Dmitriy E. Andreev

Picornaviruses are small viruses with a plus-strand RNA genome in which RNA secondary structures bind cellular proteins to support viral translation and replication. Here, we characterize tRNA anticodon stem-loop-like structures in the 5′- and 3′ untranslated regions (UTRs) of the RNA of Mengovirus, a member of the Cardiovirus group in the Picornaviridae family. These RNA elements specifically bind cellular Glycyl-tRNA synthetase (GARS). Mutation of the conserved CCA motifs in the loops of these GARS binding elements (GBEs) impairs binding, as does deletion of the anticodon binding domain of GARS. Mutation of the 3′-UTR GBE reduces Mengovirus translation early after transfection, independent of viral polymerase activity. The 3′UTR GBE is a stronger GARS binding site, and in reporter RNAs with the Mengovirus 5′- and 3′-UTRs, the 3′UTR GBE strongly contributes to recruitment of translation factors and ribosomes, thereby stimulating translation. In contrast, the 5′UTR GBE is a weaker GARS binding site, but its mutation has a stronger effect on translation. Therefore, we hypothesize that a GARS dimer binds strongly to an “anchor” site in the 3′UTR with one monomer, while the other monomer interacts with the 5′UTR to stimulate recruitment of translation factors and ribosomes.

Ribosome profiling is a powerful technique used to study gene expression on a transcriptome-wide scale. It involves sequencing of mRNA fragments protected by ribosomes from ribonuclease digestion. The initial steps commonly involve cell lysis followed by centrifugation and ribonuclease digestion. We find that centrifugation depletes 329 translated mRNAs in HEK293T cells. Many of these mRNAs encode cytoskeleton proteins. This suggests that the expression of a subset of mRNAs may be significantly underestimated in most ribosome profiling experiments. We show that omitting the centrifugation step after cell lysis can resolve this issue.

Dysregulated translation is a hallmark of cancer, and recent genome-wide studies in tumour cells have uncovered widespread translation of non-canonical reading frames that often initiate at non-AUG codons. If an upstream non-canonical start site is located within a frame with an annotated coding sequence (CDS), such translation events can lead to the production of proteoforms with altered N-termini (PANTs). Certain examples of PANTs from oncogenes (e.g. c-MYC) and tumour suppressors (e.g. PTEN) have been previously linked to cancer. We have performed a systematic computational analysis on recently identified non-AUG initiation-derived N-terminal extensions of cancer-associated proteins, and we discuss how these extended proteoforms may acquire new oncogenic properties. We identified a loss of stability for the N-terminally extended proteoforms of oncogenes TCF-4 and SOX2. Furthermore, we discovered likely functional short linear motifs within the N-terminal extensions of oncogenes and tumour suppressors (SOX2, SUFU, SFPQ, TOP1 and SPEN/SHARP) that could provide an explanation for previously described functionalities or interactions of the proteins. In all, we identify novel cases where PANTs likely show different localization, functions, partner binding or turnover rates compared to the annotated proteoforms. Therefore, we propose that alterations in the stringency of translation initiation, often seen under conditions of cellular stress, may result in reprogramming of translation to generate novel PANTs that influence cancer progression.

Objective: Survival of oocytes subjected to injections is a critical parameter affecting cost efficiency, time of experiments and their reproducibility. A technique of microinjections into oocytes can be different, and often rely on an additional equipment simplifying the procedure for operator. The demand in protocols helping to increase oocytes’ survival rate drives a constant optimization of microinjection methodology. Methods: Here, we propose a protocol of injection into mouse oocytes that significantly increases the viability of eggs in comparison to common protocols and not requiring a special equipment. The main feature of our protocol is impaling an oocyte onto the tip of fixed injection needle by moving holding capillary. Another feature of proposed protocol is a simple procedure suitable for real time volume measurements during microinjections into germinal vesicle or mouse oocytes in metaphase II. Results and Discussion: The switch between moving capillary is allowing better oocytes’ survival rate in our hands. Method of measuring injected volumes does not require preliminary calibration of microinjecting system and can be used with capillaries of different tip shapes. Conclusions: Impaling on capillary technique allows decreasing eggs’ death and could be practiced in microinjection systems without special devices. Little error of measuring volume method suggests its using in cases of indicated solution volumes introduction in oocytes.

Dual reporters encoding two distinct proteins within the same mRNA have had a crucial role in identifying and characterizing unconventional mechanisms of eukaryotic translation. These mechanisms include initiation via internal ribosomal entry sites (IRESs), ribosomal frameshifting, stop codon readthrough and reinitiation. This design enables the expression of one reporter to be influenced by the specific mechanism under investigation, while the other reporter serves as an internal control. However, challenges arise when intervening test sequences are placed between these two reporters. Such sequences can inadvertently impact the expression or function of either reporter, independent of translation-related changes, potentially biasing the results. These effects may occur due to cryptic regulatory elements inducing or affecting transcription initiation, splicing, polyadenylation and antisense transcription as well as unpredictable effects of the translated test sequences on the stability and activity of the reporters. Unfortunately, these unintended effects may lead to misinterpretation of data and the publication of incorrect conclusions in the scientific literature. To address this issue and to assist the scientific community in accurately interpreting dual-reporter experiments, we have developed comprehensive guidelines. These guidelines cover experimental design, interpretation and the minimal requirements for reporting results. They are designed to aid researchers conducting these experiments as well as reviewers, editors and other investigators who seek to evaluate published data.

Recent advances in functional genomics have allowed identification of thousands of translated short open reading frames (sORFs) in the 5′ leaders of mammalian mRNA transcripts. While most sORFs are unlikely to encode functional proteins, a small number have been shown to have evolved as protein-coding genes. As a result, dozens of these sORFs have already been annotated as protein-coding ORFs. mRNAs that contain both a protein-coding sORF and an annotated coding sequence (CDS) are referred to as bicistronic transcripts. In this study, we focus on three genes – ASNSD1, SLC35A4, and MIEF1 – which give rise to bicistronic mRNAs. We discuss recent findings regarding functional investigation of the corresponding polypeptide products, as well as how their translation is regulated, and how this unusual genetic arrangement may have evolved.

The aim of the researches is detecting and exploration of microorganisms of Terrestrial and Cosmic origin.Microorganisms are supposed to be studied in the near-earth space on space objects of manmade origin and on the space bodies of Solar system in the extraterrestrial space, including planets.For research, it is proposed to use the properties of microorganisms to emit a fluorescent glow when they irradiated with flashes of light causing their fluorescence.One of the research tasks is to search for terrestrial microorganisms that have occurred in space from Earth, as well as research of the survival of the terrestrial microorganisms in space conditions which shall be placed in special laboratories on board of the microsatellites on the Earth.The second task is to search for microorganisms on space bodies in interplanetary space by remote sensing of the surface of space bodies by flashes of light.To solve the first problem of this work is considered an example of a micro-laboratory for the study of terrestrial microorganisms located in space conditions in near-earth space on microsatellites. To solve the second problem, is considered an example of remote sensing equipment of space objects for searching for microorganisms on space bodies in interplanetary space which is installed on board of microsatellite created for far space exploration.Concerning to the first task it is shown that in automatic laboratories on microsatellites, it is possible to study the dynamics of microorganisms survival in space in conditions with a fixed habitat similar to earth's and in a changing environment that adequate the entry of microorganisms into open space and return them back to the earth conditions.Concerning to the second task it is shown that colonies of microorganisms on the surface of space bodies can be detected and studied from the orbits of their artificial satellites or from flight path trajectories near the space body at distances in order 200 km, and single microorganisms can be detected and studied at distances in order hundreds of meters

Upstream open reading frames (uORFs) are a frequent feature of eukaryotic mRNAs. Upstream ORFs govern main ORF translation in a variety of ways, but, in a nutshell, they either filter out scanning ribosomes or allow downstream translation initiation via leaky scanning or reinitiation. Previous reports concurred that eIF4G2, a long-known but insufficiently studied eIF4G1 homologue, can rescue the downstream translation, but disagreed on whether it is leaky scanning or reinitiation that eIF4G2 promotes. Here, we investigated a unique human mRNA that encodes two highly conserved proteins (POLGARF with unknown function and POLG, the catalytic subunit of the mitochondrial DNA polymerase) in overlapping reading frames downstream of a regulatory uORF. We show that the uORF renders the translation of both POLGARF and POLG mRNAs reliant on eIF4G2. Mechanistically, eIF4G2 enhances both leaky scanning and reinitiation, and it appears that ribosomes can acquire eIF4G2 during the early steps of reinitiation. This emphasizes the role of eIF4G2 as a multifunctional scanning guardian that replaces eIF4G1 to facilitate ribosome movement but not ribosome attachment to an mRNA.