FOXA1-dependent PUS1 regulates EIF3b stability in a non-enzymatic pathway mediating prostate cancer bone metastasis.

Bone metastasis is a significant contributor to the poor prognosis in prostate cancer. Recent evidence highlights the pivotal role of pseudouridine synthases in solid tumor progression, yet the specific enzyme driving prostate cancer metastasis remains unidentified. This study uncovers a novel regulatory mechanism of the FOXA1/PUS1/EIF3b signaling axis in prostate cancer bone metastasis. We identified elevated PUS1 expression in prostate cancer tissues, correlating with higher clinical grade and worse prognosis. Knockdown of PUS1 inhibited metastasis independently of its enzymatic activity, with EIF3b acting as a downstream effector, protected from ubiquitin-mediated degradation by PUS1. Overexpression of EIF3b countered the metastasis suppression due to PUS1 knockdown. Additionally, FOXA1 was shown to enhance PUS1 expression by binding to its promoter. Mogroside IV-E, a specific PUS1 inhibitor, demonstrated potent anti-metastatic effects by reducing PUS1 expression. Our findings highlight the FOXA1/PUS1/EIF3b axis as a critical mediator of prostate cancer bone metastasis and suggest that targeting this pathway could be a promising therapeutic strategy.

The molecular basis of tRNA selectivity by human pseudouridine synthase 3.

Pseudouridine (Ψ), the isomer of uridine, is ubiquitously found in RNA, including tRNA, rRNA, and mRNA. Human pseudouridine synthase 3 (PUS3) catalyzes pseudouridylation of position 38/39 in tRNAs. However, the molecular mechanisms by which it recognizes its RNA targets and achieves site specificity remain elusive. Here, we determine single-particle cryo-EM structures of PUS3 in its apo form and bound to three tRNAs, showing how the symmetric PUS3 homodimer recognizes tRNAs and positions the target uridine next to its active site. Structure-guided and patient-derived mutations validate our structural findings in complementary biochemical assays. Furthermore, we deleted PUS1 and PUS3 in HEK293 cells and mapped transcriptome-wide Ψ sites by Pseudo-seq. Although PUS1-dependent sites were detectable in tRNA and mRNA, we found no evidence that human PUS3 modifies mRNAs. Our work provides the molecular basis for PUS3-mediated tRNA modification in humans and explains how its tRNA modification activity is linked to intellectual disabilities.

Truncated protein isoforms generate diversity of protein localization and function in yeast.

Genome-wide measurement of ribosome occupancy on mRNAs has enabled empirical identification of translated regions, but high-confidence detection of coding regions that overlap annotated coding regions has remained challenging. Here, we report a sensitive and robust algorithm that revealed the translation of 388 N-terminally truncated proteins in budding yeast-more than 30-fold more than previously known. We extensively experimentally validated them and defined two classes. The first class lacks large portions of the annotated protein and tends to be produced from a truncated transcript. We show that two such cases, Yap5truncation and Pus1truncation, have condition-specific regulation and distinct functions from their respective annotated isoforms. The second class of truncated protein isoforms lacks only a small region of the annotated protein and is less likely to be produced from an alternative transcript isoform. Many display different subcellular localizations than their annotated counterpart, representing a common strategy for dual localization of otherwise functionally identical proteins. A record of this paper's transparent peer review process is included in the supplemental information.

Multi-omics analysis reveals PUS1 triggered malignancy and correlated with immune infiltrates in NSCLC.

Non-small cell lung cancer (NSCLC) is the main pathological type of lung cancer. In this study, multi-omics analysis revealed a significant increase of pseudouridine synthase 1 (PUS1) in NSCLC and the high expression of PUS1 was associated with shorter OS (Overall Survival), PFS (Progression Free Survival), and PPS (Post Progression Survival) of NSCLC patients. Clinical subgroup analysis showed that PUS1 may be involved in the occurrence and development of NSCLC. Besides, TIMER, ESTIMATE, and IPS analysis suggested that PUS1 expression was associated with immune cell infiltration, and the expression of PUS1 was significantly negatively correlated with DC cell infiltration. GESA analysis also indicated PUS1 may involve in DNA_REPAIR, E2F_TARGETS, MYC_TARGETS_V2, G2M_CHECKPOINT and MYC_TARGETS_V1 pathways and triggered NSCLC malignancy through MCM5 or XPO1. Furthermore, PUS1 may be a potential target for NSCLC therapy.

PUS1 is a novel biomarker for evaluating malignancy of human renal cell carcinoma.

Renal cell carcinoma (RCC) is one of the most common malignancies. Despite the rapid development of the oncology research and surgical treatment, the prognosis of RCC has not significantly improved. Thus, exploration of the pathological molecular mechanism and development of new therapeutic targets of RCC are of great importance. Herein, by bioinformatic analysis and in vitro cell experiments, we report that, the expression of pseudouridine synthase 1 (PUS1), belonging to the family of PUS enzymes that participate in RNA modifications, is closely associated with RCC progression. In addition, the upregulated PUS1 expression results in the elevated RCC cancer cell viability, migration, invasion and colony formation ability, whereas the decreased PUS1 expression exerts the opposite effects on RCC cells. Thus, our findings show the potential role of PUS1 in RCC cells, providing with evidence that PUS1 is involved in RCC progression, which may help contribute to RCC diagnosis and intervention in clinic.

PUS1 May Be a Potential Prognostic Biomarker and Therapeutic Target for Hepatocellular Carcinoma.

Objective: The mechanisms of pseudouridine synthase (PUS) are not definite in hepatocellular carcinoma (HCC), the objective of this study is to investigate the effect of PUS genes in HCC. Materials and Methods: Differentially expressed and prognostic gene of PUS enzymes was identified based on The Cancer Genome Atlas (TCGA), International Cancer Genome Consortium (ICGC) and Gene Expression Profiling Interactive Analysis (GEPIA) databases. For the identified gene, pseudouridine synthase 1 (PUS1), was used for further research. The clinicopathological feature of PUS1 was analyzed by Student's t-test. Prognostic significance was explored by Kaplan-Meier (KM) analysis and Cox proportional hazards regression model. Receiver operating characteristic (ROC) curve was applied to appraise diagnostic and prognostic value. The Database for Annotation, Visualization, and Integrated Discovery (DAVID) and Gene Set Enrichment Analysis (GSEA) were implemented to explore mechanism of PUS1. A Guangxi cohort was applied to verify differential expression. In vitro cell experiments were implemented to investigate the influence for proliferation, reactive oxygen species (ROS) level, migration, and invasion of HCC cells after a knockdown of PUS1. Results: PUS1 was significantly overexpressed in HCC tissues, and patients with high PUS1 were related to unpromising clinicopathological features. Survival analysis revealed high PUS1 expression was associated with a poor overall survival (OS) and 1 year-recurrence free survival (RFS), was an independent risk factor. Meanwhile, ROC curve showed that PUS1 had a diagnostic and prognostic significance to HCC. Functional enrichment analysis implied that PUS1 may be involved in metabolic pathways, mitochondrial function, non-alcoholic fatty liver disease (NAFLD), and some important carcinogenic pathways. Cell assays revealed that knockdown of PUS1 significantly constrained the migration, proliferation, invasion and improved the ROS level of HCC cells. Conclusion: PUS1 may be a prognostic biomarker and a underlying treatment target for HCC.

PUS1 is a novel biomarker for predicting poor outcomes and triple-negative status in breast cancer.

Breast cancer patients' outcomes have improved dramatically in recent years, but relapses and poor prognosis remain common due to its aggressiveness and heterogeneity. The development of reliable biomarkers is still needed for predicting prognosis and treatment effectiveness. Recently, a growing body of research suggests that pseudouridine synthases contribute to the development of many cancers, but their contribution to breast cancer remains largely unknown. Using an integrative analysis, we selected pseudouridine synthase1(PUS1) as the candidate biomarker. A tissue microarray of 131 breast cancer patients was then utilized to determine the clinical significance and prognostic value of PUS1. RNA sequencing analysis was conducted to identify downstream genes that differ between control and PUS1 knockdown cells. The effect of PUS1 on phenotypes of cells was assessed using cell proliferation, colony formation, and transwell invasion assays. We found that breast tumors overexpressed PUS1 compared with paired normal tissues. PUS1 expression was positively correlated with triple-negative breast cancer (TNBC) status (P= 0.020) and tumor grade (P <0.0001), but not with age (P= 0.736), tumor size (P= 0.608), lymph node (P= 0.742), oestrogen receptor (ER) (P= 0.162), progesterone receptor (PR) (P= 0.901), human epidermal growth factor receptor 2 (HER2) (P= 0.608) or tumor stage (P= 0.411). Comparatively, patients with high PUS1 levels had shorter overall survival time (P=0.0001) and relapse-free survival time (P = 0.0093). A univariate and multivariate survival analysis suggested that the overall survival of patients was independently influenced by the PUS1 score (Univariate Cox P <0.0001, HR=5.176, 95% CI =2.420-11.07; Multivariate Cox P = 0.001, HR = 5.291, 95% CI =1.893-14.78). RNA sequencing data revealed the PUS1 knockdown significantly affects a series of cancer related biological process such as regulation of cell proliferation and cell migration, as well as KEGG pathways including Mitophagy and PI3K-Akt signaling. In vitro, knockdown of PUS1 significantly suppressed the proliferation and colony formation abilities of MDA-MB-231 cells and BT-549 cells. Additionally, the ability of tumor cells to invade was remarkably attenuated in low PUS1 expression groups compared with the corresponding control groups. Our results suggested that PUS1 is a novel biomarker that predicts poor outcomes in patients with breast cancer and may prove to be a promising treatment target.

Impact of Pus1 Pseudouridine Synthase on Specific Decoding Events in Saccharomyces cerevisiae.

Pus1-dependent pseudouridylation occurs in many tRNAs and at multiple positions, yet the functional impact of this modification is incompletely understood. We analyzed the consequences of PUS1 deletion on the essential decoding of CAG (Gln) codons by tRNAGlnCUG in yeast. Synthetic lethality was observed upon combining the modification defect with destabilized variants of tRNAGlnCUG, pointing to a severe CAG-decoding defect of the hypomodified tRNA. In addition, we demonstrated that misreading of UAG stop codons by a tRNAGlnCUG variant is positively affected by Pus1. Genetic approaches further indicated that mildly elevated temperature decreases the decoding efficiency of CAG and UAG via destabilized tRNAGlnCAG variants. We also determined the misreading of CGC (Arg) codons by tRNAHisGUG, where the CGC decoder tRNAArgICG contains Pus1-dependent pseudouridine, but not the mistranslating tRNAHis. We found that the absence of Pus1 increased CGC misreading by tRNAHis, demonstrating a positive role of the modification in the competition against non-synonymous near-cognate tRNA. Part of the in vivo decoding defects and phenotypes in pus1 mutants and strains carrying destabilized tRNAGlnCAG were suppressible by additional deletion of the rapid tRNA decay (RTD)-relevant MET22, suggesting the involvement of RTD-mediated tRNA destabilization.

Myopathy, lactic acidosis and sideroblastic anemia 1 (MLASA1): A 25-year follow-up.

Mitochondrial myopathy, lactic acidosis and sideroblastic anemia 1 (MLASA1) is a rare disease caused by biallelic pathogenic variants in the PUS1 gene. There are eleven MLASA1 patients reported worldwide with the majority of the patients originating from the Shiraz region of Iran. The rarity of this disease poses challenges to counseling patients due to a lack of natural history data. This report reviews what is known regarding MLASA1 and describes two brothers with MLASA1 who were cared for over the course of 10 years at the University of California Los Angeles. The brothers suffered from chronic anemia, transfusion dependency and muscle wasting that lead to respiratory insufficiency and death in one of the brothers.

Dual nature of pseudouridylation in U2 snRNA: Pus1p-dependent and Pus1p-independent activities in yeasts and higher eukaryotes.

The pseudouridine at position 43 in vertebrate U2 snRNA is one of the most conserved post-transcriptional modifications of spliceosomal snRNAs; the equivalent position is pseudouridylated in U2 snRNAs in different phyla including fungi, insects, and worms. Pseudouridine synthase Pus1p acts alone on U2 snRNA to form this pseudouridine in yeast Saccharomyces cerevisiae and mouse. Furthermore, in S. cerevisiae, Pus1p is the only pseudouridine synthase for this position. Using an in vivo yeast cell system, we tested enzymatic activity of Pus1p from the fission yeast Schizosaccharomyces pombe, the worm Caenorhabditis elegans, the fruit fly Drosophila melanogaster, and the frog Xenopus tropicalis We demonstrated that Pus1p from C. elegans has no enzymatic activity on U2 snRNA when expressed in yeast cells, whereas in similar experiments, position 44 in yeast U2 snRNA (equivalent to position 43 in vertebrates) is a genuine substrate for Pus1p from S. cerevisiae, S. pombe, Drosophila, Xenopus, and mouse. However, when we analyzed U2 snRNAs from Pus1 knockout mice and the pus1Δ S. pombe strain, we could not detect any changes in their modification patterns when compared to wild-type U2 snRNAs. In S. pombe, we found a novel box H/ACA RNA encoded downstream from the RPC10 gene and experimentally verified its guide RNA activity for positioning Ψ43 and Ψ44 in U2 snRNA. In vertebrates, we showed that SCARNA8 (also known as U92 scaRNA) is a guide for U2-Ψ43 in addition to its previously established targets U2-Ψ34/Ψ44.

Clinical and molecular study in a long-surviving patient with MLASA syndrome due to novel PUS1 mutations.

Myopathy-lactic acidosis-sideroblastic anemia (MLASA) syndrome is a rare autosomal recessive disease. We studied a 43-year-old female presenting since childhood with mild cognitive impairment and sideroblastic anemia. She later developed hepatopathy, cardiomyopathy, and insulin-dependent diabetes. Muscle weakness appeared in adolescence and, at age 43, she was unable to walk. Two novel different mutations in the PUS1 gene were identified: c.487delA (p.I163Lfs*4) and c.884 G>A (p.R295Q). Quantitative analysis of DNA from skeletal muscle biopsies showed a significant increase in mitochondrial DNA (mtDNA) content in the patient compared to controls. Clinical and molecular findings of this patient widen the genotype-phenotype spectrum in MLASA syndrome.