[Functional Characteristics of Long Noncoding RNAs Containing Sequences of Mobile Genetic Elements].

Long nonconding RNAs (lncRNAs) perform a variety of functions: they are involved in chromatin organization, regulation of gene expression at the transcriptional and post-transcriptional levels, and regulation of activity and stability of some proteins. The majority of known lncRNAs contain sequences of mobile genetic elements (MGEs) in a sense or antisense orientation. According to several studies, MGE may serve as functional modules responsible for interactions between the lncRNA and certain proteins, DNA regions, or other RNAs. The available data make it possible to describe groups of lncRNAs that possess common structural features and contain certain MGEs and to predict the characteristics of new lncRNAs. The review summarizes the data on the role that MGE sequences play in lncRNA functions.

[RNA Degradation in Eukaryotic Cells].

RNA is a crucial component of every living organism and is necessary for gene expression and its regulation in the cell. Mechanisms of RNA synthesis (especially mRNA synthesis) were a subject of extensive study for a long time. More recently, RNA degradation pathways began to be considered as equally important part of eukaryotic cell metabolism. These pathways have been studied intensely, and ample information accumulated about RNA degradation systems and their role in cell life. It is currently obvious that RNA decay is of no less importance as RNA synthesis and contributes to regulating the RNA level in the cell. The review considers the main RNA degradation enzymes, the decay pathways of various coding and non-coding RNAs, the mechanisms providing RNA quality control in the nucleus and cytoplasm, and certain structural elements responsible for RNA stability or short life in the cell.

[Polyadenylation of Sine Transcripts Generated by RNA Polymerase III Dramatically Prolongs Their Lifetime in Cells].

Short Interspersed Elements (SINEs) are mobile genetic elements of higher eukaryotes, which originated during evolution from various tRNAs and less often from 5S rRNA and 7SL RNA. Similar to the genes of these RNAs, SINEs are transcribed by RNA polymerase III. The transcripts of some mammalian SINEs have an ability to undergo AAUAAA-dependent polyadenylation, which is unique for the RNA polymerase III transcripts. It is well known that this polyadenylation of many RNA polymerase II transcripts (e.g., mRNAs) increases their lifetime in the cell. The aim of this work is to examine whether the stability of SINE transcripts increases as a result of AAUAAA-dependent polyadenylation. HeLa cells were transfected with SINE DNA, both containing and not containing the polyadenylation signal (AATAAA). One day later, the transcription was inhibited by actinomycin D, and the decrease in the level of the SINE transcripts was monitored by northern hybridization. For all the eight studied SINEs, the half-life of nonpolyadenylated transcripts was 20-30 minutes, and for polyadenylated transcripts, this parameter exceeded 3 hours. Interestingly, the insertion of an additional 80-bp DNA fragment into the middle region of B2 SINE did not significantly reduce the stability of the polyadenylated transcripts. It is most likely that the increase in the lifetime of the polyadenylated SINE transcripts is due to the fact that the poly(A) tail interacts with the poly(A)-binding proteins (PABPs), thus protecting the RNA from degradation by the exonucleases acting from the 3'-end. The results make it possible to design SINE-based vectors intended for the expression of short noncoding RNAs, which are stable in a cell due to polyadenylation.

Constitutive Expression of NRAS with Q61R Driver Mutation Activates Processes of Epithelial-Mesenchymal Transition and Leads to Substantial Transcriptome Change of Nthy-ori 3-1 Thyroid Epithelial Cells.

The Q61R mutation of the NRAS gene is one of the most frequent driver mutations of thyroid cancer. Tumors with this mutation are characterized by invasion into blood vessels and formation of distant metastases. To study the role of this mutation in the growth of thyroid cancer, we developed a model system on the basis of thyroid epithelial cell line Nthy-ori 3-1 transduced by a lentiviral vector containing the NRAS gene with the Q61R mutation. It was found that the expression of NRAS(Q61R) in thyroid epithelial cells has a profound influence on groups of genes involved in the formation of intercellular contacts, as well as in processes of epithelial-mesenchymal transition and cell invasion. The alteration in the expression of these genes affects the phenotype of the model cells, which acquire traits of mesenchymal cells and demonstrate increased ability for survival and growth without attachment to the substrate. The key regulators of these processes are transcription factors belonging to families SNAIL, ZEB, and TWIST, and in different types of tumors the contribution of each individual factor can vary greatly. In our model system, phenotype change correlates with an increase in the expression of SNAIL2 and TWIST2 factors, which indicates their possible role in regulating invasive growth of thyroid cancer with the mutation of NRAS(Q61R).

[The Novel Short Isoform of Securin Stimulates the Expression of Cyclin D3 and Angiogenesis Factors VEGFA and FGF2, but Does Not Affect the Expression of MYC Transcription Factor].

Pituitary tumor-transforming gene-1 (PTTG1) encodes securin, a multifunctional protein involved in development of various types of cancer. Securin participates in the regulation of sister chromatids separation and the expression of multiple genes involved in the control of the cell cycle, metabolism, and angiogenesis. In several human cell lines, we have found a novel short isoform of securin mRNA, which does not contain exons 3 and 4. After the translation of this new mRNA, a shortened protein is produced that, like the full-size form, is able to activate the transcription of cyclin D3 gene (CCND3), which controls the G1/S transition and angiogenesis factors VEGFA (vascular endothelial growth factor), and FGF2 (fibroblast growth factor 2) in HEK293 cells. However, unlike the full-size protein, the short isoform of PTTG1 does not affect the MYC gene expression because it lacks the DNA-binding domain, which is needed for its interactions with the MYC promoter. Furthermore, the short form of securin does not influence the expression of MYC transcriptional targets, such as TP53 and IL-8. Thus, we found a novel isoform of securin which is able to activate a more restricted repertoire of genes compared to the full-size protein.

Potential Markers of Autoimmune Diseases, Alleles rs115662534(T) and rs548231435(C), Disrupt the Binding of Transcription Factors STAT1 and EBF1 to the Regulatory Elements of Human CD40 Gene.

CD40 receptor is expressed on B lymphocytes and other professional antigen-presenting cells. The binding of CD40 to its ligand CD154 on the surface of T helper cells plays an important role in the activation of B lymphocytes required for production of antibodies, in particular, against autoantigens. Association of several single nucleotide polymorphisms (SNPs) located in the non-coding areas of human CD40 locus with the elevated risk of autoimmune diseases has been demonstrated. The most studied of these SNPs is rs4810485 located in the first intron of the CD40 gene. Expression of the CD40 gene in B lymphocytes of donors homozygous for the common allelic variant of this polymorphism (G) is higher than in B cells from donors carrying the minor (T) variant. We investigated the enhancer activity of this fragment of the CD40 locus in human B cell lines and showed that it is independent on the rs4810485 alleles. However, the minor allelic variants of the rs4810485-linked SNPs rs548231435 and rs115662534 were associated with a significant decrease in the activity of the CD40 promoter due to the impairments in the binding of EBF1 and STAT1 transcription factors, respectively.

[Small Noncoding 4.5SH and 4.5SI RNAs and Their Binding to Proteins].

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.

[4.5SI and 4.5SH RNAs: Expression in various rodent organs and abundance and distribution in the cell].

Studying the structure, functions, and cell physiology of small RNAs remains important. The 4.5SI and 4.5SH small RNAs, which were among the first to be discovered and sequenced, share several features, i.e., they are both approximately 100 nt in size, are synthesized by RNA polymerase III, and are found only in rodents of several related families. Genes coding for these RNAs are evolutionarily related to short interspersed elements (SINEs). However, the two RNAs differ in nucleotide sequence, half-life in the cell, and the organization of their genes in the genome. Although the 4.5SI and 4.5SH RNAs have been identified more than three decades ago, several aspects of their metabolism in the cell are still poorly understood. The 4.5SI and 4.5SH RNA levels were measured in various organs of three rodent species (mouse, rat, and hamster). Both of the RNAs were found to occur at high levels, which were much the same in different organs in the case of the 4.5SI RNA and varied among organs in the case of the 4.5SH RNA. Both 4.5SI and 4.5SH RNAs demonstrated a predominantly nuclear localization with a detectable presence in the cytoplasm. The copy number per cell for the RNAs was estimated at 0.4-2.4 × 10^(6). A quantitative study for the 4.5SI and 4.5SH RNAs was performed for the first time and resolved a number of contradictions in data from other studies.

Modification of human fibroblast properties in microtransplant.

We studied the properties of human skin fibroblast in filamentous polyglycolic microtransplant. Fibroblast adhesion to the microtransplant filaments is followed by the formation of a network cross-linked with fibroblasts. The cells rapidly proliferate during the first few days; after transfer of the microtransplant to the standard culture flask, the cells migrate to the plastic and continue proliferation. The cells are uniform and exhibit high colony-formation capacity. The bundles of microtransplant filaments persist in the culture for several days and through the cells completely leave them, the area around these filaments remains the most populated for 40 days. Mitotic cells are seen in the immediate proximity to the degrading filaments of the transplant. The effect of cell "rejuvenation" in the microtransplant can be explained by selection of cells by their adhesion to relatively thin (about 15 µ) filaments, which excludes large old cells.