Identification of mRNA binding proteins and determination of mRNA stability in AHCY deficiency

Nedostatna aktivnost S-adenozilhomocistein-hidrolaze (SAHH) je nasljedno metaboliĉko oboljenje uzrokovano mutacijom u genu koja dovodi do inaktivacije proteina SAHH te poremećaja staniĉnih metilacijskih procesa. Nedavno je u ovom genu otkrivena nova mutacija koja kodira za verziju proteina SAHH_Y328D i koja uzrokuje gotovo potpuni nedostatak molekule mRNA te smanjenu ekspresiju proteina iz dosad nepoznatog razloga. Navedena mutacija ne uvodi preuranjeni STOP-kodon niti dokida postojeći, stoga su staniĉni mehanizmi koji uklanjaju takve transkripte iskljuĉeni. Hipoteza ovog rada ukljuĉuje moguće promjene u sekundarnoj strukturi mutiranog transkripta uzrokovane toĉkastom mutacijom koje dovode do aktivacije manje istraženog mehanizma degradacije uslijed zastajanja ribosoma tijekom translacije (eng. no-go decay, NGD). U cilju utvrđivanja mehanizma degradacije, in vitro mutagenezom uvedena je mutacija u molekulu cDNA koja kodira za protein SAHH, te je korištenjem lanĉane reakcije polimerazom (PCR) dobiven kalup za in vitro transkripciju. Instrumentnim metodama cirkularnog dikroizma (CD) i diferencijalno skenirajućom kalorimetrijom (DSC) utvrđene su razlike u sekundarnim strukturama i temperaturnoj stabilnosti sintetiziranih molekula mRNA. Molekule mRNA divljeg tipa odnosno verzije mRNA koja kodira za protein SAHH_Y328D, obilježene su detiobiotinom te su iskorištenje za izdvajanje RNA-vezujućih proteina iz staniĉnog lizata HepG2 stanica. Izolirani proteini identificirani su spektrometrijom masa (MS) a iz diferencijalnih proteinskih profila predložen je najvjerojatniji mehanizam razgradnje molekule mRNA kod pacijenta s navedenom mutacijom.S-adenosylhomocysteine hydrolase (AHCY) deficiency is an inherited metabolic disorder caused by a mutation of the AHCY gene, which significantly reduces enzymatic activity and alters the cellular methylation potential. Recently, a novel mutation (p.Y328D) was discovered that causes its transcript to degrade leading to highly reduced expression of AHCY protein. The single nucleotide polymorphism (SNP) does not introduce a premature STOP codon nor does it affect the actual STOP codon so the cellular mechanisms that degrade such transcripts can be excluded. Main hypothesis of this paper includes changes in the secondary structure of the transcript caused by the SNP. Those changes of the overall structure could cause stalling of ribosomes during translation of the mutant transcript and activate a no-go decay (NGD) mechanism. Identical SNP was introduced into cDNA sequence of AHCY using site-directed mutagenesis. Templates for in vitro transcription were synthesized using polymerase chain reaction (PCR). Circular dichroism (CD) and differential scanning calorimetry (DSC) measurements were performed to show that the SNP introduces changes to the secondary structure and influences thermal stability of the mutant transcript. mRNAs were labeled on their 3' end with desthiobiotin and used for RNA-protein pull-down assay using HepG2 cell lysates. After enrichment of the RNA-binding proteins (RBPs) samples were analyzed using mass spectrometry (MS). On the basis of the identified proteins a most probable degradation mechanism was proposed for the mutant AHCY mRNA

msBiodat analysis tool, big data analysis for high-throughput experiments

Background Mass spectrometry (MS) are a group of a high-throughput techniques used to increase knowledge about biomolecules. They produce a large amount of data which is presented as a list of hundreds or thousands of proteins. Filtering those data efficiently is the first step for extracting biologically relevant information. The filtering may increase interest by merging previous data with the data obtained from public databases, resulting in an accurate list of proteins which meet the predetermined conditions. Results In this article we present msBiodat Analysis Tool, a web-based application thought to approach proteomics to the big data analysis. With this tool, researchers can easily select the most relevant information from their MS experiments using an easy-to-use web interface. An interesting feature of msBiodat analysis tool is the possibility of selecting proteins by its annotation on Gene Ontology using its Gene Id, ensembl or UniProt codes. Conclusion The msBiodat analysis tool is a web-based application that allows researchers with any programming experience to deal with efficient database querying advantages. Its versatility and user-friendly interface makes easy to perform fast and accurate data screening by using complex queries. Once the analysis is finished, the result is delivered by e-mail. msBiodat analysis tool is freely available at http://msbiodata.irb.h

Geographical and temporal distribution of SARS-CoV-2 clades in the WHO European Region, January to June 2020

We show the distribution of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) genetic clades over time and between countries and outline potential genomic surveillance objectives. We applied three genomic nomenclature systems to all sequence data from the World Health Organization European Region available until 10 July 2020. We highlight the importance of real-time sequencing and data dissemination in a pandemic situation, compare the nomenclatures and lay a foundation for future European genomic surveillance of SARS-CoV-2

Diverse SARS-CoV-2 variants preceded the initial COVID-19 outbreak in Croatia

We developed a next-generation SARS-CoV-2 sequencing platform and obtained the first SARS-CoV-2 sequences from patients in Croatia at the beginning of the COVID-19 outbreak in the spring of 2020. Integrating the sequencing and the epidemiological data, we show that patients were infected with different SARS-CoV-2 variants belonging to different clades (mostly G and GH). This result confirms that there was widespread virus transmission early in 2020. Interestingly, we identified a unique mutation resulting in a V13I substitution in Nsp5A, the main viral protease, in a patient who had not received antiviral therapy

Herpes simplex virus 1 miRNA sequence variations in latently infected human trigeminal ganglia

Human herpes simplex virus 1 (HSV-1) expresses numerous miRNAs, the function of which is not well understood. Several qualitative and quantitative analyses of HSV-1 miRNAs have been performed on infected cells in culture and animal models, however, there is very limited knowledge of their expression in human samples. We sequenced small-RNA libraries of RNA derived from human trigeminal ganglia latently infected with HSV-1 and Varicella zoster virus (VZV) and detected only a small subset of HSV-1 miRNA. The most abundantly expressed miRNAs are miR- H2, miRNA that regulates the expression of immediate early gene ICP0, and miR-H3 and -H4, both miRNAs expressed antisense to the transcript encoding the major neurovirulence factor ICP34.5. The sequence of many HSV-1 miRNAs detected in human samples was different from the sequences deposited in miRBase, which might significantly affect targeted functional analyses

Analysis of HIV-1 diversity, primary drug resistance and transmission networks in Croatia

Abstract Molecular epidemiology of HIV-1 infection in treatment-naive HIV-1 infected persons from Croatia was investigated. We included 403 persons, representing 92.4% of all HIV-positive individuals entering clinical care in Croatia in 2014–2017. Overall prevalence of transmitted drug resistance (TDR) was estimated at 16.4%. Resistance to nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside RTI (NNRTIs) and protease inhibitors (PIs) was found in 11.4%, 6.7% and 2.5% of persons, respectively. Triple-class resistance was determined in 2.2% of individuals. In addition, a single case (1.0%) of resistance to integrase strand-transfer inhibitors (InSTIs) was found. Deep sequencing was performed on 48 randomly selected samples and detected additional TDR mutations in 6 cases. Phylogenetic inference showed that 347/403 sequences (86.1%) were part of transmission clusters and identified forward transmission of resistance in Croatia, even that of triple-class resistance. The largest TDR cluster of 53 persons with T215S was estimated to originate in the year 1992. Our data show a continuing need for pre-treatment HIV resistance testing in Croatia. Even though a low prevalence of resistance to InSTI was observed, surveillance of TDR to InSTI should be continued

The Virus-Induced Upregulation of the miR-183/96/182 Cluster and the FoxO Family Protein Members Are Not Required for Efficient Replication of HSV-1

Herpes simplex virus 1 (HSV-1) expresses a large number of miRNAs, and their function is still not completely understood. In addition, HSV-1 has been found to deregulate host miRNAs, which adds to the complexity of the regulation of efficient virus replication. In this study, we comprehensively addressed the deregulation of host miRNAs by massive-parallel sequencing. We found that only miRNAs expressed from a single cluster, miR-183/96/182, are reproducibly deregulated during productive infection. These miRNAs are predicted to regulate a great number of potential targets involved in different cellular processes and have only 33 shared targets. Among these, members of the FoxO family of proteins were identified as potential targets for all three miRNAs. However, our study shows that the upregulated miRNAs do not affect the expression of FoxO proteins, moreover, these proteins were upregulated in HSV-1 infection. Furthermore, we show that the individual FoxO proteins are not required for efficient HSV-1 replication. Taken together, our results indicate a complex and redundant response of infected cells to the virus infection that is efficiently inhibited by the virus

Knock-down of AHCY and depletion of adenosine induces DNA damage and cell cycle arrest

Recently, functional connections between S-adenosylhomocysteine hydrolase (AHCY) activity and cancer have been reported. As the properties of AHCY include the hydrolysis of S-adenosylhomocysteine and maintenance of the cellular methylation potential, the connection between AHCY and cancer is not obvious. The mechanisms by which AHCY influences the cell cycle or cell proliferation have not yet been confirmed. To elucidate AHCY-driven cancer-specific mechanisms, we pursued a multi-omics approach to investigate the effect of AHCY-knockdown on hepatocellular carcinoma cells. Here, we show that reduced AHCY activity causes adenosine depletion with activation of the DNA damage response (DDR), leading to cell cycle arrest, a decreased proliferation rate and DNA damage. The underlying mechanism behind these effects might be applicable to cancer types that have either significant levels of endogenous AHCY and/or are dependent on high concentrations of adenosine in their microenvironments. Thus, adenosine monitoring might be used as a preventive measure in liver disease, whereas induced adenosine depletion might be the desired approach for provoking the DDR in diagnosed cancer, thus opening new avenues for targeted therapy. Additionally, including AHCY in mutational screens as a potential risk factor may be a beneficial preventive measure.This work was supported by FP7-REGPOT-2012-2013-1, grant agreement number 316289-InnoMol, and the FP7 PRIME-XS- project - grant no. 262067. The CRG/UPF Proteomics Unit is part of the “Plataforma de Recursos Biomoleculares y Bioinformáticos (ProteoRed)” supported by grant PT13/0001 of the Instituto de Salud Carlos III (ISCIII) and the Spanish Ministry of Economy and Competitiveness. We acknowledge support from the Spanish Ministry of Economy and Competitiveness, “Centro de Excelencia Severo Ochoa 2013–2017”, SEV-2012-0208, and “Secretaria d’Universitats i Recerca del Departament d’Economia i Coneixement de la Generalitat de Catalunya” (2014SGR678). JHP is supported by the grant NRF 2016R1A2A1A05005295. We acknowledge the kind support of staff at the University Clinic Freiburg, Germany, Zentrum für Kinder- und Jugendmedizin, Labor für Klinische Biochemie und Stoffwechsel (LKBS), especially B.Sc. Sidney Behringer, and Dr. Malkanthi Fernando, for performing adenosine measurements. We also thank Dr. Marko Marjanović for help with the flow cytometry analysis