Том 52, № 6 (2018)

The noncoding part of the human genome, which was previously considered nonfunctional or junk DNA, has been the subject of extensive research this decade. Nevertheless, long noncoding RNAs still represent one of the least investigated fields because of their complexity, multiplicity, and diversity. While some long noncoding RNAs have been characterized fairly well, the functions of many others remain poorly understood. Long noncoding RNAs play an essential role in the regulation of gene expression in all tissues and on all developmental stages. They are involved in a number of signaling pathways, and their aberrant functioning can be pathogenic. This review aims to summarize current state-of-the-art structures of these transcripts in this research field, their genomic localization, their functions, and underlying mechanisms. It also focuses on cancer-associated aberrations of long noncoding RNAs, as well as on prospects of their application in tumor diagnostics and therapy. Examples of decreasing the levels of oncogenic long noncoding RNAs via silencing with short interfering RNAs, antisense oligonucleotides, or low molecular-weight inhibitors are also described.

The origin of bioluminescence in living organisms was first mentioned by Charles Darwin (1859) and remains obscure despite significant success achieved over the past decades. Here we discuss the mechanisms of bacterial bioluminescence. We have the main results from structural and functional analysis of the genes of lux operons, enzymes (luciferase), and mechanisms of bioluminescence in several species of marine bacteria, which belong to three genera, Vibrio, Aliivibrio, and Photobacterium (A. fischeri, V. harveyi, P. leiognathi, and P. phosphoreum), and in terrestrial bacteria of the genus Photorhabdus (Ph. luminescens). The structure and mechanisms for the regulation of the expression of the lux operons are discussed. The fundamental characteristics of luciferase and luciferase-catalyzed reactions (stages of FMNH₂ and tetradecanal oxidation, dimensional structure, as well as folding and refolding of the macromolecule) are described. We also discuss the main concepts of the origin of bacterial bioluminescence and its role in the ecology of modern marine fauna, including its involvement in the processes of detoxification of the reactive oxygen species and DNA repair, as well as the bait hypothesis.

Reverse genetics approach, involving genome editing, makes it possible not only to establish the nonredundant and unique functions of genes and their products, but also to construct animal models for biomedical research. Interleukin 6 (IL-6) is an important immunoregulatory and proinflammatory cytokine that differs from many related proteins in having a rather complicated signal transduction scheme. Apart from the multiple functions of IL-6, the most relevant biological problem of recent years was establishing what cells produce IL-6, in what form IL-6 is produced, what cells are recipients of the IL-6 signal, and what are the downstream events and physiological consequences of the IL-6 signaling cascade. Because IL-6 is involved in the pathogenesis of many diseases and is a drug target, understanding the mechanisms of its normal and pathogenic effects is important for the clinics. The review summarizes the recent data available in the field.

A modification of the enzymatic method for the preparation of combinatorial random DNA libraries, which combines amplification in isolated microvolumes with the simultaneous incorporation of modified nucleotides and subsequent separation of DNA strands, was developed. Deoxyuridine triphosphate with hydrophobic substituents such as structural analogues of amino acid side chains in the C5 position of the pyrimidine ring was used to introduce modifications into DNA. To prevent competitive amplification, which reduces the representativeness of combinatorial libraries, PCR in inverse emulsion was used. The separation of the strands of PCR products was carried out. There were six single-stranded DNA libraries with complete substitution of deoxythymidine via modified analogues with various functional groups. These DNA libraries are suitable for generating aptamers to protein targets through additional hydrophobic interactions from the introductions of appropriate modifications, and are completely compatible with the SELEX aptamer selection methodology.

The heritable component of susceptibility to myocardial infraction (MI) remains unexplained, possibly due to the minor effects of genes, which are not obviously associated with the disease. These genes may be integrated in miRNA regulated networks associated with myocardial infarction. A systematic review of the literature led us to selecting rs2910164 (MIR146A), rs11614913 (MIR196A2), and rs3746444 (MIR499А) variants to study the association with the MI phenotype. In ethnic Russians, variant rs11614913*C (MIR196A2) was found to be associated with the risk of myocardial infraction (p = 0.023, OR = 1.74) for the first time; this association was validated in an independent cohort. The gene-gene interaction network for experimentally validated miR-196a2 target genes was built and analyzed. One of its four topological clusters contained the majority of miR-196a2 target genes associated with atherosclerosis, coronary artery disease or myocardial infarction and was enriched with the genes regulating the TGFβ and class I MHC signaling pathways, platelet activation/aggregation, and the cell cycle control. This analysis points towards the role of miR-196a2 in the pathological coronary phenotypes and opens up an avenue for further investigations.

Continuous low-intensity laser irradiation (LILI) affects the state of cells in culture, including their proliferation rate. Data collected with various cell models vary significantly, but most studies have reported positive effects of LILI on cell proliferation. The effects of continuous infrared LILI (835 nm) was studied using three independent different melanoma cell lines. The LILI effect was shown to strongly depend on the irradiation dose. Higher doses (230 kJ/m²) significantly suppressed the cell growth. A further increase in LILI dose led to a significant cytotoxic effect, which increased disproportionately quickly with the increasing light intensity. Human mesenchymal stem cells (MSCs) were found to be significantly more resistant to the cytotoxic effect of higher-dose LILI. Importantly, the effects were not due to the difference in culture conditions. Control experiments showed that 15 non-melanoma tumor cell lines were more resistant to LILI than melanoma cells. Selective sensitivity of melanoma cells to LILI in vitro was assumed to provide a basis for LILI-based approaches to melanoma treatment.

The functions of small noncoding RNAs 4.5SH and 4.5SI found in murine-like rodents are unclear. These RNAs synthesized by RNA polymerase III are widely expressed in rodent organs and tissues. Using crosslinking assays, it was shown that approximately half of all 4.5SI and 4.5SH RNA molecules were bound to proteins provisionally called X and Y, respectively. An immunoprecipitation experiment showed that both these RNAs were associated with the La protein, which did not crosslink to them. The termini of 4.5SI RNA form a long duplex stem, which makes the molecule more stable than 4.5SH RNA. Modification of the 5'-end sequence destructing the stem of 4.5SI RNA altered its protein-binding properties; after the 3'-end sequence was changed to the complementary, both the stem structure and the RNA binding to protein X were restored. Presumably, this protein plays a role in increasing the half-life of 4.5SI RNA.

The ability of a series of novel modified external guide sequences (EGS oligonucleotides) to induce the hydrolysis of target RNA with bacterial ribonuclease P has been studied; the most efficient modification variants have been selected. We have found patterns of the oligonucleotide sugar-phosphate backbone modi-fications that enhance oligonucleotide stability in the biological environment and do not violate the ability to interact with the enzyme and induce the RNA hydrolysis. It has been shown that analogues of EGS oligonucleotides selectively modified at 2'-position (2'-O-methyl and 2'-fluoro) or at internucleotide phosphates (phosphoryl guanidines) can be used for the addressed cleavage of a model RNA target by bacterial RNase P. The ability of new phosphoryl guanidine analogues of oligodeoxyribonucleotides that are stable in biological media to induce the hydrolysis of target RNA with bacterial ribonuclease P has been shown for the first time. The modified EGS oligonucleotides with an optimal balance between functional activity and stability in biological media can be considered as potential antibacterial agents.

We have studied the excision efficiency of human apurinic/apyrimidinic endonuclease 1 (APE1) and tyrosyl-DNA phosphodiesterase 1 (TDP1) on matched or mismatched bases located at the 3' end of DNA primers. We have used model DNA duplexes, which mimic DNA structures that occur during either replication (DNA with a 3' recessed end) or repair (DNA with a single-strand break). Both APE1 and TDP1 are more efficient in removing ribose-modified dNMP residues from mismatched pairs rather than canonical pairs. Thus, both of these enzymes may act as proofreading factors during the repair synthesis catalyzed by DNA polymerases including DNA polymerase β (Polβ). The design of new DNA polymerase inhibitors, which act as DNA or RNA chain terminators, is one of the main strategies in the development of antiviral agents. The excision efficacy of APE1 and TDP1 has also been studied for 3'-modified DNA duplexes that contain ddNMP or phosphorylated morpholino nucleosides (MorB) commonly used as terminators in the DNA synthesis. We have also investigated the insertion of ddNTP and morpholino nucleotides catalyzed by Polβ and human immunodeficiency virus reverse transcriptase. This experiment has pointed to MorCyt, cytosine-containing morpholino nucleoside, as a potential antiviral agent.

The accumulation and aggregation of β-amyloids are major molecular events underlying the progression of Alzheimer’s disease. In neural cells, recombinant HSP70 reduces the toxic effect of Aβ and its isomeric forms. Here we describe the proteome of the neuroblastoma cell line after incubation with amyloid peptides Aβ42 and isomerized Asp7 (isoAβ42) without and with human recombinant heat shock protein 70 (HSP70). Incubation of SH-SY5Y cell culture with the synthetic Aβ-peptides leads to a decrease in the levels of several cytoskeleton proteins (e.g., ACTN1, VIME, TPM3) and several chaperonines involved in the folding of actin and tubulin (TCPQ, TCPG, TCPE, TCPB). These changes are accompanied by an increase in the expression of calmodulin and the proteins involved in folding in the endoplasmic reticulum and endoplasmic cell stress response. The presence of exogenous HSP70 has led to an increase in expression of several chaperones and a few other proteins including endogenous HSP70. A combined effect of recombinant HSP70 with Aβ peptides reduced cell apoptosis and significantly decreased the level of tubulin phosphorylation caused by the addition of Aβ peptides.