Global analysis of aging-related protein structural changes uncovers enzyme-polymerization-based control of longevity.

Aging is associated with progressive phenotypic changes. Virtually all cellular phenotypes are produced by proteins, and their structural alterations can lead to age-related diseases. However, we still lack comprehensive knowledge of proteins undergoing structural-functional changes during cellular aging and their contributions to age-related phenotypes. Here, we conducted proteome-wide analysis of early age-related protein structural changes in budding yeast using limited proteolysis-mass spectrometry (LiP-MS). The results, compiled in online ProtAge catalog, unraveled age-related functional changes in regulators of translation, protein folding, and amino acid metabolism. Mechanistically, we found that folded glutamate synthase Glt1 polymerizes into supramolecular self-assemblies during aging, causing breakdown of cellular amino acid homeostasis. Inhibiting Glt1 polymerization by mutating the polymerization interface restored amino acid levels in aged cells, attenuated mitochondrial dysfunction, and led to lifespan extension. Altogether, this comprehensive map of protein structural changes enables identifying mechanisms of age-related phenotypes and offers opportunities for their reversal.

Bacteriophage Infection of the Marine Bacterium Shewanella glacialimarina Induces Dynamic Changes in tRNA Modifications.

Viruses are obligate intracellular parasites that, throughout evolution, have adapted numerous strategies to control the translation machinery, including the modulation of post-transcriptional modifications (PTMs) on transfer RNA (tRNA). PTMs are critical translation regulators used to further host immune responses as well as the expression of viral proteins. Yet, we lack critical insight into the temporal dynamics of infection-induced changes to the tRNA modification landscape (i.e., 'modificome'). In this study, we provide the first comprehensive quantitative characterization of the tRNA modificome in the marine bacterium Shewanella glacialimarina during Shewanella phage 1/4 infection. Specifically, we show that PTMs can be grouped into distinct categories based on modification level changes at various infection stages. Furthermore, we observe a preference for the UAC codon in viral transcripts expressed at the late stage of infection, which coincides with an increase in queuosine modification. Queuosine appears exclusively on tRNAs with GUN anticodons, suggesting a correlation between phage codon usage and PTM modification. Importantly, this work provides the basis for further studies into RNA-based regulatory mechanisms employed by bacteriophages to control the prokaryotic translation machinery.

An improved RT-qPCR method for direct quantification of enveloped RNA viruses.

Reverse transcription quantitative PCR (RT-qPCR) has emerged as the gold standard for virus detection and quantification, being utilized in numerous diagnostic and research applications. However, the direct detection of viruses has so far posed a challenge as the viral genome is often encapsidated by a proteinaceous layer surrounded by a lipid envelope. This necessitates an additional and undesired RNA extraction step prior to RT-qPCR amplification. To circumvent this limitation, we have developed a direct RT-qPCR method for the detection of RNA viruses. In our method, we provide a proof-of-concept using phage phi6, a safe-to-use proxy for pathogenic enveloped RNA viruses that is commonly utilized in e.g. aerosolization studies. First, the phage phi6 envelope is removed by 1% chloroform treatment and the virus is then directly quantified by RT-qPCR. To identify false negative results, firefly luciferase is included as a synthetic external control. Thanks to the duplex format, our direct RT-qPCR method reduces the reagents needed and provides an easy to implement and broadly applicable, fast, and cost-effective tool for the quantitative analysis of enveloped RNA viruses.•One-step direct RT-qPCR quantification of phage phi6 virus without prior RNA isolation.•Reduced reaction volume for sustainable and cost-effective analysis.

The comprehensive interactomes of human adenosine RNA methyltransferases and demethylases reveal distinct functional and regulatory features.

N6-methyladenosine (m6A) and N6,2'-O-dimethyladenosine (m6Am) are two abundant modifications found in mRNAs and ncRNAs that can regulate multiple aspects of RNA biology. They function mainly by regulating interactions with specific RNA-binding proteins. Both modifications are linked to development, disease and stress response. To date, three methyltransferases and two demethylases have been identified that modify adenosines in mammalian mRNAs. Here, we present a comprehensive analysis of the interactomes of these enzymes. PCIF1 protein network comprises mostly factors involved in nascent RNA synthesis by RNA polymerase II, whereas ALKBH5 is closely linked with most aspects of pre-mRNA processing and mRNA export to the cytoplasm. METTL16 resides in subcellular compartments co-inhabited by several other RNA modifiers and processing factors. FTO interactome positions this demethylase at a crossroad between RNA transcription, RNA processing and DNA replication and repair. Altogether, these enzymes share limited spatial interactomes, pointing to specific molecular mechanisms of their regulation.

Broad-range RNA modification analysis of complex biological samples using rapid C18-UPLC-MS.

Post-transcriptional RNA modifications play an important role in cellular metabolism with homoeostatic disturbances manifesting as a wide repertoire of phenotypes, reduced stress tolerance and translational perturbation, developmental defects, and diseases, such as type II diabetes, leukaemia, and carcinomas. Hence, there has been an intense effort to develop various methods for investigating RNA modifications and their roles in various organisms, including sequencing-based approaches and, more frequently, liquid chromatography-mass spectrometry (LC-MS)-based methods. Although LC-MS offers numerous advantages, such as being highly sensitive and quantitative over a broad detection range, some stationary phase chemistries struggle to resolve positional isomers. Furthermore, the demand for detailed analyses of complex biological samples often necessitates long separation times, hampering sample-to-sample turnover and making multisample analyses time consuming. To overcome this limitation, we have developed an ultra-performance LC-MS (UPLC-MS) method that uses an octadecyl carbon chain (C18)-bonded silica matrix for the efficient separation of 50 modified ribonucleosides, including positional isomers, in a single 9-min sample-to-sample run. To validate the performance and versatility of our method, we analysed tRNA modification patterns of representative microorganisms from each domain of life, namely Archaea (Methanosarcina acetivorans), Bacteria (Pseudomonas syringae), and Eukarya (Saccharomyces cerevisiae). Additionally, our method is flexible and readily applicable for detection and relative quantification using stable isotope labelling and targeted approaches like multiple reaction monitoring (MRM). In conclusion, this method represents a fast and robust tool for broad-range exploration and quantification of ribonucleosides, facilitating future homoeostasis studies of RNA modification in complex biological samples.

N6-methyladenosine demethylase FTO targets pre-mRNAs and regulates alternative splicing and 3'-end processing.

N6-methyladenosine (m6A) is the most abundant base modification found in messenger RNAs (mRNAs). The discovery of FTO as the first m6A mRNA demethylase established the concept of reversible RNA modification. Here, we present a comprehensive transcriptome-wide analysis of RNA demethylation and uncover FTO as a potent regulator of nuclear mRNA processing events such as alternative splicing and 3΄ end mRNA processing. We show that FTO binds preferentially to pre-mRNAs in intronic regions, in the proximity of alternatively spliced (AS) exons and poly(A) sites. FTO knockout (KO) results in substantial changes in pre-mRNA splicing with prevalence of exon skipping events. The alternative splicing effects of FTO KO anti-correlate with METTL3 knockdown suggesting the involvement of m6A. Besides, deletion of intronic region that contains m6A-linked DRACH motifs partially rescues the FTO KO phenotype in a reporter system. All together, we demonstrate that the splicing effects of FTO are dependent on the catalytic activity in vivo and are mediated by m6A. Our results reveal for the first time the dynamic connection between FTO RNA binding and demethylation activity that influences several mRNA processing events.

Contraceptive behaviour of Czech and Romanian women: comparison of representative national samples.

OBJECTIVE: To investigate the contraceptive behaviour of Czech and Romanian women and their attitudes towards different forms of contraception. STUDY DESIGN: Anonymous questionnaire research among 1011 Czech and 1001 Romanian women between 15 and 50 years of age. The samples were representative of both the Czech and Romanian female populations with respect to age, education and the size of their place of residence. RESULTS: The majority of women use contraception when in a stable sexual relationship. We found a higher percentage of women using contraception among Czech women. Romanian women more often use less reliable methods in a stable relationship, and condoms. Czech women use hormonal contraception much more. Women in the Czech Republic would more often agree to use a condom and, in particular, to use hormonal and intrauterine contraception. More Romanian women than Czech women prefer the method of infertile days. The use of contraception is influenced by the factors of age, education and size of the place of residence. CONCLUSION: The majority of Czech and Romanian women protect themselves against unwanted pregnancy. Czech women are more frequent users of contraceptives in general and, in particular, of hormonal contraception. Romanian women tend to use less reliable methods of contraception but on the other hand, they more often use condoms as a means of protection.