Human adenoviruses (HAdVs) are small DNA viruses that generally cause mild disease. Certain strains, particularly those belonging to species B HAdVs, can cause severe pneumonia and have a relatively high mortality rate. Little is known about the molecular aspects of how these highly pathogenic species affect the infected cell and how they suppress innate immunity. The present study provides molecular insights into how species B adenoviruses suppress the interferon signaling pathway. Our study shows that these viruses, unlike HAdV-C2, are resistant to type I interferon. This resistance likely arises due to the highly efficient suppression of interferon-stimulated gene expression. Unlike in HAdV-C2, HAdV-B7 and B14 sequester STAT2 and RNA polymerase II from interferon-stimulated gene promoters in infected cells. This results in suppressed interferon- stimulated gene activation. In addition, we show that RuvBL1 and RuvBL2, cofactors important for RNA polymerase II recruitment to promoters and interferon-stimulated gene activation, are redirected to the cytoplasm forming high molecular weight complexes that, likely, are unable to associate with chromatin. Proteomic analysis also identified key differences in the way these viruses affect the host cell, providing insights into species B-associated high pathogenicity. Curiously, we observed that at the level of protein expression changes to the infected cell, HAdV-C2 and B7 were more similar than those of the same species, B7 and B14. Collectively, our study represents the first such study of innate immune suppression by the highly pathogenic HAdV-B7 and B14, laying an important foundation for future investigations.IMPORTANCEHuman adenoviruses form a large family of double-stranded DNA viruses known for a variety of usually mild diseases. Certain strains of human adenovirus cause severe pneumonia leading to much higher mortality and morbidity than most other strains. The reasons for this enhanced pathogenicity are unknown. Our study provides a molecular investigation of how these highly pathogenic strains might inactivate the interferon signaling pathway, highlighting the lack of sensitivity of these viruses to type I interferon in general while providing a global picture of how viral changes in cellular proteins drive worse disease outcomes.

This study investigates the roles of RUVBL1 and HIF1A in ccRCC development and explores their clinical significance as prognostic biomarkers. mRNA and protein expressions were analyzed using TCGA data and an institutional tissue cohort, respectively. Correlations with clinicopathological parameters and patient outcomes were assessed. TCGA data revealed significantly elevated RUVBL1 mRNA expression in ccRCC tissues, associated with advanced histological grade, T stage, lymph node metastasis, and clinical stage. High RUVBL1 mRNA expression correlated with inferior overall survival and served as an adverse prognostic factor. Similarly, HIF1A mRNA expression was significantly higher in ccRCC tissues, correlating with worse overall survival and acting as an adverse prognostic factor for treatment outcomes. Simultaneous evaluation of RUVBL1 and HIF1A mRNA expression demonstrated enhanced prognostic capacity, surpassing the predictive power of individual markers. Immunohistochemical staining confirmed substantial upregulation of both RUVBL1 and HIF-1α proteins in ccRCC tissues. Furthermore, high expression of both RUVBL1 and HIF-1α proteins was significantly associated with shorter patient survival time. Our findings underscore the significance of RUVBL1 and HIF-1α as potential prognostic markers in ccRCC, paving the way for further research to translate these insights into clinically relevant applications.

Macromolecular complexes play essential roles in various cellular processes. The assembly of macromolecular assemblies within the cell must overcome barriers imposed by a crowded cellular environment which is characterized by an estimated concentration of biological macromolecules amounting to 100-450 g/L that take up approximately 5-40% of the cytoplasmic volume. The formation of the macromolecular assemblies is facilitated by molecular chaperones in cooperation with their co-chaperones. The R2TP protein complex has emerged as a co-chaperone of Hsp90 that plays an important role in macromolecular assembly. The R2TP complex is composed of a heterodimer of RPAP3:P1H1DI that is in turn complexed to members of the ATPase associated with diverse cellular activities (AAA +), RUVBL1 and RUVBL2 (R1 and R2) families. What makes the R2TP co-chaperone complex particularly important is that it is involved in a wide variety of cellular processes including gene expression, translation, co-translational complex assembly, and posttranslational protein complex formation. The functional versatility of the R2TP co-chaperone complex makes it central to cellular development; hence, it is implicated in various human diseases. In addition, their roles in the development of infectious disease agents has become of interest. In the current review, we discuss the roles of these proteins as co-chaperones regulating Hsp90 and its partnership with Hsp70. Furthermore, we highlight the structure-function features of the individual proteins within the R2TP complex and describe their roles in various cellular processes.

The Ruvb-like AAA ATPase1 (Ruvbl1; also known as Pontin) is an evolutionary conserved protein belonging to the adenosine triphosphates associated with diverse cellular activities (AAA+) superfamily of ATPases. Ruvbl1 is a component of various protein supercomplexes and is involved in a variety of cellular activities, including chromatin remodeling, DNA damage repair, and mitotic spindle assembly however, the developmental significance of this protein is unknown and needs detailed investigation. We investigated the developmental significance of Ruvbl1 in multiciliated cells of the Xenopus laevis epidermis since ruvbl1 is expressed in the multiciliated cells and pronephros during X. laevis embryogenesis. The knockdown of ruvbl1 significantly impaired cilia-driven fluid flow and basal body polarity in the X. laevis epidermis compared to control embryos, but did not affect cilia morphology. Our results suggest that Ruvbl1 plays a significant role in embryonic development by regulating ciliary beating; however, further investigation is needed to determine the mechanisms involved.

The zebrafish is amenable to a variety of genetic approaches. However, lack of conditional deletion alleles limits stage- or cell-specific gene knockout. Here, we applied an existing protocol to establish a floxed allele for gata2a but failed to do so due to off-target integration and incomplete knockin. To address these problems, we applied simultaneous co-targeting with Cas12a to insert loxP sites in cis, together with transgenic counterscreening and comprehensive molecular analysis, to identify off-target insertions and confirm targeted knockins. We subsequently used our approach to establish endogenously floxed alleles of foxc1a, rasa1a, and ruvbl1, each in a single generation. We demonstrate the utility of these alleles by verifying Cre-dependent deletion, which yielded expected phenotypes in each case. Finally, we used the floxed gata2a allele to demonstrate an endothelial autonomous requirement in lymphatic valve development. Together, our results provide a framework for routine generation and application of endogenously floxed alleles in zebrafish.

Genome-wide association studies along with expression quantitative trait locus (eQTL) mapping have identified hundreds of single-nucleotide polymorphisms (SNPs) and their target genes in prostate cancer (PCa), yet functional characterization of these risk loci remains challenging. To screen for potential regulatory SNPs, we designed a CRISPRi library containing 9,133 guide RNAs (gRNAs) to cover 2,166 candidate SNP loci implicated in PCa and identified 117 SNPs that could regulate 90 genes for PCa cell growth advantage. Among these, rs60464856 was covered by multiple gRNAs significantly depleted in screening (FDR < 0.05). Pooled SNP association analysis in the PRACTICAL and FinnGen cohorts showed significantly higher PCa risk for the rs60464856 G allele (p value = 1.2 × 10-16 and 3.2 × 10-7, respectively). Subsequent eQTL analysis revealed that the G allele is associated with increased RUVBL1 expression in multiple datasets. Further CRISPRi and xCas9 base editing confirmed that the rs60464856 G allele leads to elevated RUVBL1 expression. Furthermore, SILAC-based proteomic analysis demonstrated allelic binding of cohesin subunits at the rs60464856 region, where the HiC dataset showed consistent chromatin interactions in prostate cell lines. RUVBL1 depletion inhibited PCa cell proliferation and tumor growth in a xenograft mouse model. Gene-set enrichment analysis suggested an association of RUVBL1 expression with cell-cycle-related pathways. Increased expression of RUVBL1 and activation of cell-cycle pathways were correlated with poor PCa survival in TCGA datasets. Our CRISPRi screening prioritized about one hundred regulatory SNPs essential for prostate cell proliferation. In combination with proteomics and functional studies, we characterized the mechanistic role of rs60464856 and RUVBL1 in PCa progression.

Lung adenocarcinoma (LUAD) is the leading cause of death among cancer diseases. The tumorigenic functions of AHNAK2 in LUAD have attracted more attention in recent years, while there are few studies which have reported its high molecular weight.
The mRNA-seq data of AHNAK2 and corresponding clinical data from UCSC Xena and GEO was analyzed. LUAD cell lines were transfected with sh-NC and sh-AHNAK2, and cell proliferation, migration and invasion were then detected by in vitro experiments. We performed RNA sequencing and mass spectrometry analysis to explore the downstream mechanism and interacting proteins of AHNAK2. Finally, western blot, cell cycle analysis and CO-IP were used to confirm our assumptions regarding previous experiments.
Our study revealed that AHNAK2 expression was significantly higher in tumors than in normal lung tissues and higher AHNAK2 expression led to a poor prognosis, especially in patients with advanced tumors. AHNAK2 suppression via shRNA reduced the LUAD cell lines proliferation, migration and invasion and induced significant changes in DNA replication, NF-kappa B signaling pathway and cell cycle. AHNAK2 knockdown also caused G1/S phase cell cycle arrest, which could be attributed to the interaction of AHNAK2 and RUVBL1. In addition, the results from gene set enrichment analysis (GSEA) and RNA sequencing suggested that AHNAK2 probably plays a part in the mitotic cell cycle.
AHNAK2 promotes proliferation, migration and invasion in LUAD and regulates the cell cycle via the interaction with RUVBL1. More studies of AHNAK2 are still needed to reveal its upstream mechanism.

Epigenetic dysregulation is reported in multiple cancers including Ewing sarcoma (EwS). However, the epigenetic networks underlying the maintenance of oncogenic signaling and therapeutic response remain unclear. Using a series of epigenetics- and complex-focused CRISPR screens, RUVBL1, the ATPase component of NuA4 histone acetyltransferase complex, is identified to be essential for EwS tumor progression. Suppression of RUVBL1 leads to attenuated tumor growth, loss of histone H4 acetylation, and ablated MYC signaling. Mechanistically, RUVBL1 controls MYC chromatin binding and modulates the MYC-driven EEF1A1 expression and thus protein synthesis. High-density CRISPR gene body scan pinpoints the critical MYC interacting residue in RUVBL1. Finally, this study reveals the synergism between RUVBL1 suppression and pharmacological inhibition of MYC in EwS xenografts and patient-derived samples. These results indicate that the dynamic interplay between chromatin remodelers, oncogenic transcription factors, and protein translation machinery can provide novel opportunities for combination cancer therapy.

Rationale: Hepatocellular carcinoma (HCC) is one of the most severe cancers worldwide, with few effective targeted therapies for HCC. Lipid metabolic reprogramming is emerged as a hallmark of cancer metabolism that guides response to antitumoral therapies. Such lipid metabolic alteration in cancers is critically regulated by the mammalian target of rapamycin mTOR, which is considered as a promising therapeutic target. Despite efforts, mTOR inhibitors (mTORi) have produced limited response clinically, partly due to incomplete knowledge of mTORC1 addiction in cancers. Methods: CRISPR-Cas9 system was used to establish Hpcal1 null mice. The liver cancer model in mice was generated using Hpcal1-deficient mice with diethylnitrosamine (DEN) /CCL4 or MYC/Trp53-/- via hydrodynamic tail-vein injection. RNA-sequencing (RNA-seq) was used to identify potential signaling pathways. The expression of HPCAL1 and mTOR signaling were determined using quantitative polymerase chain reaction (qPCR), western blot and immunohistochemistry. The role of Hpcal1 in liver tumorigenesis and its response to mTORi was assessed by CCK-8 measurements, colony formation assay and in mouse model. Results: In this study, we identified hippocalcin-like protein 1 (HPCAL1) as an important negative regulator of de novo lipid biosynthesis and mTOR signaling activation, limiting liver tumorigenesis and establishing a metabolic vulnerability of HCC in mice. Genetic loss of HPCAL1 rendered HCC mTORC1-addicted and sensitive to mTORi AZD-8055 in vitro and in vivo. Importantly, HPCAL1 expression was inversely correlated with the levels of mTOR phosphorylation and several critical lipid biosynthesis enzymes in human specimens. Mechanistically, HPCAL1 directly bound to RuvB Like AAA ATPase 1 (RUVBL1), inhibiting the assembly of TEL2-TTI1-TTI2 (TTT)-RUVBL complex and subsequent leading the mTOR signaling suppression. Conclusion: We uncover a metabolic vulnerability and mTOR addiction in HCC with HPCAL1 loss that provides a selective therapeutic window for HCC with mTORC1 hyperactivation using mTORi.

Although histone H3K4 methyltransferase SETD1A is overexpressed in various cancer types, the molecular mechanism underlying its overexpression and its target genes in pancreatic ductal adenocarcinoma (PDAC) remain unclarified. We conducted immunohistochemical staining for SETD1A in 105 human PDAC specimens to assess the relationship between SETD1A overexpression and clinicopathological features. The function and target genes of SETD1A were investigated using human pancreatic cancer cell lines. SETD1A expression was upregulated in 51.4% of patients with PDAC and was an independent prognostic factor associated with shorter disease-free survival after resection (p < 0.05). Knockdown and overexpression of SETD1A showed that SETD1A plays a crucial role in increasing the proliferation and motility of PDAC cells. SETD1A overexpression increased tumorigenicity. RNA sequencing of SETD1A-knockdown cells revealed downregulation of RUVBL1, an oncogenic protein ATP-dependent DNA helicase gene. ChIP analysis revealed that SETD1A binds to the RUVBL1 promoter region, resulting in increased H3K4me3 levels. Knockdown of RUVBL1 showed inhibition of cell proliferation, migration, and invasion of PDAC cells, which are similar biological effects to SETD1A knockdown. High expression of both SETD1A and RUVBL1 was an independent prognostic factor not only for disease-free survival but also for overall survival (p < 0.05). In conclusion, we identified RUVBL1 as a novel downstream target gene of the SETD1A-H3K4me3 pathway. Co-expression of SETD1A and RUVBL1 is an important factor for predicting the prognosis of patients with PDAC.