Light Affects Mood and Learning through Distinct Retina-Brain Pathways.

Light exerts a range of powerful biological effects beyond image vision, including mood and learning regulation. While the source of photic information affecting mood and cognitive functions is well established, viz. intrinsically photosensitive retinal ganglion cells (ipRGCs), the central mediators are unknown. Here, we reveal that the direct effects of light on learning and mood utilize distinct ipRGC output streams. ipRGCs that project to the suprachiasmatic nucleus (SCN) mediate the effects of light on learning, independently of the SCN's pacemaker function. Mood regulation by light, on the other hand, requires an SCN-independent pathway linking ipRGCs to a previously unrecognized thalamic region, termed perihabenular nucleus (PHb). The PHb is integrated in a distinctive circuitry with mood-regulating centers and is both necessary and sufficient for driving the effects of light on affective behavior. Together, these results provide new insights into the neural basis required for light to influence mood and learning.

Arginine deprivation enriches lung cancer proteomes with cysteine by inducing arginine-to-cysteine substitutants.

Many types of human cancers suppress the expression of argininosuccinate synthase 1 (ASS1), a rate-limiting enzyme for arginine production. Although dependency on exogenous arginine can be harnessed by arginine-deprivation therapies, the impact of ASS1 suppression on the quality of the tumor proteome is unknown. We therefore interrogated proteomes of cancer patients for arginine codon reassignments (substitutants) and surprisingly identified a strong enrichment for cysteine (R>C) in lung tumors specifically. Most R>C events did not coincide with genetically encoded R>C mutations but were likely products of tRNA misalignments. The expression of R>C substitutants was highly associated with oncogenic kelch-like epichlorohydrin (ECH)-associated protein 1 (KEAP1)-pathway mutations and suppressed by intact-KEAP1 in KEAP1-mutated cancer cells. Finally, functional interrogation indicated a key role for R>C substitutants in cell survival to cisplatin, suggesting that regulatory codon reassignments endow cancer cells with more resilience to stress. Thus, we present a mechanism for enriching lung cancer proteomes with cysteines that may affect therapeutic decisions.

Proteome diversification by mRNA translation in cancer.

mRNA translation is a highly conserved and tightly controlled mechanism for protein synthesis and is well known to be altered by oncogenes to promote cancer development. This distorted mRNA translation is accompanied by the vulnerability of cancer to inhibitors of key mRNA translation components. Novel studies also suggest that these alternations could be utilized for immunotherapy. Ribosome heterogeneity and alternative responses to nutrient shortages, which aid cancer growth and spread, are proposed to elicit aberrant protein production but may also result in previously unidentified therapeutic targets, such as the presentation of cancer-specific peptides at the surface of cancer cells (neoepitopes). This review will assess the driving forces in tRNA and ribosome function that underlie proteome diversification due to alterations in mRNA translation in cancer cells.

Oncogene-dependent sloppiness in mRNA translation.

mRNA translation is a highly conserved and tightly controlled mechanism for protein synthesis. Despite protein quality control mechanisms, amino acid shortage in melanoma induces aberrant proteins by ribosomal frameshifting. The extent and the underlying mechanisms related to this phenomenon are yet unknown. Here, we show that tryptophan depletion-induced ribosomal frameshifting is a widespread phenomenon in cancer. We termed this event sloppiness and strikingly observed its association with MAPK pathway hyperactivation. Sloppiness is stimulated by RAS activation in primary cells, suppressed by pharmacological inhibition of the oncogenic MAPK pathway in sloppy cells, and restored in cells with acquired resistance to MAPK pathway inhibition. Interestingly, sloppiness causes aberrant peptide presentation at the cell surface, allowing recognition and specific killing of drug-resistant cancer cells by T lymphocytes. Thus, while oncogenes empower cancer progression and aggressiveness, they also expose a vulnerability by provoking the production of aberrant peptides through sloppiness.

Loss of Dynamic RNA Interaction and Aberrant Phase Separation Induced by Two Distinct Types of ALS/FTD-Linked FUS Mutations.

FUS is a nuclear RNA-binding protein, and its cytoplasmic aggregation is a pathogenic signature of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). It remains unknown how the FUS-RNA interactions contribute to phase separation and whether its phase behavior is affected by ALS-linked mutations. Here we demonstrate that wild-type FUS binds single-stranded RNA stoichiometrically in a length-dependent manner and that multimers induce highly dynamic interactions with RNA, giving rise to small and fluid condensates. In contrast, mutations in arginine display a severely altered conformation, static binding to RNA, and formation of large condensates, signifying the role of arginine in driving proper RNA interaction. Glycine mutations undergo rapid loss of fluidity, emphasizing the role of glycine in promoting fluidity. Strikingly, the nuclear import receptor Karyopherin-ß2 reverses the mutant defects and recovers the wild-type FUS behavior. We reveal two distinct mechanisms underpinning potentially disparate pathogenic pathways of ALS-linked FUS mutants.

Misexpression of inactive genes in whole blood is associated with nearby rare structural variants.

Gene misexpression is the aberrant transcription of a gene in a context where it is usually inactive. Despite its known pathological consequences in specific rare diseases, we have a limited understanding of its wider prevalence and mechanisms in humans. To address this, we analyzed gene misexpression in 4,568 whole-blood bulk RNA sequencing samples from INTERVAL study blood donors. We found that while individual misexpression events occur rarely, in aggregate they were found in almost all samples and a third of inactive protein-coding genes. Using 2,821 paired whole-genome and RNA sequencing samples, we identified that misexpression events are enriched in cis for rare structural variants. We established putative mechanisms through which a subset of SVs lead to gene misexpression, including transcriptional readthrough, transcript fusions, and gene inversion. Overall, we develop misexpression as a type of transcriptomic outlier analysis and extend our understanding of the variety of mechanisms by which genetic variants can influence gene expression.

Alu insertion-mediated dsRNA structure formation with pre-existing Alu elements as a disease-causing mechanism.

We previously identified a homozygous Alu insertion variant (Alu_Ins) in the 3'-untranslated region (3'-UTR) of SPINK1 as the cause of severe infantile isolated exocrine pancreatic insufficiency. Although we established that Alu_Ins leads to the complete loss of SPINK1 mRNA expression, the precise mechanisms remained elusive. Here, we aimed to elucidate these mechanisms through a hypothesis-driven approach. Initially, we speculated that, owing to its particular location, Alu_Ins could independently disrupt mRNA 3' end formation and/or affect other post-transcriptional processes such as nuclear export and translation. However, employing a 3'-UTR luciferase reporter assay, Alu_Ins was found to result in only an ∼50% reduction in luciferase activity compared to wild type, which is insufficient to account for the severe pancreatic deficiency in the Alu_Ins homozygote. We then postulated that double-stranded RNA (dsRNA) structures formed between Alu elements, an upstream mechanism regulating gene expression, might be responsible. Using RepeatMasker, we identified two Alu elements within SPINK1's third intron, both oriented oppositely to Alu_Ins. Through RNAfold predictions and full-length gene expression assays, we investigated orientation-dependent interactions between these Alu repeats. We provide compelling evidence to link the detrimental effect of Alu_Ins to extensive dsRNA structures formed between Alu_Ins and pre-existing intronic Alu sequences, including the restoration of SPINK1 mRNA expression by aligning all three Alu elements in the same orientation. Given the widespread presence of Alu elements in the human genome and the potential for new Alu insertions at almost any locus, our findings have important implications for detecting and interpreting Alu insertions in disease genes.

Improved detection of aberrant splicing with FRASER 2.0 and the intron Jaccard index.

Detection of aberrantly spliced genes is an important step in RNA-seq-based rare-disease diagnostics. We recently developed FRASER, a denoising autoencoder-based method that outperformed alternative methods of detecting aberrant splicing. However, because FRASER's three splice metrics are partially redundant and tend to be sensitive to sequencing depth, we introduce here a more robust intron-excision metric, the intron Jaccard index, that combines the alternative donor, alternative acceptor, and intron-retention signal into a single value. Moreover, we optimized model parameters and filter cutoffs by using candidate rare-splice-disrupting variants as independent evidence. On 16,213 GTEx samples, our improved algorithm, FRASER 2.0, called typically 10 times fewer splicing outliers while increasing the proportion of candidate rare-splice-disrupting variants by 10-fold and substantially decreasing the effect of sequencing depth on the number of reported outliers. To lower the multiple-testing correction burden, we introduce an option to select the genes to be tested for each sample instead of a transcriptome-wide approach. This option can be particularly useful when prior information, such as candidate variants or genes, is available. Application on 303 rare-disease samples confirmed the relative reduction in the number of outlier calls for a slight loss of sensitivity; FRASER 2.0 recovered 22 out of 26 previously identified pathogenic splicing cases with default cutoffs and 24 when multiple-testing correction was limited to OMIM genes containing rare variants. Altogether, these methodological improvements contribute to more effective RNA-seq-based rare diagnostics by drastically reducing the amount of splicing outlier calls per sample at minimal loss of sensitivity.

The novel pre-rRNA detection workflow "Riboprobing" allows simple identification of undescribed RNA species.

Ribosomes translate mRNA into proteins and are essential for every living organism. In eukaryotes, both ribosomal subunits are rapidly assembled in a strict hierarchical order, starting in the nucleolus with the transcription of a common precursor ribosomal RNA (pre-rRNA). This pre-rRNA encodes three of the four mature rRNAs, which are formed by several, consecutive endonucleolytic and exonucleolytic processing steps. Historically, northern blots are used to analyze the variety of different pre-rRNA species, only allowing rough length estimations. Although this limitation can be overcome with primer extension, both approaches often use radioactivity and are time-consuming and costly. Here, we present "Riboprobing," a linker ligation-based workflow followed by reverse transcription and PCR for easy and fast detection and characterization of pre-rRNA species and their 5' as well as 3' ends. Using standard molecular biology laboratory equipment, "Riboprobing" allows reliable discrimination of pre-rRNA species not resolved by northern blot (e.g., 27SA2, 27SA3, and 27SB pre-rRNA). The method can successfully be used for the analysis of total cell extracts as well as purified pre-ribosomes for a straightforward evaluation of the impact of mutant gene versions or inhibitors. In the course of method development, we identified and characterized a hitherto undescribed aberrant pre-rRNA arising from LiCl inhibition. This pre-rRNA fragment spans from processing site A1 to E, forming a small RNP that lacks most early joining assembly factors. This finding expands our knowledge of how the cell deals with severe pre-rRNA processing defects and demonstrates the strict requirement for the 5'ETS (external transcribed spacer) for the assembly process.

Slippy-Sloppy translation: a tale of programmed and induced-ribosomal frameshifting.

Programmed ribosomal frameshifting (PRF) is a key mechanism that viruses use to generate essential proteins for replication, and as a means of regulating gene expression. PRF generally involves recoding signals or frameshift stimulators to elevate the occurrence of frameshifting at shift-prone 'slippery' sequences. Given its essential role in viral replication, targeting PRF was envisioned as an attractive tool to block viral infection. However, in contrast to controlled-PRF mechanisms, recent studies have shown that ribosomes of many human cancer cell types are prone to frameshifting upon amino acid shortage; thus, these cells are deemed to be sloppy. The resulting products of a sloppy frameshift at the 'hungry' codons are aberrant proteins the degradation and display of which at the cell surface can trigger T cell activation. In this review, we address recent discoveries in ribosomal frameshifting and their functional consequences for the proteome in human cancer cells.

Computational approaches for detecting disease-associated alternative splicing events.

Alternative splicing (AS) is a key transcriptional regulation pathway. Recent studies have shown that AS events are associated with the occurrence of complex diseases. Various computational approaches have been developed for the detection of disease-associated AS events. In this review, we first describe the metrics used for quantitative characterization of AS events. Second, we review and discuss the three types of methods for detecting disease-associated splicing events, which are differential splicing analysis, aberrant splicing detection and splicing-related network analysis. Third, to further exploit the genetic mechanism of disease-associated AS events, we describe the methods for detecting genetic variants that potentially regulate splicing. For each type of methods, we conducted experimental comparison to illustrate their performance. Finally, we discuss the limitations of these methods and point out potential ways to address them. We anticipate that this review provides a systematic understanding of computational approaches for the analysis of disease-associated splicing.

Comprehensive splicing analysis of the alternatively spliced CHEK2 exons 8 and 10 reveals three enhancer/silencer-rich regions and 38 spliceogenic variants.

Splicing is controlled by a large set of regulatory elements (SREs) including splicing enhancers and silencers, which are involved in exon recognition. Variants at these motifs may dysregulate splicing and trigger loss-of-function transcripts associated with disease. Our goal here was to study the alternatively spliced exons 8 and 10 of the breast cancer susceptibility gene CHEK2. For this purpose, we used a previously published minigene with exons 6-10 that produced the expected minigene full-length transcript and replicated the naturally occurring events of exon 8 [Δ(E8)] and exon 10 [Δ(E10)] skipping. We then introduced 12 internal microdeletions of exons 8 and 10 by mutagenesis in order to map SRE-rich intervals by splicing assays in MCF-7 cells. We identified three minimal (10-, 11-, 15-nt) regions essential for exon recognition: c.863_877del [ex8, Δ(E8): 75%] and c.1073_1083del and c.1083_1092del [ex10, Δ(E10): 97% and 62%, respectively]. Then 87 variants found within these intervals were introduced into the wild-type minigene and tested functionally. Thirty-eight of them (44%) impaired splicing, four of which (c.883G>A, c.883G>T, c.884A>T, and c.1080G>T) induced negligible amounts (<5%) of the minigene full-length transcript. Another six variants (c.886G>A, c.886G>T, c.1075G>A, c.1075G>T, c.1076A>T, and c.1078G>T) showed significantly strong impacts (20-50% of the minigene full-length transcript). Thirty-three of the 38 spliceogenic variants were annotated as missense, three as nonsense, and two as synonymous, underlying the fact that any exonic change is capable of disrupting splicing. Moreover, c.883G>A, c.883G>T, and c.884A>T were classified as pathogenic/likely pathogenic variants according to ACMG/AMP (American College of Medical Genetics and Genomics/Association for Molecular Pathology)-based criteria. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Exon 1-targeting miRNA reduces the pathogenic exon 1 HTT protein in Huntington disease models.

Huntington disease (HD) is a fatal neurodegenerative disease caused by a trinucleotide repeat expansion in exon 1 of the huntingtin gene (HTT) resulting in toxic gain-of-function and cell death. Despite its monogenic cause, the pathogenesis of HD is highly complex and increasing evidence indicates that, in addition to the full-length (FL) mutant HTT protein, the expanded exon 1 HTT (HTTexon1) protein that is translated from the HTT1a transcript generated by aberrant splicing is prone to aggregate and may contribute to HD pathology. This finding suggests that reducing the expression of HTT1a may achieve a greater therapeutic benefit than targeting only FL mutant HTT. Conversely, strategies that exclusively target FL HTT may not fully prevent the pathogenesis of HD. We have developed an engineered microRNA targeting the HTT exon 1 sequence (miHTT), delivered via adeno-associated virus serotype 5 (AAV5). The target sequence of miHTT is present in both FL HTT and HTT1a transcripts. Preclinical studies with AAV5-miHTT have demonstrated efficacy in several rodent and large animal models by reducing FL HTT mRNA and protein and rescuing HD-like phenotypes, and have been the rationale for phase I/II clinical studies now ongoing in the US and Europe. In the present study, we evaluated the ability of AAV5-miHTT to reduce the levels of aberrantly spliced HTT1a mRNA and the HTTexon1 protein in the brain of two mouse models of HD (heterozygous zQ175 knock-in mice and humanized Hu128/21 mice). Polyadenylated HTT1a mRNA and HTTexon1 protein were detected in the striatum and cortex of heterozygous zQ175 knock-in mice, but not in wild-type, littermate control mice. Intrastriatal administration of AAV5-miHTT resulted in dose-dependent expression of mature miHTT microRNA in cortical brain regions, accompanied by significant lowering of both FL HTT and HTT1a mRNA expression at two months post-injection. Mutant HTT and HTTexon1 protein levels were also significantly reduced in the striatum and cortex of heterozygous zQ175 knock-in at 2 months after AAV5-miHTT treatment and in humanized Hu128/21 mice 7 months post-treatment. The effects were confirmed in primary Hu128/21 neuronal cultures. These results demonstrate that AAV5-miHTT gene therapy is an effective approach to lower both FL HTT and the pathogenic HTTexon1 levels, which could potentially have an additive therapeutic benefit compared to other HTT-targeting modalities.

Chronically activated microglia in ALS gradually lose their immune functions and develop unconventional proteome.

Neuroinflammation and chronic activation of microglial cells are the prominent features of amyotrophic lateral sclerosis (ALS) pathology. While alterations in the mRNA profile of diseased microglia have been well documented, the actual microglia proteome remains poorly characterized. Here we performed a functional characterization together with proteome analyses of microglial cells at different stages of disease in the SOD1-G93A model of ALS. Functional analyses of microglia derived from the lumbar spinal cord of symptomatic mice revealed: (i) remarkably high mitotic index (close to 100% cells are Ki67+) (ii) significant decrease in phagocytic capacity when compared to age-matched control microglia, and (iii) diminished response to innate immune challenges in vitro and in vivo. Proteome analysis revealed a development of two distinct molecular signatures at early and advanced stages of disease. While at early stages of disease, we identified several proteins implicated in microglia immune functions such as GPNMB, HMBOX1, at advanced stages of disease microglia signature at protein level was characterized with a robust upregulation of several unconventional proteins including rootletin, major vaults proteins and STK38. Upregulation of GPNMB and rootletin has been also found in the spinal cord samples of sporadic ALS. Remarkably, the top biological functions of microglia, in particular in the advanced disease, were not related to immunity/immune response, but were highly enriched in terms linked to RNA metabolism. Together, our results suggest that, over the course of disease, chronically activated microglia develop unconventional protein signatures and gradually lose their immune identity ultimately turning into functionally inefficient immune cells.

Loss-of-function variants affecting the STAGA complex component SUPT7L cause a developmental disorder with generalized lipodystrophy.

Generalized lipodystrophy is a feature of various hereditary disorders, often leading to a progeroid appearance. In the present study we identified a missense and a frameshift variant in a compound heterozygous state in SUPT7L in a boy with intrauterine growth retardation, generalized lipodystrophy, and additional progeroid features. SUPT7L encodes a component of the transcriptional coactivator complex STAGA. By transcriptome sequencing, we showed the predicted missense variant to cause aberrant splicing, leading to exon truncation and thereby to a complete absence of SUPT7L in dermal fibroblasts. In addition, we found altered expression of genes encoding DNA repair pathway components. This pathway was further investigated and an increased rate of DNA damage was detected in proband-derived fibroblasts and genome-edited HeLa cells. Finally, we performed transient overexpression of wildtype SUPT7L in both cellular systems, which normalizes the number of DNA damage events. Our findings suggest SUPT7L as a novel disease gene and underline the link between genome instability and progeroid phenotypes.

Multi-omics to characterize the functional relationships of R-loops with epigenetic modifications, RNAPII transcription and gene expression.

Abnormal accumulation of R-loops results in replication stress, genome instability, chromatin alterations and gene silencing. Little research has been done to characterize functional relationships among R-loops, histone marks, RNA polymerase II (RNAPII) transcription and gene regulation. We built extremely randomized trees (ETs) models to predict the genome-wide R-loops using RNAPII and multiple histone modifications chromatin immunoprecipitation (ChIP)-seq, DNase-seq, Global Run-On sequencing (GRO-seq) and R-loop profiling data. We compared the performance of ET models to multiple machine learning approaches, and the proposed ET models achieved the best and extremely robust performances. Epigenetic profiles are highly predictive of R-loops genome-widely and they are strongly associated with R-loop formation. In addition, the presence of R-loops is significantly correlated with RNAPII transcription activity, H3K4me3 and open chromatin around the transcription start site, and H3K9me1 and H3K9me3 around the transcription termination site. RNAPII pausing defects were correlated with 5'R-loops accumulation, and transcriptional termination defects and read-throughs were correlated with 3'R-loops accumulation. Furthermore, we found driver genes with 5'R-loops and RNAPII pausing defects express significantly higher and genes with 3'R-loops and read-through transcription express significantly lower than genes without R-loops. These driver genes are enriched with chromosomal instability, Hippo-Merlin signaling Dysregulation, DNA damage response and TGF-ß pathways, indicating R-loops accumulating at the 5' end of genes play oncogenic roles, whereas at the 3' end of genes play tumor-suppressive roles in tumorigenesis.

Therapeutic strategies for aberrant splicing in cancer and genetic disorders.

Accurate pre-mRNA splicing is essential for proper protein translation; however, aberrant splicing is commonly observed in the context of cancer and genetic disorders. Notably, in genetic diseases, these splicing abnormalities often play a pivotal role. Substantial challenges persist in accurately identifying and classifying disease-induced aberrant splicing, as well as in development of targeted therapeutic strategies. In this review, we examine prevalent forms of aberrant splicing and explore potential therapeutic approaches aimed at addressing these splicing-related diseases. This summary contributes to a deeper understanding of the complexities about aberrant splicing and provide a foundation for the development of effective therapeutic interventions in the field of genetic disorders and cancer.

Genetic Variants Supporting the Diagnosis of Primary Ciliary Dyskinesia in Japan.

Primary ciliary dyskinesia (PCD; OMIM 244400) is a rare genetic disorder affecting motile cilia and is characterized by impaired mucociliary clearance in the airway epithelium that leads to chronic oto-sinopulmonary manifestations. To date, over 50 PCD-causing genes have been identified, with these genes and their variants varying globally across populations. We performed targeted resequencing of 42 PCD-causative genes in 150 Japanese patients suspected of having PCD and identified pathogenic or likely pathogenic variants in 51 patients. Among these, 24 patients exhibited a homozygous deletion of DRC1 exons 1-4, the most common cause of PCD in Japan. The allele frequency of this deletion was estimated at 0.0034 (95% CI: 0.0025-0.0044), based on bioinformatic analysis of 7906 whole-genome sequences from the general Japanese population. Additionally, RNA sequencing of nasal samples supplemented in silico variant predictions, aiding in the identification of causative variants. Considering potential ethnic differences, it is essential to accumulate global data on these variants and their functional impacts.

Comparative study of left vertebral artery revascularization in patients with and without aberrant left vertebral anatomy.

OBJECTIVE: Left vertebral artery revascularization is indicated in surgery involving zone 2 of the aortic arch and is typically accomplished indirectly via subclavian artery revascularization. For aberrant left vertebral anatomy, direct revascularization is indicated. Our objective was to compare the outcomes of direct vertebral artery revascularization with indirect subclavian artery revascularization for treating aortic arch pathology and to identify predictors of mortality. METHODS: A retrospective cohort study was conducted at a single tertiary hospital, including patients who underwent open or endovascular vertebral artery revascularization from 2005 to 2022. Those who underwent direct vertebral revascularization were compared with those who were indirectly revascularized via subclavian artery revascularization. The outcomes of interest were a composite outcome (any of death, stroke, nerve injury, and thrombosis) and mortality. Univariate logistic regression models were fitted to quantify the strength of differences between the direct and indirect revascularization cohorts. Cox regression was used to identify mortality predictors. RESULTS: Of 143 patients who underwent vertebral artery revascularization, 21 (14.7%) had a vertebral artery originating from the aortic arch. The median length of stay was 10 days (interquartile range, 6-20 days), and demographics were similar between cohorts. The incidence of composite outcome, bypass thrombosis, and hoarseness was significantly higher in the direct group (42.9% vs 18.0%, P = .019; 33.3% vs 0.8%, P < .0001; 57.1% vs 18.0%, P < .001, respectively). The direct group was approximately three times more likely to experience the composite outcome (odds ratio, 3.41; 95% confidence interval, 1.28, 9.08); similarly, this group was approximately six times more likely to have hoarseness (odds ratio, 5.88; 95% confidence interval, 2.21, 15.62). There was no significant difference in mortality rates at 30 days, 1, 3, 5, and 10 years of follow-up. Age, length of hospital stay, and congestive heart failure were identified as predictors of higher mortality. After adjusting for these covariates, the group itself was not an independent predictor of mortality. CONCLUSIONS: Direct vertebral revascularization was associated with higher rates of composite outcome (death, stroke, nerve injury, and thrombosis), bypass thrombosis and hoarseness. Patients with aberrant vertebral anatomy are at higher risks of these complications compared with patients with standard arch anatomy. However, after adjusting for other factors, mortality rates were not significantly different between the groups.

Aberrant expression of GATA3 in metastatic adenocarcinoma of the prostate: an important pitfall.

AIMS: The distinction of high-grade prostate cancer (PCa) from poorly differentiated urothelial carcinoma (UC) can be somewhat challenging on clinical and morphological grounds alone, yet it is of great importance for prognostication and choice of treatment. GATA3 is a useful immunohistochemical marker to confirm urothelial origin. However, recent works report strong GATA3 immunoexpression in primary high-grade PCa. The aim of this study was to explore GATA3 expression specifically in metastatic PCa. METHODS AND RESULTS: The pathology databases of four tertiary institutions were queried for cases of metastatic PCa. Available slides and clinical records were reviewed by experienced genitourinary pathologists. Prostatic markers (PSA, PSAP, NKX3.1) and GATA3 immunohistochemistry were performed. A total of 163 metastatic PCa cases were included. At least one prostate marker was positive in each case of non-regional distant metastasis, confirming prostatic origin. GATA3 strong staining was found in four (2.5%) cases: two liver, one bone and one non-regional lymph-node metastases. All four patients had Grade Group 5 PCa at the initial diagnosis. The metastatic prostatic adenocarcinomas were solid, either with no gland formation (n = 3) or with only focal cribriforming (n = 1). CONCLUSIONS: To our knowledge, this is the first study exploring GATA3 expression specifically in metastatic PCa. Despite being infrequent, GATA3 positivity in high-grade PCa may lead to misdiagnosis, with clinical implications. We recommend a panel of immunohistochemical markers, both prostatic and urothelial, for ruling out UC, either in primary tumour samples or in the event of metastases of unknown primary, when a genitourinary origin is suspected.