenvironment. Interestingly, spatial transcriptome annotation and multispectral fluorescence showed that an abundance of mature plasma cells within mature TLS has the capacity to produce IgA and IgG, which demonstrate significantly higher objective response rates and a superior prognosis for ccRCC patients subjected to immunotherapy. In conclusion, this study revealed the implications of TLS spatial and maturation heterogeneity on the immunological status and clinical responses, allowing the improvement of precise immunotherapies of ccRCC.

Electrosynthesis of buckyballs with fused-ring systems from PCBM and its analogue.

[6,6]-Phenyl-C61-butyric acid methyl ester (PCBM), a star molecule in the fullerene field, has found wide applications in materials science. Herein, electrosynthesis of buckyballs with fused-ring systems has been achieved through radical α-C-H functionalization of the side-chain ester for both PCBM and its analogue, [6,6]-phenyl-C61-propionic acid methyl ester (PCPM), in the presence of a trace amount of oxygen. Two classes of buckyballs with fused bi- and tricyclic carbocycles have been electrochemically synthesized. Furthermore, an unknown type of a bisfulleroid with two tethered [6,6]-open orifices can also be efficiently generated from PCPM. All three types of products have been confirmed by single-crystal X-ray crystallography. A representative intramolecularly annulated isomer of PCBM has been applied as an additive to inverted planar perovskite solar cells and boosted a significant enhancement of power conversion efficiency from 15.83% to 17.67%.

Construction of cuproptosis signature based on bioinformatics and experimental validation in clear cell renal cell carcinoma.

BACKGROUND: Cuproptosis was defined as a novel nonapoptotic cell death pathway and its potential function in clear cell renal cell carcinoma (ccRCC) remains unclear. METHODS: We obtained gene expression profiles, somatic mutation and corresponding clinical information of 881 ccRCC samples from 3 cohorts including the cancer genome atlas cohort, GSE29609 cohort and CheckMate 025 cohort. As described in the latest published article, we enrolled 16 genes as cuproptosis-related genes (CRGs). We explored the expression level, variants and copy number variation of the CRGs. Univariate and multi-variate regression were utilized to assess the prognostic significance of the CRGs. Non-negative matrix factorization was used to identify potential subgroup and gene set variation analysis was used to explore the potential biological functions. CIBERSORT, ESTIMATE algorithm and single sample gene set enrichment analysis were used to evaluate the tumor microenvironment. In vitro experiments including CCK-8, transwell and wound healing assays were utilized to explore the potential biological function of DLAT in ccRCC. RESULTS: We found that except for CDKN2A, the CRGs were positively associated with patients' OS. Cuproptosis cluster, cuproptosis gene cluster and cuproptosis score were established, respectively, and higher cuproptosis score was significantly associated with a worse OS in ccRCC (p < 0.001). The area under the receiver operating characteristic curve of the cuproptosis-related nomogram at 1 year, 3 years, 5 years was 0.858, 0.821 and 0.78, respectively. In addition, we found that the cuproptosis score was positively associated with PDCD1, CTLA4 expression level, thus the cuproptosis score may also reflect the dysfunction of tumor infiltrating immune cells. In vitro experiments indicated that overexpression of DLAT could inhibited the migration and proliferation ability of ccRCC cells. CONCLUSION: Our findings identify a novel cuproptosis-related signature and the cuproptosis characteristics may influence the anti-tumor immunity though complex regulating networks, and thus cuproptosis may play a role in developing novel therapeutic target of ccRCC.

Protein Arginine Methyltransferases Refine the Classification of Clear Cell Renal Cell Carcinoma with Distinct Prognosis and Tumor Microenvironment Characteristics.

Background: Clear cell renal cell carcinoma (ccRCC) is an aggressive urological cancer that originates from the proximal tubular epithelium. As one of the most common post-translational modification, protein arginine methylation plays a pivotal role in various cancer-associated biological functions, especially in cancer immunity. Therefore, constructing a protein arginine methylation-related prognostic signature would be beneficial in guiding better personalized clinical management for patients with ccRCC. Methods: Based on the multi-omics profiling of the expression levels of eight protein arginine methyltransferases (PRMTs) in 763 ccRCC samples (from TCGA, CPTAC, EMBL, and ICGC databases), we established a scoring system with machine-learning algorithms to quantify the modification patterns on clinical and immunological characterizations of individual ccRCC patient, which was termed as PRMTScore. Moreover, we utilized two external clinical cohorts receiving immunotherapy (n=302) to validate the reliability of the PRMTScore system. Multiplex immunohistochemistry (mIHC) was performed to characterize the cellular composition of 30 paired ccRCC samples. The proteomic profiling of 232 ccRCC samples obtained from Fudan University Shanghai Cancer Center (FUSCC) was analyzed to validate the protein expression of PRMT5 in ccRCC. Finally, CCK-8, transwell, and wound healing assays were conducted to elucidate the role of PRMT5 in ccRCC in vitro. Results: A total of 763 ccRCC patients with available multi-omics profiling were stratified into two clusters (PRMTCluster A and B) with distinctive prognosis, genomic alterations, tumor microenvironment (TME) characteristics, and fundamental biological mechanisms. Subsequently, protein arginine methylation-related prognostic signature (PRMTScore) was constructed and consisted of SLC16A12, HRH2, F2RL3, and SAA1. The PRMTScore showed remarkable differences in outcomes, immune and stromal fractions, expressions of immune checkpoints, the abundance of immune cells, and immunotherapy response in ccRCC patients. Additionally, preliminary insights unveiled the tumor-suppressive role of PRMT5 in ccRCC, and the signal of PRMT5low significantly predicted aggressive prognosis and the high abundance of PD1+ CD8+ cells in ccRCC. Conclusion: We constructed a PRMTScore system, which showed the potent ability to assess the prognosis, TME characteristics, and immunotherapy response for patients with ccRCC. Moreover, this is the first study to propose that PRMT5 acts as a cancer suppressor in ccRCC.

Plasma proteomic profiling discovers molecular features associated with upper tract urothelial carcinoma.

Upper tract urothelial carcinoma (UTUC) is often diagnosed late and exhibits poor prognosis. Limited data are available on potential non-invasive biomarkers for disease monitoring. Here, we investigate the proteomic profile of plasma in 362 UTUC patients and 239 healthy controls. We present an integrated tissue-plasma proteomic approach to infer the signature proteins for identifying patients with muscle-invasive UTUC. We discover a protein panel that reflects lymph node metastasis, which is of interest in identifying UTUC patients with high risk and poor prognosis. We also identify a ten-protein classifier and establish a progression clock predicting progression-free survival of UTUC patients. Finally, we further validate the signature proteins by parallel reaction monitoring assay in an independent cohort. Collectively, this study portrays the plasma proteomic landscape of a UTUC cohort and provides a valuable resource for further biological and diagnostic research in UTUC.

The CHCHD2/Sirt1 corepressors involve in G9a-mediated regulation of RNase H1 expression to control R-loop.

R-loops are regulators of many cellular processes and are threats to genome integrity. Therefore, understanding the mechanisms underlying the regulation of R-loops is important. Inspired by the findings on RNase H1-mediated R-loop degradation or accumulation, we focused our interest on the regulation of RNase H1 expression. In the present study, we report that G9a positively regulates RNase H1 expression to boost R-loop degradation. CHCHD2 acts as a repressive transcription factor that inhibits the expression of RNase H1 to promote R-loop accumulation. Sirt1 interacts with CHCHD2 and deacetylates it, which functions as a corepressor that suppresses the expression of downstream target gene RNase H1. We also found that G9a methylated the promoter of RNase H1, inhibiting the binding of CHCHD2 and Sirt1. In contrast, when G9a was knocked down, recruitment of CHCHD2 and Sirt1 to the RNase H1 promoter increased, which co-inhibited RNase H1 transcription. Furthermore, knockdown of Sirt1 led to binding of G9a to the RNase H1 promoter. In summary, we demonstrated that G9a regulates RNase H1 expression to maintain the steady-state balance of R-loops by suppressing the recruitment of CHCHD2/Sirt1 corepressors to the target gene promoter.

Genome-wide analysis of the CSN genes in land plants and their expression under various abiotic stress and phytohormone conditions in rice.

The CONSTITUTIVE PHOTOMORPHOGENIC9 (COP9) signalosome (CSN) is a multi-functional protein complex, which is involved in plant growth and abiotic stress response. However, the evolution and function of the CSN genes in land plants are still largely unclear. Here, we have identified 124 CSN genes and constructed phylogenetic trees of these CSN proteins to elucidate their feature and evolution in twelve land plants. Analysis of gene structure, protein property and protein motif composition shows the evolutional conservation and variation of the CSN proteins in land plants. These CSN genes might evolve through whole genome duplication (WGD)/segmental duplication (SD) and tandem duplication (TD). Analysis of promoter cis-elements shows that the CSN genes are implicated in diverse biological processes and different signaling pathways. RT-qPCR indicates that the transcript abundance of the OsCSN genes is up-regulated or down-regulated in response to abiotic stress and treatment with various hormones in rice. These results provide new insights into the CSN gene evolution and its possible function in land plants.

The expression and prognostic value of transporter 1, ATP binding cassette subfamily B member in clear cell renal cell cancer with experimental validation.

Transporter associated with antigen processing 1(TAP1) serves as a protein to transport antigenic peptides from the surface of the endoplasmic reticulum to the lumen of the endoplasmic reticulum when the antigens are presented by major histocompatibility complex type I (MHC-I), which has been identified to play a critical role in antigen presentation in innate immunity. In tumors, the role of TAP1 seems to remain controversial. On the one hand, given the role of TAP1 in antigen presentation, it is indicated that high TAP1 expression corresponds to the emergence of more neoantigens epitopes that facilitate the recognition for phagocytes, T cells and other cells. On the other hand, the genetic ablation of transporter associated with antigen processing (TAP) results in the presentation of new class I-restricted epitopes encoded in house-keeping products. Opposite result has been revealed by studies in other tumors suggest, which implies a more complex function of TAP1. Therefore, it's significant to clarify the role of TAP1 in clear cell renal cell carcinoma (ccRCC). In this study, we found the elevated expression levels in mRNA and protein of TAP1 in ccRCC tissues, which indicated a relatively worse prognosis. Transwell assay and Scratch assay in vitro demonstrated the promotive role of TAP1 in ccRCC migration as well as a significant role in metastasis. And the increased expression of TAP1 resulted in more immune cells infiltrated in cancer tissues. TAP1 was also demonstrated to be related to immune regulator genes, as gene set enrichment analysis (GSEA) indicated its significant role in immune regulation. The results of CancerSEA indicated the positive association of the high-level TAP1 expression with epithelial-mesenchymal transition (EMT) and the inverse association with Cell Cycle. The effective drugs were also predicted based on TAP1 expression, of which the high level was indeed associated with resistance to multiple drugs, but some effective drugs still identified based on high TAP1 expression. According to the analysis of various databases, the role of TAP1 in ccRCC was explored, especially in relationship of TAP1 with tumor microenvironment. These results indicate that TAP1 can serve as a potential target for treatment of ccRCC.

A novel amino acid metabolism-related gene risk signature for predicting prognosis in clear cell renal cell carcinoma.

Background: Renal cancer is one of the most lethal cancers because of its atypical symptoms and metastatic potential. The metabolism of amino acids and their derivatives is essential for cancer cell survival and proliferation. Thus, the construction of the amino acid metabolism-related risk signature might enhance the accuracy of the prognostic model and shed light on the treatments of renal cancers. Methods: RNA expression and clinical data were downloaded from Santa Cruz (UCSC) Xena, GEO, and ArrayExpress databases. The "DESeq2" package identified the differentially expressed genes. Univariate COX analysis selected prognostic genes related to the metabolism of amino acids. Patients were divided into two clusters using the "ConsensusClusterPlus" package, and the CIBERSORT, ESTIMATE methods were explored to assess the immune infiltrations. The LASSO regression analysis constructed a risk model which was evaluated the prediction accuracy in two independent cohorts. The genomic alterations and drug sensitivity of 18-LASSO-genes were assessed. The differentially expressed genes between two clusters were used to perform functional enrichment analysis and weighted gene co-expression network analysis (WGCNA). Furthermore, external validation of TMEM72 expression was conducted in the FUSCC cohort containing 33 ccRCC patients. Results: The amino acid metabolism-related genes had significant correlations with prognosis. The patients in Cluster A demonstrated better survival, lower Treg cell proportion, higher ESTIMATE scores, and higher cuproptosis-related gene expressions. Amino acid metabolism-related genes with prognostic values were used to construct a risk model and patients in the low risk group were associated with improved outcomes. The Area Under Curve of the risk model was 0.801, 0.777, and 0.767 at the first, second, and third year respectively. The external validation cohort confirmed the stable prognostic value of the risk model. WGCNA identified four gene modules correlated with immune cell infiltrations and cuproptosis. We found that TMEM72 was downregulated in tumors by using TCGA, GEO datasets (p<0.001) and the FUSCC cohort (p=0.002). Conclusion: Our study firstly constructed an 18 amino acid metabolism related signature to predict the prognosis in clear cell renal cell carcinoma. We also identified four potential gene modules potentially correlated with cuproptosis and identified TMEM72 downregulation in ccRCC which deserved further studies.

The Inflammasomes Adaptor Protein PYCARD Is a Potential Pyroptosis Biomarker Related to Immune Response and Prognosis in Clear Cell Renal Cell Carcinoma.

PYCARD is a protein engaged in inflammation, pyroptosis, and apoptosis. However, the function of PYCARD in human cancers remains unclear. The objective of our study was to explore PYCARD expression and prognostic value in human cancers. Public databases were used to assess PYCARD expression and prognostic value. The TISIDB database was used to explore the associations between PYCARD expression and different immune subtypes. The correlations between PYCARD expression and ICP genes, MMR genes, MSI, and TMB were also investigated. The immunotherapy response was assessed using the TIDE database. Single-cell RNA databases evaluated the PYCARD expression of immune cells. External datasets and immunohistochemical staining were conducted to validate PYCARD expression and prognostic value. The results showed that PYCARD expression varied in several cancers and was associated with prognosis, immune-related genes, published biomarkers, and immunotherapy response. Of note, PYCARD expression was upregulated in renal cancers with high diagnostic ability. Upregulation of PYCARD was correlated with worse prognosis in KIRC and external validation cohorts. In conclusion, PYCARD demonstrated strong correlations with prognosis, immune response, and disease progression in pan-cancer analysis. In ccRCC, PYCARD might serve as a biomarker for diagnosis and therapeutic target-boosting immunotherapy response.

Electrochemically Promoted Benzylation of [60]Fullerooxazolidinone.

Benzylation of the electrochemically generated dianion from N-p-tolyl-[60]fullerooxazolidinone with benzyl bromide provides three products with different addition patterns. The product distribution can be dramatically altered by varying the reaction conditions. Based on spectral characterizations, these products have been assigned as mono-benzylated 1,4-adduct and bis-benzylated 1,2,3,16- and 1,4,9,25-adducts, respectively. The assigned 1,2,3,16-adduct has been further established by X-ray diffraction analysis. It is believed that the 1,4-adduct is obtained by decarboxylative benzylation of the dianionic species, while bis-benzylated 1,2,3,16- and 1,4,9,25-adducts are achieved via a rearrangement process. In addition, the electrochemical properties of these products have been studied.

Excess S-adenosylmethionine inhibits methylation via catabolism to adenine.

The global dietary supplement market is valued at over USD 100 billion. One popular dietary supplement, S-adenosylmethionine, is marketed to improve joints, liver health and emotional well-being in the US since 1999, and has been a prescription drug in Europe to treat depression and arthritis since 1975, but recent studies questioned its efficacy. In our body, S-adenosylmethionine is critical for the methylation of nucleic acids, proteins and many other targets. The marketing of SAM implies that more S-adenosylmethionine is better since it would stimulate methylations and improve health. Previously, we have shown that methylation reactions regulate biological rhythms in many organisms. Here, using biological rhythms to assess the effects of exogenous S-adenosylmethionine, we reveal that excess S-adenosylmethionine disrupts rhythms and, rather than promoting methylation, is catabolized to adenine and methylthioadenosine, toxic methylation inhibitors. These findings further our understanding of methyl metabolism and question the safety of S-adenosylmethionine as a supplement.

Publisher Correction: Methylation deficiency disrupts biological rhythms from bacteria to humans.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Methylation deficiency disrupts biological rhythms from bacteria to humans.

The methyl cycle is a universal metabolic pathway providing methyl groups for the methylation of nuclei acids and proteins, regulating all aspects of cellular physiology. We have previously shown that methyl cycle inhibition in mammals strongly affects circadian rhythms. Since the methyl cycle and circadian clocks have evolved early during evolution and operate in organisms across the tree of life, we sought to determine whether the link between the two is also conserved. Here, we show that methyl cycle inhibition affects biological rhythms in species ranging from unicellular algae to humans, separated by more than 1 billion years of evolution. In contrast, the cyanobacterial clock is resistant to methyl cycle inhibition, although we demonstrate that methylations themselves regulate circadian rhythms in this organism. Mammalian cells with a rewired bacteria-like methyl cycle are protected, like cyanobacteria, from methyl cycle inhibition, providing interesting new possibilities for the treatment of methylation deficiencies.

Comprehensive Analysis and Functional Studies of WRKY Transcription Factors in Nelumbo nucifera.

The WRKY family is one of the largest transcription factor (TF) families in plants and plays central roles in modulating plant stress responses and developmental processes, as well as secondary metabolic regulations. Lotus (Nelumbo nucifera) is an aquatic crop that has significant food, ornamental and pharmacological values. Here, we performed an overview analysis of WRKY TF family members in lotus, and studied their functions in environmental adaptation and regulation of lotus benzylisoquinoline alkaloid (BIA) biosynthesis. A total of 65 WRKY genes were identified in the lotus genome and they were well clustered in a similar pattern with their Arabidopsis homologs in seven groups (designated I, IIa-IIe, and III), although no lotus WRKY was clustered in the group IIIa. Most lotus WRKYs were functionally paired, which was attributed to the recently occurred whole genome duplication in lotus. In addition, lotus WRKYs were regulated dramatically by salicilic acid (SA), jasmonic acid (JA), and submergence treatments, and two lotus WRKYs, NnWRKY40a and NnWRKY40b, were significantly induced by JA and promoted lotus BIA biosynthesis through activating BIA biosynthetic genes. The investigation of WRKY TFs for this basal eudicot reveals new insights into the evolution of the WRKY family, and provides fundamental information for their functional studies and lotus breeding.

Dual inhibitors of RAF-MEK-ERK and PI3K-PDK1-AKT pathways: Design, synthesis and preliminary anticancer activity studies of 3-substituted-5-(phenylamino) indolone derivatives.

The dysfunction and mutual compensatory activation of RAF-MEK-ERK and PI3K-PDK1-AKT pathways have been demonstrated as the hallmarks in several primary and recurrent cancers. The strategy of concurrent blocking of these two pathways shows clinical merits on effective cancer therapy, such as combinatory treatments and dual-pathway inhibitors. Herein, we report a novel prototype of dual-pathway inhibitors by means of merging the core structural scaffolds of a MEK1 inhibitor and a PDK1 inhibitor. A library of 43 compounds that categorized into three series (Series I-III) was synthesized and tested for antitumor activity in lung cancer cells. The results from structure-activity relationship (SAR) analysis showed the following order of antitumor activity that 3-hydroxy-5-(phenylamino) indolone (Series III) > 3-alkenyl-5-(phenylamino) indolone (Series I) > 3-alkyl-5-(phenylamino) indolone (Series II). A lead compound 9za in Series III showed most potent antitumor activity with IC50 value of 1.8 ±â€¯0.8 µM in A549 cells. Moreover, antitumor mechanism study demonstrated that 9za exerted significant apoptotic effect, and cellular signal pathway analysis revealed the potent blockage of phosphorylation levels of ERK and AKT in RAF-MEK-ERK and PI3K-PDK1-AKT pathways, respectively. The results reported here provide robust experimental basis for the discovery and optimization of dual pathway agents for anti-lung cancer therapy.

Two Ck1δ transcripts regulated by m6A methylation code for two antagonistic kinases in the control of the circadian clock.

The N6-methylation of internal adenosines (m6A) in mRNA has been quantified and localized throughout the transcriptome. However, the physiological significance of m6A in most highly methylated mRNAs is unknown. It was demonstrated previously that the circadian clock, based on transcription-translation negative feedback loops, is sensitive to the general inhibition of m6A. Here, we show that the Casein Kinase 1 Delta mRNA (Ck1δ), coding for a critical kinase in the control of circadian rhythms, cellular growth, and survival, is negatively regulated by m6A. Inhibition of Ck1δ mRNA methylation leads to increased translation of two alternatively spliced CK1δ isoforms, CK1δ1 and CK1δ2, uncharacterized until now. The expression ratio between these isoforms is tissue-specific, CK1δ1 and CK1δ2 have different kinase activities, and they cooperate in the phosphorylation of the circadian clock protein PER2. While CK1δ1 accelerates the circadian clock by promoting the decay of PER2 proteins, CK1δ2 slows it down by stabilizing PER2 via increased phosphorylation at a key residue on PER2 protein. These observations challenge the previously established model of PER2 phosphorylation and, given the multiple functions and targets of CK1δ, the existence of two isoforms calls for a re-evaluation of past research when CK1δ1 and CK1δ2 were simply CK1δ.