AtVQ25 promotes salicylic acid-related leaf senescence by fine-tuning the self-repression of AtWRKY53.

Most mechanistic details of chronologically ordered regulation of leaf senescence are unknown. Regulatory networks centered on AtWRKY53 are crucial for orchestrating and integrating various senescence-related signals. Notably, AtWRKY53 binds to its own promoter and represses transcription of AtWRKY53, but the biological significance and mechanism underlying this self-repression remain unclear. In this study, we identified the VQ motif-containing protein AtVQ25 as a cooperator of AtWRKY53. The expression level of AtVQ25 peaked at mature stage and was specifically repressed after the onset of leaf senescence. AtVQ25-overexpressing plants and atvq25 mutants displayed precocious and delayed leaf senescence, respectively. Importantly, we identified AtWRKY53 as an interacting partner of AtVQ25. We determined that interaction between AtVQ25 and AtWRKY53 prevented AtWRKY53 from binding to W-box elements on the AtWRKY53 promoter and thus counteracted the self-repression of AtWRKY53. In addition, our RNA-sequencing data revealed that the AtVQ25-AtWRKY53 module is related to the salicylic acid (SA) pathway. Precocious leaf senescence and SA-induced leaf senescence in AtVQ25-overexpressing lines were inhibited by an SA pathway mutant, atsid2, and NahG transgenic plants; AtVQ25-overexpressing/atwrky53 plants were also insensitive to SA-induced leaf senescence. Collectively, we demonstrated that AtVQ25 directly attenuates the self-repression of AtWRKY53 during the onset of leaf senescence, which is substantially helpful for understanding the timing of leaf senescence onset modulated by AtWRKY53.

JASMONATE ZIM-domain protein 3 regulates photomorphogenesis and thermomorphogenesis through inhibiting PIF4 in Arabidopsis.

Light and temperature are 2 major environmental factors that affect the growth and development of plants during their life cycle. Plants have evolved complex mechanisms to adapt to varying external environments. Here, we show that JASMONATE ZIM-domain protein 3 (JAZ3), a jasmonic acid signaling component, acts as a factor to integrate light and temperature in regulating seedling morphogenesis. JAZ3 overexpression transgenic lines display short hypocotyls under red, far-red, and blue light and warm temperature (28 °C) conditions compared to the wild type in Arabidopsis (Arabidopsis thaliana). We show that JAZ3 interacts with the transcription factor PHYTOCHROME-INTERACTING FACTOR4 (PIF4). Interestingly, JAZ3 spontaneously undergoes liquid-liquid phase separation (LLPS) in vitro and in vivo and promotes LLPS formation of PIF4. Moreover, transcriptomic analyses indicate that JAZ3 regulates the expression of genes involved in many biological processes, such as response to auxin, auxin-activated signaling pathway, regulation of growth, and response to red light. Finally, JAZ3 inhibits the transcriptional activation activity and binding ability of PIF4. Collectively, our study reveals a function and molecular mechanism of JAZ3 in regulating plant growth in response to environmental factors such as light and temperature.

LINC00887 regulates malignant progression and T-cell chemotaxis in clear cell renal cell carcinoma by activating CD70 via recruitment of SPI1.

BACKGROUND: LINC00887 has been mentioned in several articles regarding its involvement in various cancers like nasopharyngeal carcinoma, lung cancer and glioma. However, the mechanism of LINC00887 in the malignant progression of clear cell renal cell carcinoma (ccRCC) is still unclear. The topic of our study is mainly centered on exploring how LINC00887 exactly affects ccRCC malignant progression. METHODS: The bioinformatics method predicted the downstream TF and target genes of LINC00887 by the "LncRNA-transcription factor (TF)-Gene" triplet model. RNA immunoprecipitation, chromatin immunoprecipitation analysis, and Dual-luciferase reporter assay determined the regulatory relationship between LINC00887 and its downstream genes. The LINC00887 expression and its downstream gene expression in ccRCC cells were examined by qRT-PCR and Western blot. The effect of LINC00887-SPI1-CD70 modulation axis on proliferative transfer, cell stemness and T cell chemotaxis of ccRCC cells was examined in cellular and animal experiments. RESULTS: Our research demonstrated an upregulation of LINC00887 in ccRCC, which facilitated tumor growth and stemness in vivo. In addition, LINC00887 could upregulate the CD70 expression by recruiting transcriptional factor SPI1. The results of in vitro experiments illustrated that the LINC00887-SPI1-CD70 regulatory axis facilitated ccRCC malignant progression by promoting cell stemness and hindering T-cell chemotaxis. CONCLUSION: LINC00887, by recruiting SPI1, activated CD70 transcription, thereby propelling malignant progression and cell stemness and suppressing T cell chemotaxis in ccRCC. Based on our findings, we believed that the LINC00887-SPI1-CD70 regulatory axis had the potential to be a critical breakthrough for treating ccRCC.

The PIF1/PIF3-MED25-HDA19 transcriptional repression complex regulates phytochrome signaling in Arabidopsis.

Light signals are perceived by photoreceptors, triggering the contrasting developmental transition in dark-germinated seedlings. Phytochrome-interacting factors (PIFs) are key regulators of this transition. Despite their prominent functions in transcriptional activation, little is known about PIFs' roles in transcriptional repression. Here, we provide evidence that histone acetylation is involved in regulating phytochrome-PIFs signaling in Arabidopsis. The histone deacetylase HDA19 interacts and forms a complex with PIF1 and PIF3 and the Mediator subunit MED25. The med25/hda19 double mutant mimics and enhances the phenotype of pif1/pif3 in both light and darkness. HDA19 and MED25 are recruited by PIF1/PIF3 to the target loci to reduce histone acetylation and chromatin accessibility, providing a mechanism for PIF1/PIF3-mediated transcriptional repression. Furthermore, MED25 forms liquid-like condensates, which can compartmentalize PIF1/PIF3 and HDA19 in vitro and in vivo, and the number of MED25 puncta increases in darkness. Collectively, our study establishes a mechanism wherein PIF1/PIF3 interact with HDA19 and MED25 to mediate transcriptional repression in the phytochrome signaling pathway and suggests that condensate formation with Mediator may explain the distinct and specific transcriptional activity of PIF proteins.

Regulation of chloroplast protein degradation.

Chloroplasts are unique organelles that not only provide sites for photosynthesis and many metabolic processes, but also are sensitive to various environmental stresses. Chloroplast proteins are encoded by genes from both nuclear and chloroplast genomes. During chloroplast development and responses to stresses, the robust protein quality control systems are essential for regulation of protein homeostasis and the integrity of chloroplast proteome. In this review, we summarize the regulatory mechanisms of chloroplast protein degradation refer to protease system, ubiquitin-proteasome system, and the chloroplast autophagy. These mechanisms symbiotically play a vital role in chloroplast development and photosynthesis under both normal or stress conditions.

Bladder Cancer Progression Is Suppressed Through the Heart and Neural Crest Derivatives Expressed 2-Antisense RNA 1/microRNA-93-5p/Defective in Cullin Neddylation 1 Domain Containing 3 Axis.

MicroRNAs (miRNAs) are critical in progression of bladder cancer (BCa). miRNA-93-5p is increased in cancers and is positively correlated with an unfavorable prognosis. But its effects on BCa remain rarely understood. This investigation aimed to dig out miRNA-93-5p affecting biological behaviors of BCa. In this research, mRNA and protein expression in cancer cells were assessed via quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. Cell Counting Kit-8 (CCK-8), colony formation, scratch healing, and transwell assays were utilized to analyze cancer cell viability, colony-forming, migration, and invasion, respectively. Bioinformatics analysis predicted upstream regulatory genes and downstream target genes of miRNA-93-5p, with the targeting relationship being verified through a dual-luciferase assay. The BCa xenograft model in nude mice further investigated the effect of miRNA-93-5p and AND2-AS1 on tumor size and quality, and validated the relationship between HAND2-AS1/miRNA-93-5p/DCUN1D3. Our results displayed that miRNA-93-5p was increased in BCa cell lines. Knockdown miRNA-93-5p constrained BCa cell malignant phenotypes. HAND2-AS1 targeted miRNA-93-5p, thus restraining malignant progression of BCa cells. DCUN1D3 was found downstream of miRNA-93-5p. miRNA-93-5p modulated proliferation, migration, and invasion of BCa cells by targeting DCUN1D3. In vivo experiments disclosed that forced expression of lncRNA HAND2-AS1, and inhibited miRNA-93-5p regressed tumor growth. Meanwhile, the same as the results of cell experiments, the expression of miRNA-93-5p was downregulated, and DCUN1D3 expression was advanced in tumor tissues. To conclude, lncRNA HAND2-AS1 exerted anti-tumor effects and regulated BCa cell proliferation, invasion, and migration by targeting miRNA-93-5p/DCUN1D3.

The tumour-associated stroma correlates with poor clinical outcomes and immunoevasive contexture in patients with upper tract urothelial carcinoma: results from a multicenter real-world study (TSU-01 Study).

BACKGROUND: In this real-world study, we aimed to elucidate the predictive value of tumour-associated stroma for clinical prognostic and therapeutic response in upper tract urothelial carcinoma (UTUC) by reviewing the clinicopathologic characteristics of 1015 UTUC patients through a nationwide multicenter analysis. METHODS: The tumour-stroma ratio (TSR) was assessed based on tissue sections stained for hematoxylin and eosin (H&E), and patients were further stratified into stroma-high (>50% stroma) and stroma-low group (≤50% stroma). Kaplan-Meier curve and Cox regression hazard analysis were conducted to assess the survival outcomes of UTUC patients. Bioinformatics analysis and immunostaining analysis were applied to portray the tumour microenvironment (TME). RESULTS: Stroma-high UTUC was significantly associated with poorer survival outcomes and inferior chemotherapeutic responsiveness. Our established nomogram achieved a high prognostic accuracy in predicting overall survival and cancer-specific survival in both of the discovery cohort (area under the curve [AUC] 0.663 and 0.712) and the validation cohort (AUC 0.741 and 0.747). Moreover, stroma-high UTUC was correlated with immunoevasive TME accompanied by increased cancer-associated fibroblasts, tumour-associated macrophages and, conspicuously a cluster of highly exhausted CD8+ T cells. CONCLUSION: Our results showed stroma-high UTUC was associated with an inferior prognosis and an immunoevasive TME with exhausted CD8+ T cells in UTUC patients. Our TSR-based nomogram could be used to refine prognosis and inform treatment decisions of patients with UTUC.

Arabidopsis EXECUTER1 interacts with WRKY transcription factors to mediate plastid-to-nucleus singlet oxygen signaling.

Chloroplasts produce singlet oxygen (1O2), which causes changes in nuclear gene expression through plastid-to-nucleus retrograde signaling to increase plant fitness. However, the identity of this 1O2-triggered pathway remains unclear. Here, we identify mutations in GENOMES UNCOUPLED4 (GUN4) and GUN5 as suppressors of phytochrome-interacting factor1 (pif1) pif3 in regulating the photo-oxidative response in Arabidopsis thaliana. GUN4 and GUN5 specifically interact with EXECUTER1 (EX1) and EX2 in plastids, and this interaction is alleviated by treatment with Rose Bengal (RB) or white light. Impaired expression of GUN4, GUN5, EX1, or EX2 leads to insensitivity to excess light and overexpression of EX1 triggers photo-oxidative responses. Strikingly, upon light irradiation or RB treatment, EX1 transiently accumulates in the nucleus and the nuclear fraction of EX1 shows a similar molecular weight as the plastid-located protein. Point mutagenesis analysis indicated that nuclear localization of EX1 is required for its function. EX1 acts as a transcriptional co-activator and interacts with the transcription factors WRKY18 and WRKY40 to promote the expression of 1O2-responsive genes. This study suggests that EX1 may act in plastid-to-nucleus signaling and establishes a 1O2-triggered retrograde signaling pathway that allows plants adapt to changing light environments during chloroplast development.

Condensation of SEUSS promotes hyperosmotic stress tolerance in Arabidopsis.

Osmotic stress imposed by drought and high salinity inhibits plant growth and crop yield. However, our current knowledge on the mechanism by which plants sense osmotic stress is still limited. Here, we identify the transcriptional regulator SEUSS (SEU) as a key player in hyperosmotic stress response in Arabidopsis. SEU rapidly coalesces into liquid-like nuclear condensates when extracellular osmolarity increases. The intrinsically disordered region 1 (IDR1) of SEU is responsible for its condensation. IDR1 undergoes conformational changes to adopt more compact states after an increase in molecular crowding both in vitro and in cells, and two predicted α-helical peptides are required. SEU condensation is indispensable for osmotic stress tolerance, and loss of SEU dramatically compromises the expression of stress tolerance genes. Our work uncovers a critical role of biomolecular condensates in cellular stress perception and response and expands our understanding of the osmotic stress pathway.

LINC00887 Fosters Development of Clear Cell Renal Cell Carcinoma via Inhibiting CD8+ T Cell Immune Infiltration.

Background: lncRNAs affect adaptive and innate immunity of cancer via mediating functional states of immune cells, genes, and pathways. Nonetheless, little is known about the molecular mechanism of lncRNA-mediated CD8+ T cell immune infiltration in progression of clear cell renal cell carcinoma (ccRCC). We designed this work to investigate the role of LINC00887 in regulating CD8+ T cell immune infiltration in ccRCC. Methods: Correlation between LINC00887 and immune factors and the expression level of LINC00887 in ccRCC were analyzed by bioinformatics methods (TCGA-KIRC database, "edgeR" package, "clusterProfiler" package, and "CIBERSORT" package). LINC00887 expression in ccRCC was examined via RT-qPCR. The cytokilling capacity of CD8+ T cells was evaluated by the lactate dehydrogenase assay. The apoptotic ability of CD8+ T cells was measured by flow cytometry. The chemotactic ability of CD8+ T cells was revealed by chemotaxis assay. CXCR3, CXCL9, and CXCL10 levels were assessed by RT-qPCR. Results: As suggested by bioinformatics analysis, LINC00887 was markedly upregulated in ccRCC patients and associated with expression of immune-suppression molecule, thereby abating the immune infiltration level of CD8+ cells in tumor tissue. As revealed by cellular assay, LINC00887 was upregulated in ccRCC cells, and knockdown of LINC00887 resulted in a decreased PD-L1 expression, increased CD8+ T cell toxicity, decreased apoptotic levels, and enhanced chemotaxis. Moreover, we found that LINC00887 exhibited inhibitory effect on immune infiltration of CD8+ cells in clinical tissues. Conclusions: The results of this study suggested that LINC00887 promoted ccRCC progression by inhibiting immune infiltration of CD8+ T cells, providing new insights into pathogenesis of ccRCC and suggesting LINC00887 being a promising immunotherapy target for ccRCC.

The RNA helicase UAP56 and the E3 ubiquitin ligase COP1 coordinately regulate alternative splicing to repress photomorphogenesis in Arabidopsis.

Light is a key environmental signal that regulates plant growth and development. While posttranscriptional regulatory mechanisms of gene expression include alternative splicing (AS) of pre-messenger RNA (mRNA) in both plants and animals, how light signaling affects AS in plants is largely unknown. Here, we identify DExD/H RNA helicase U2AF65-associated protein (UAP56) as a negative regulator of photomorphogenesis in Arabidopsis thaliana. UAP56 is encoded by the homologs UAP56a and UAP56b. Knockdown of UAP56 led to enhanced photomorphogenic responses and diverse developmental defects during vegetative and reproductive growth. UAP56 physically interacts with the central light signaling repressor constitutive photomorphogenic 1 (COP1) and U2AF65. Global transcriptome analysis revealed that UAP56 and COP1 co-regulate the transcription of a subset of genes. Furthermore, deep RNA-sequencing analysis showed that UAP56 and COP1 control pre-mRNA AS in both overlapping and distinct manners. Ribonucleic acid immunoprecipitation assays showed that UAP56 and COP1 bind to common small nuclear RNAs and mRNAs of downstream targets. Our study reveals that both UAP56 and COP1 function as splicing factors that coordinately regulate AS during light-regulated plant growth and development.

Multi-responsive mesoporous polydopamine composite nanorods cooperate with nano-enzyme and photosensitiser for intensive immunotherapy of bladder cancer.

Bladder cancer is a common malignancy in the urinary system. Defects of drug molecules in bladder during treatment, such as passive diffusion, rapid clearance of periodic urination, poor adhesion and permeation abilities, lead to low delivery efficiency of conventional drugs and high recurrence rate of disease. In this study, we designed multi-responsive mesoporous polydopamine (PDA) composite nanorods cooperating with nano-enzyme and photosensitiser for intensive immunotherapy of bladder cancer. The strongly adhesive mesoporous PDA with wheat germ agglutinin on nanoparticles could specifically adhere to epithelial glycocalyx and made the nanoparticles aggregate in urinary pathways. Meanwhile, 2,3-dimethylmaleic anhydride could be hydrolysed in acidic conditions of tumour microenvironment, giving it a positive charge (charge reversal), which is more amenable to enter cancer cells. Afterwards, manganese dioxide nanorods could catalyse the reaction of excess H2 O2 in tumour microenvironment to generate active oxygen, so as to change the hypoxic environment in tumour, and achieve a pH-responsive for slow release of PD-L1. After the ICG was irradiated by infrared light, a large amount of singlet oxygen was generated, thereby enhancing the therapeutic effect and reducing toxicity in vivo. Besides, mesoporous PDA with indocyanine green photothermal agent could have a local heat up quickly under the near-infrared light to kill cancer cells, thereby enhancing therapeutic efficacy. Accordingly, this mesoporous PDA composite nanorods shed a light on bladder tumour treatment.

Two chloroplast-localized MORF proteins act as chaperones to maintain tetrapyrrole biosynthesis.

Tetrapyrroles have essential functions as pigments and cofactors during plant growth and development, and the tetrapyrrole biosynthesis pathway is tightly controlled. Multiple organellar RNA editing factors (MORFs) are required for editing of a wide variety of RNA sites in chloroplasts and mitochondria, but their biochemical properties remain elusive. Here, we uncovered the roles of chloroplast-localized MORF2 and MORF9 in modulating tetrapyrrole biosynthesis and embryogenesis in Arabidopsis thaliana. The lack or reduced transcripts of MORF2 or MORF9 significantly affected biosynthesis of the tetrapyrrole precursor 5-aminolevulinic acid and accumulation of Chl and other tetrapyrrole intermediates. MORF2 directly interacts with multiple tetrapyrrole biosynthesis enzymes and regulators, including NADPH:PROTOCHLOROPHYLLIDE OXIDOREDUCTASE B (PORB) and GENOMES UNCOUPLED4 (GUN4). Strikingly, MORF2 and MORF9 display holdase chaperone activity, alleviate the aggregation of PORB in vitro, and are essential for POR accumulation in vivo. Moreover, both MORF2 and MORF9 significantly stimulate magnesium chelatase activity. Our findings reveal a previously unknown biochemical property of MORF proteins as chaperones and point to a new layer of post-translational control of the tightly regulated tetrapyrrole biosynthesis in plants.

Overexpression of miRNA-93-5p Promotes Proliferation and Migration of Bladder Urothelial Carcinoma via Inhibition of KLF9.

We focused on studying the effects of a key miRNA-mRNA axis in bladder urothelial carcinoma (BUC). Firstly, miRNAs and mRNAs differentially expressed in BUC were analyzed. Clinical information in the TCGA database was used for survival analysis, and the regulator of miRNA-93-5p was predicted. miRNA-93-5p and KLF9 mRNA expression were detected by qRT-PCR. Protein level detection and targeting measurement were, respectively, achieved by western blot and dual-luciferase approaches. The proliferative, invasive, and migratory abilities were tested through CCK-8, Transwell, and wound healing methods. Cell apoptosis in each group was detected through flow cytometry. As discovered, miRNA-93-5p level was markedly high in BUC cells while KLF9 expression was remarkably low. miRNA-93-5p overexpression promoted BUC cell abilities. Besides, miRNA-93-5p inhibited KLF9 expression. Furthermore, KLF9 overexpression dramatically attenuated such promotion on cancer cell abilities. On the whole, miRNA-93-5p/KLF9 axis facilitated BUC progression, offering a new potential target for BUC patients.

The CDC48 complex mediates ubiquitin-dependent degradation of intra-chloroplast proteins in plants.

Chloroplasts are the site of numerous biochemical reactions including photosynthesis, but they also produce reactive oxygen species (ROS) that negatively affect chloroplast integrity. The chaperone-like CDC48 complex plays critical roles in ubiquitin-dependent protein degradation in yeast and mammals, but its function in plants is largely unknown. Here, we show that defects in CDC48A and its cofactors UFD1 and NPL4 lead to the accumulation of ubiquitinated chloroplast proteins in Arabidopsis thaliana. We reveal that two plastid genome-encoded proteins, RbcL and AtpB, associate with the CDC48 complex. Strikingly, RbcL and AtpB are ubiquitinated and degraded by the 26S proteasome pathway upon ROS stress, and these processes are impaired by defects of the CDC48 complex. Functional analysis demonstrates that the CDC48 complex is required for plant tolerance to ROS. This study reveals a role for the plant CDC48 complex in modulating ubiquitin-dependent degradation of intra-chloroplast proteins in response to oxidative stress.