Penile squamous cell carcinoma (pSCC) represents an uncommon malignancy characterized by stagnant mortality, psychosexual distress, and a highly variable prognosis. Currently, the World Health Organization distinguishes between human papillomavirus (HPV)-related and HPV-independent pSCC. Recently, there has been an evolving line of research documenting the enrichment of HPV-independent pSCC with a high tumor mutational burden (TMB) and programmed death ligand-1 expression, as well as clusters of genes associated with HPV status. In this study, we conducted comprehensive next-generation sequencing DNA profiling of 146 pSCC samples using a panel consisting of 355 genes associated with tumors. This profiling was correlated with immunohistochemical markers and prognostic clinical data. A survival analysis of recurrent genomic events (found in ≥10 cases) was performed. TP53, CDKN2A, ATM, EPHA7, POT1, CHEK1, GRIN2A, and EGFR alterations were associated with significantly shortened overall survival in univariate and multivariate analysis. HPV positivity, diagnosed through both p16 immunohistochemistry and HPV DNA analysis, displayed no impact on survival but was associated with high-grade, lymphatic invasion, programmed death ligand-1 negativity/weak expression, and low TMB. FAT1, TP53, CDKN2A, CASP8, and HRAS were more often mutated in HPV-independent pSCC. In contrast, HPV-associated pSCCs were enriched by EPHA7, ATM, GRIN2A, and CHEK1 mutations. PIK3CA, FAT1, FBXW7, and KMT2D mutations were associated with high TMB. NOTCH1, TP53, CDKN2A, POT1, KMT2D, ATM, CHEK1, EPHA3, and EGFR alterations were related to adverse clinicopathologic signs, such as advanced stage, high tumor budding, and lymphovascular invasion. We detected 160 alterations with potential treatment implications, with 21.2% of samples showing alterations in the homologous recombination repair pathway. To the best of our knowledge, this study describes the largest cohort of pSCC with complex molecular pathologic, clinical, and prognostic analysis correlating with prognosis.

• MeSH
• ATM protein genetika   MeSH
• dospělí MeSH
• erbB receptory genetika   MeSH
• infekce papilomavirem MeSH
• inhibitor p16 cyklin-dependentní kinasy genetika   MeSH
• lidé středního věku MeSH
• lidé MeSH
• mutace MeSH
• nádorové biomarkery * genetika   analýza   MeSH
• nádorový supresorový protein p53 genetika   MeSH
• nádory penisu * genetika   mortalita   patologie   virologie   MeSH
• prognóza MeSH
• proteiny vázající telomery MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• shelterinový komplex MeSH
• spinocelulární karcinom * genetika   mortalita   patologie   virologie   MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• Publikační typ
• časopisecké články MeSH

In this study, we employed short- and long-read sequencing technologies to delineate the transcriptional architecture of the human monkeypox virus and to identify key regulatory elements that govern its gene expression. Specifically, we conducted a transcriptomic analysis to annotate the transcription start sites (TSSs) and transcription end sites (TESs) of the virus by utilizing Cap Analysis of gene expression sequencing on the Illumina platform and direct RNA sequencing on the Oxford Nanopore technology device. Our investigations uncovered significant complexity in the use of alternative TSSs and TESs in viral genes. In this research, we also detected the promoter elements and poly(A) signals associated with the viral genes. Additionally, we identified novel genes in both the left and right variable regions of the viral genome.IMPORTANCEGenerally, gaining insight into how the transcription of a virus is regulated offers insights into the key mechanisms that control its life cycle. The recent outbreak of the human monkeypox virus has underscored the necessity of understanding the basic biology of its causative agent. Our results are pivotal for constructing a comprehensive transcriptomic atlas of the human monkeypox virus, providing valuable resources for future studies.

• MeSH
• genom virový MeSH
• lidé MeSH
• počátek transkripce * MeSH
• promotorové oblasti (genetika) MeSH
• RNA virová genetika   MeSH
• sekvenční analýza RNA * metody   MeSH
• stanovení celkové genové exprese MeSH
• transkriptom * MeSH
• virus opičích neštovic genetika   MeSH
• vysoce účinné nukleotidové sekvenování MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Protein phosphatase magnesium-dependent 1 delta (PPM1D) terminates the cell cycle checkpoint by dephosphorylating the tumour suppressor protein p53. By targeting additional substrates at chromatin, PPM1D contributes to the control of DNA damage response and DNA repair. Using proximity biotinylation followed by proteomic analysis, we identified a novel interaction between PPM1D and the shelterin complex that protects telomeric DNA. In addition, confocal microscopy revealed that endogenous PPM1D localises at telomeres. Further, we found that ATR phosphorylated TRF2 at S410 after induction of DNA double strand breaks at telomeres and this modification increased after inhibition or loss of PPM1D. TRF2 phosphorylation stimulated its interaction with TIN2 both in vitro and at telomeres. Conversely, induced expression of PPM1D impaired localisation of TIN2 and TPP1 at telomeres. Finally, recruitment of the DNA repair factor 53BP1 to the telomeric breaks was strongly reduced after inhibition of PPM1D and was rescued by the expression of TRF2-S410A mutant. Our results suggest that TRF2 phosphorylation promotes the association of TIN2 within the shelterin complex and regulates DNA repair at telomeres.

• MeSH
• fosforylace MeSH
• lidé MeSH
• poškození DNA MeSH
• protein TRF2 * MeSH
• proteiny vázající telomery * metabolismus   MeSH
• proteomika MeSH
• shelterinový komplex * MeSH
• telomery metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The role of alternative promoter usage in tissue-specific gene expression has been well established; however, its role in complex diseases is poorly understood. We performed cap analysis of gene expression (CAGE) sequencing from the left ventricle of a rat model of hypertension, the spontaneously hypertensive rat (SHR), and a normotensive strain, Brown Norway to understand the role of alternative promoter usage in complex disease. We identified 26,560 CAGE-defined transcription start sites in the rat left ventricle, including 1,970 novel cardiac transcription start sites. We identified 28 genes with alternative promoter usage between SHR and Brown Norway, which could lead to protein isoforms differing at the amino terminus between two strains and 475 promoter switching events altering the length of the 5' UTR. We found that the shift in Insr promoter usage was significantly associated with insulin levels and blood pressure within a panel of HXB/BXH recombinant inbred rat strains, suggesting that hyperinsulinemia due to insulin resistance might lead to hypertension in SHR. Our study provides a preliminary evidence of alternative promoter usage in complex diseases.

• MeSH
• genetická transkripce genetika   MeSH
• hypertenze * genetika   metabolismus   MeSH
• krysa rodu rattus MeSH
• potkani inbrední SHR MeSH
• promotorové oblasti (genetika) genetika   MeSH
• sekvenční analýza RNA metody   MeSH
• stanovení celkové genové exprese metody   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

The ongoing COVID-19 pandemic exemplifies the general need to better understand viral infections. The positive single-strand RNA genome of its causative agent, the SARS coronavirus 2 (SARS-CoV-2), encodes all viral enzymes. In this work, we focused on one particular methyltransferase (MTase), nsp16, which, in complex with nsp10, is capable of methylating the first nucleotide of a capped RNA strand at the 2'-O position. This process is part of a viral capping system and is crucial for viral evasion of the innate immune reaction. In light of recently discovered non-canonical RNA caps, we tested various dinucleoside polyphosphate-capped RNAs as substrates for nsp10-nsp16 MTase. We developed an LC-MS-based method and discovered four types of capped RNA (m7Gp3A(G)- and Gp3A(G)-RNA) that are substrates of the nsp10-nsp16 MTase. Our technique is an alternative to the classical isotope labelling approach for the measurement of 2'-O-MTase activity. Further, we determined the IC50 value of sinefungin to illustrate the use of our approach for inhibitor screening. In the future, this approach may be an alternative technique to the radioactive labelling method for screening inhibitors of any type of 2'-O-MTase.

• MeSH
• chromatografie kapalinová MeSH
• COVID-19 virologie   MeSH
• hmotnostní spektrometrie MeSH
• lidé MeSH
• methyltransferasy genetika   metabolismus   MeSH
• metylace MeSH
• regulace exprese virových genů MeSH
• RNA čepičky MeSH
• RNA virová genetika   MeSH
• SARS-CoV-2 enzymologie   genetika   MeSH
• substrátová specifita MeSH
• virové nestrukturální proteiny genetika   metabolismus   MeSH
• virové regulační a přídatné proteiny genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

In somatotroph pituitary tumours, somatostatin analogue (SSA) therapy outcomes vary throughout the studies. We performed an analysis of cohort of patients with acromegaly from the Czech registry to identify new prognostic and predictive factors. Clinical data of patients were collected, and complex immunohistochemical assessment of tumour samples was performed (SSTR1-5, dopamine D2 receptor, E-cadherin, AIP). The study included 110 patients. In 31, SSA treatment outcome was evaluated. Sparsely granulated tumours (SGST) differed from the other subtypes in expression of SSTR2A, SSTR3, SSTR5 and E-cadherin and occurred more often in young. No other clinical differences were observed. Trouillas grading system showed association with age, tumour size and SSTR2A expression. Factors significantly associated with SSA treatment outcome included age, IGF1 levels, tumour size and expression of E-cadherin and SSTR2A. In the group of SGST, poor SSA response was observed in younger patients with larger tumours, lower levels of SSTR2A and higher Ki67. We observed no relationship with expression of other proteins including AIP. No predictive value of E-cadherin was observed when tumour subtype was considered. Multiple additional factors apart from SSTR2A expression can predict treatment outcome in patients with acromegaly.

• MeSH
• akromegalie komplikace   genetika   metabolismus   MeSH
• biologické markery MeSH
• dospělí MeSH
• imunohistochemie MeSH
• kadheriny genetika   MeSH
• klinické rozhodování MeSH
• kombinovaná terapie MeSH
• lidé středního věku MeSH
• lidé MeSH
• management nemoci MeSH
• mladý dospělý MeSH
• nádory hypofýzy diagnóza   etiologie   terapie   MeSH
• prognóza MeSH
• protein - isoformy MeSH
• receptory somatostatinu genetika   metabolismus   MeSH
• regulace genové exprese * MeSH
• ROC křivka MeSH
• výsledek terapie MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Deregulation of microRNA (miRNA) expression plays a critical role in the transition from a physiological to a pathological state. The accurate miRNA promoter identification in multiple cell types is a fundamental endeavor towards understanding and characterizing the underlying mechanisms of both physiological as well as pathological conditions. DIANA-miRGen v4 (www.microrna.gr/mirgenv4) provides cell type specific miRNA transcription start sites (TSSs) for over 1500 miRNAs retrieved from the analysis of >1000 cap analysis of gene expression (CAGE) samples corresponding to 133 tissues, cell lines and primary cells available in FANTOM repository. MiRNA TSS locations were associated with transcription factor binding site (TFBSs) annotation, for >280 TFs, derived from analyzing the majority of ENCODE ChIP-Seq datasets. For the first time, clusters of cell types having common miRNA TSSs are characterized and provided through a user friendly interface with multiple layers of customization. DIANA-miRGen v4 significantly improves our understanding of miRNA biogenesis regulation at the transcriptional level by providing a unique integration of high-quality annotations for hundreds of cell specific miRNA promoters with experimentally derived TFBSs.

• MeSH
• anotace sekvence MeSH
• buněčné linie MeSH
• databáze nukleových kyselin * MeSH
• genetická transkripce MeSH
• genom * MeSH
• internet MeSH
• lidé MeSH
• mikro RNA genetika   metabolismus   MeSH
• počátek transkripce MeSH
• primární buněčná kultura MeSH
• promotorové oblasti (genetika) * MeSH
• sekvence nukleotidů MeSH
• software * MeSH
• transkripční faktory genetika   metabolismus   MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

In the absence of transcription, the regulation of gene expression in oocytes is controlled almost exclusively at the level of transcriptome and proteome stabilization, and translation. A subset of maternal transcripts is stored in a translationally dormant state in the oocyte, and temporally driven translation of specific mRNAs propel meiotic progression, oocyte-to-embryo transition and early embryo development. We identified Ank2.3 as the only transcript variant present in the mouse oocyte and discovered that it is translated after nuclear envelope breakdown. Here we show that Ank2.3 mRNA is localized in higher concentration in the oocyte nucleoplasm and, after nuclear envelope breakdown, in the newly forming spindle where its translation occurs. Furthermore, we reveal that Ank2.3 mRNA contains an oligo-pyrimidine motif at 5'UTR that predetermines its translation through a cap-dependent pathway. Lastly, we show that prevention of ANK2 translation leads to abnormalities in oocyte cytokinesis.

• MeSH
• ankyriny genetika   metabolismus   MeSH
• časoprostorová analýza * MeSH
• cytokineze * MeSH
• embryo savčí cytologie   fyziologie   MeSH
• meióza * MeSH
• messenger RNA genetika   metabolismus   MeSH
• myši MeSH
• oocyty cytologie   fyziologie   MeSH
• oogeneze MeSH
• vývojová regulace genové exprese * MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Haloalkane dehalogenases (HLDs) convert halogenated aliphatic pollutants to less toxic compounds by a hydrolytic mechanism. Owing to their broad substrate specificity and high enantioselectivity, haloalkane dehalogenases can function as biosensors to detect toxic compounds in the environment or can be used for the production of optically pure compounds. Here, the structural analysis of the haloalkane dehalogenase DpcA isolated from the psychrophilic bacterium Psychrobacter cryohalolentis K5 is presented at the atomic resolution of 1.05 Å. This enzyme exhibits a low temperature optimum, making it attractive for environmental applications such as biosensing at the subsurface environment, where the temperature typically does not exceed 25°C. The structure revealed that DpcA possesses the shortest access tunnel and one of the most widely open main tunnels among structural homologs of the HLD-I subfamily. Comparative analysis revealed major differences in the region of the α4 helix of the cap domain, which is one of the key determinants of the anatomy of the tunnels. The crystal structure of DpcA will contribute to better understanding of the structure-function relationships of cold-adapted enzymes.

• MeSH
• bakteriální proteiny chemie   genetika   metabolismus   MeSH
• Escherichia coli genetika   metabolismus   MeSH
• exprese genu MeSH
• genetické vektory chemie   metabolismus   MeSH
• halogenované uhlovodíky chemie   metabolismus   MeSH
• hydrolasy chemie   genetika   metabolismus   MeSH
• interakční proteinové domény a motivy MeSH
• klonování DNA MeSH
• konformace proteinů, alfa-helix MeSH
• konformace proteinů, beta-řetězec MeSH
• krystalografie rentgenová MeSH
• nízká teplota MeSH
• Psychrobacter chemie   enzymologie   MeSH
• rekombinantní fúzní proteiny chemie   genetika   metabolismus   MeSH
• sekvence aminokyselin MeSH
• simulace molekulového dockingu MeSH
• strukturní homologie proteinů MeSH
• substrátová specifita MeSH
• termodynamika MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• Publikační typ
• časopisecké články MeSH

Alternative polyadenylation is an important and pervasive mechanism that generates heterogeneous 3'-termini of mRNA and is considered an important regulator of gene expression. We performed bioinformatics analyses of ESTs and the 3'-UTRs of the main transcript splice variants of the translational initiation factor eIF4E1 and its family members, eIF4E2 and eIF4E3. This systematic analysis led to the prediction of new polyadenylation signals. All identified polyadenylation sites were subsequently verified by 3'RACE of transcripts isolated from human lymphoblastic cell lines. This led to the observation that multiple simultaneous polyadenylation site utilization occurs in single cell population. Importantly, we described the use of new polyadenylation site in the eIF4E1 mRNA, which lacked any known polyadenylation signal. The proportion of eIF4E1 transcripts derived from the first two polyadenylation sites in eIF4E1 mRNA achieved 15% in a wide range of cell lines. This result demonstrates the ubiquitous presence of ARE-lacking transcripts, which escape HuR/Auf1-mediated control, the main mechanism of eIF4E1 gene expression regulation. We found many EST clones documenting the significant production of transcript variants 2-4 of eIF4E2 gene that encode proteins with C-termini that were distinct from the mainly studied prototypical isoform A. Similarly, eIF4E3 mRNAs are produced as two main variants with the same very long 3'-UTR with potential for heavy post-transcriptional regulation. We identified sparsely documented transcript variant 1 of eIF4E3 gene in human placenta. eIF4E3 truncated transcript variants were found mainly in brain. We propose to elucidate the minor splice variants of eIF4E2 and eIF4E3 in great detail because they might produce proteins with modified features that fulfill different cellular roles from their major counterparts.

• MeSH
• 3' nepřekládaná oblast MeSH
• buněčné linie MeSH
• eukaryotický iniciační faktor 4E genetika   metabolismus   MeSH
• exprimované sekvenční adresy MeSH
• lidé MeSH
• messenger RNA genetika   metabolismus   MeSH
• mozek metabolismus   MeSH
• placenta metabolismus   MeSH
• polyadenylace genetika   MeSH
• proteiny vázající čepičku mRNA genetika   MeSH
• regulace genové exprese MeSH
• sestřih RNA genetika   MeSH
• těhotenství MeSH
• Check Tag
• lidé MeSH
• těhotenství MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH