Cut from the same cloth: RNAs transcribed from regulatory elements.

A certain degree of chromatin openness is necessary for the activity of transcription-regulating regions within the genome, facilitating accessibility to RNA polymerases and subsequent synthesis of regulatory element RNAs (regRNAs) from these regions. The rapidly increasing number of studies underscores the significance of regRNAs across diverse cellular processes and diseases, challenging the paradigm that these transcripts are non-functional transcriptional noise. This review explores the multifaceted roles of regRNAs in human cells, encompassing rather well-studied entities such as promoter RNAs and enhancer RNAs (eRNAs), while also providing insights into overshadowed silencer RNAs and insulator RNAs. Furthermore, we assess notable examples of shorter regRNAs, like miRNAs, snRNAs, and snoRNAs, playing important roles. Expanding our discourse, we deliberate on the potential usage of regRNAs as biomarkers and novel targets for cancer and other human diseases.

[Full and D-Box-Deficient PTTG1 Isoforms: Effects on Cell Proliferation].

Human securin (PTTG1) is a protooncogene whose expression is elevated in many types of malignant cells. We previously discovered a minor short isoform of securin lacking exons 3 and 4. The missing exons encode the main recognition site (D-box) of the anaphase-promoting complex (APC/C). We show that these two PTTG1 isoforms have different effects on transcription. Here, we have studied the effects of overexpression and selective knockdown of the short and complete securin isoforms on cell proliferation using the xCELLigence system. Notably, selective knockdown of the short isoform mRNA led to a dramatic decrease in cell growth, while overexpression of both isoforms accelerated cell growth. To search for genes with alternative isoforms similar to securin, we analyzed the GENCODE database and found that 54 of 128 genes with a PTTG1-like set of APC/C recognition sites have known isoforms without the D-box. Overall, the data obtained indicate the existence of a new class of alternative isoforms and reinstates the importance of minor isoforms.

[The Minor T Allele of the Single Nucleotide Polymorphism rs 13360222 Decreases the Activity of the HAVCR2 Gene Enhancer in a Cell Model of Human Macrophages].

The TIM-3 receptor, encoded by the Hepatitis A Virus Cellular Receptor 2 (HAVCR2) gene, is an immune checkpoint and plays an important role in preventing the development of autoimmune reactions. This receptor is expressed on the surface of various immunocytes and its functions in myeloid cells remain poorly understood, compared to the role of T cell specific TIM-3 that is actively studied in the context of the search for promising therapeutic targets in cancer immunotherapy. During this study, we performed deletion analysis of the promoter region of the HAVCR2 gene, as well as functional characterization of its enhancer, and studied the effect of a number of single nucleotide polymorphisms (SNPs) on the activity of these regulatory elements in the relevant model of human macrophage-like cells-U937 activated monocytes. We have shown that the SNPs rs10515746(A) and rs4704853(A) located in the HAVCR2 gene promoter and associated with the development of a number of pathologies, do not affect the activity of the promoter in activated monocytes. However, a minor T variant of SNP rs13360222 located in the enhancer in the third intron of the gene, significantly reduces the ability of the enhancer to activate the HAVCR2 promoter, presumably due to weakening of the binding of nuclear receptor ESR2 to the respective region.

[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.

[The Influence of the Minor Short Isoform of Securin (PTTG1) on Transcription is Significantly Different from the Impact of the Full Isoform].

PTTG1 (vertebrate securin) is a separation inhibitor and regulates DNA repair and transcription. The protein is predominantly expressed in the second half of the S phase and at the G2 stage. With the onset of anaphase, securin is ubiquitinated by the APC/C complex and degraded rapidly. Increased expression of PTTG1 is associated with enhanced tumor cell growth and metastasis. Recently, we found a short securin isoform lacking the main APC/C recognition site (D-box) and the DNA-binding domain encoded by exons 3 and 4. The mRNA level of the short isoform in unsynchronized cells is 0.4-2% of the full-length one. We reported earlier on the ability of the short PTTG1 isoform to activate some of the genes controlled by the full-length protein. In this work, groups of genes, whose expression is altered by the action of the short and complete securin isoforms, were determined using RNA sequencing. Groups of genes whose mRNA levels are regulated by both protein isoforms and only one of the isoforms were identified. For a more detailed study of the effect of securin isoforms on the transcriptional program of cells, the NFYB gene, encoding the NF-Y transcription regulator subunit, was chosen. Our data showed that with overexpression of the short isoform, the level of NFYB mRNA decreased 2.4 ± 0.7 times, while the complete isoform did not significantly affect the expression of NFYB. 2.2-fold suppression of the short isoform of securin led to an increase in the expression of NFYB mRNA by 2.7 ± 0.3 times. Moreover, the mRNA expression of full-length securin increased by 2.7 ± 0.4 times. Since NFYB is associated with the PTTG1 promoter region, we suggest that the short isoform may be involved in regulation of the expression of the main isoform of securin by changing the level of this transcription factor. Since NFYB and PTTG1 are involved in the development of tumors and the formation of drug resistance, we assume that the short isoform of securin may play an important role in these processes. Thus, we showed the functional significance of the minor short isoform of securin.

PTPN11 Knockdown Prevents Changes in the Expression of Genes Controlling Cell Cycle, Chemotherapy Resistance, and Oncogene-Induced Senescence in Human Thyroid Cells Overexpressing BRAF V600E Oncogenic Protein.

The MAPK (RAS/BRAF/MEK/ERK) signaling pathway is a kinase cascade involved in the regulation of cell proliferation, differentiation, and survival in response to external stimuli. The V600E mutation in the BRAF gene has been detected in various tumors, resulting in a 500-fold increase in BRAF kinase activity. However, monotherapy with selective BRAF V600E inhibitors often leads to reactivation of MAPK signaling cascade and emergence of drug resistance. Therefore, new targets are being developed for the inhibition of components of the aberrantly activated cascade. It was recently discovered that resistance to BRAF V600E inhibitors may be associated with the activity of the tyrosine phosphatase SHP-2 encoded by the PTPN11 gene. In this paper, we analyzed transcriptional effects of PTPN11 gene knockdown and selective suppression of BRAF V600E in a model of thyroid follicular epithelium. We found that the siRNA-mediated knockdown of PTPN11 after vemurafenib treatment prevented an increase in the expression CCNA1 and NOTCH4 genes involved in the formation of drug resistance of tumors. On the other hand, downregulation of PTPN11 expression blocked the transcriptional activation of genes (p21, p15, p16, RB1, and IGFBP7) involved in cell cycle regulation and oncogene-induced senescence in response to BRAF V600E expression. Therefore, it can be assumed that SHP-2 participates not only in emergence of drug resistance in cancer cells, but also in oncogene-induced cell senescence.

The diversity and evolution of odorant receptors in beetles (Coleoptera).

The insect odorant receptors (ORs) are amongst the largest gene families in insect genomes and the primary means by which insects recognize volatile compounds. The evolution of ORs is thus instrumental in explaining the chemical ecology of insects and as a model of evolutionary biology. However, although ORs have been described from numerous insect species, their analysis within and amongst the insect orders has been hindered by a combination of limited genomic information and a tendency of the OR family toward rapid divergence, gain, and loss. We addressed these issues in the insect order Coleoptera through a targeted genomic annotation effort that included 1181 ORs from one species of the sister order Strepsiptera and 10 species representing the four coleopteran suborders. The numbers of ORs in each species varied from hundreds to fewer than 10, but coleopteran ORs could nevertheless be represented within a scheme of nine monophyletic subfamilies. We observed many radiations and losses of genes amongst OR subfamilies, and the diversity of ORs appeared to parallel the host breadth of the study species. However, some small lineages of ORs persisted amongst many coleopteran families, suggesting receptors of key function that underlie the olfactory ecology of beetles.

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 Effect of Season and Weather on Orthopaedic Trauma: Consult Volume Is Significantly Correlated with Daily Weather.

INTRODUCTION: On-call orthopedic clinicians have long speculated that daily consult volume is closely correlated with weather. While prior studies have demonstrated a relationship between weather and certain fracture types, the effect of weather on total orthopaedic consult volume has not yet been examined. The aim of this study was to investigate this relationship. METHODS: We retrospectively reviewed orthopaedic consult data from 405 consecutive days at an urban, level one trauma center. The number, mechanism of injury, and type of consult were collected, along with daily weather data (temperature, wind, and precipitation). Statistical analysis was then performed to determine the relationship between weather and orthopaedic trauma consults. RESULTS: A total of 4543 consults were received during the study period. There was a significant difference in total number of consults between months of the year (p<0.001). A post hoc analysis revealed that this was due to increased volume in the summer months relative to the winter months (i.e., August 13.7 consults/day; January 9.3 consults/day). Average daily temperature and consult volume were also positively correlated (p<0.001, r= 0.30). While there was no significant association between precipitation and total consult volume, when there was over 0.25 inches of rain, there were less penetrating trauma (p=0.034) and motorcycle collision consults (p=0.013). CONCLUSION: Weather parameters, specifically average temperature and precipitation, were found to be associated with daily orthopedic consult type and volume. Additionally, consult volume varies significantly between months of the year. Because trauma centers are often resource scarce, this is an important relationship to understand for proper resource allocation.

[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.

Isolation of Large Amounts of Highly Pure Mitochondria for "Omics" Studies.

Ultracentrifugation on a density gradient remains the only reliable way to obtain highly pure mitochondria preparations. However, it is not readily available for any laboratory and has a serious disadvantage of providing low mitochondria yield, which can be critical when working with limited starting material. Here we describe a combined method for isolation of mitochondria for proteomic studies that includes cell disruption by sonication, differential centrifugation, and magnetic separation. Our method provides remarkable enrichment of mitochondrial proteins as compared to differential centrifugation, magnetic separation, or their combination, and it enables the strongest depletion of cytoplasmic components, as assessed by two-dimensional electrophoresis, mass spectrometry, and Western blot. It also doubles the yield of mitochondria. However, our method should not be used for functional studies as most of the isolated organelles demonstrate disturbed structure in electron microphotographs.

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.

The Minor Variant of the Single-Nucleotide Polymorphism rs3753381 Affects the Activity of a SLAMF1 Enhancer.

The SLAMF1 gene encodes CD150, a transmembrane glycoprotein expressed on the surface of T and B-lymphocytes, NK-cells, dendritic cells, and subpopulations of macrophages and basophils. We investigated the functional regulatory polymorphisms of the SLAMF1 locus associated with autoimmune processes, using bioinformatics and a mutational analysis of the regulatory elements overlapping with polymorphic positions. In the reporter gene assay in MP-1 and Raji B-cell lines, the enhancer activity of the regulatory region of the locus containing the rs3753381 polymorphism demonstrated a twofold increase upon the introduction of the rs3753381 minor variant (G → A) associated with myasthenia gravis. An analysis of the nucleotide context in the vicinity of rs3753381 revealed that the minor version of this polymorphism improves several binding sites for the transcription factors of FOX and NFAT, and RXR nuclear receptors. All mutations that disrupt any of these sites lead to a decrease in the enhancer activity both in MP1 and in Raji cells, and each of the two B-cell lines expresses a specific set of these factors. Thus, the minor variant of the rs3753381 polymorphism may contribute to the development of myasthenia gravis by modulating SLAMF1 expression, presumably in pathogenic B-lymphocytes.

[Preparation of recombinant serpins B3 and B4 and investigation of their specific interactions with antibodies using hydrogel-based microarrays].

The objective of this work was to obtain preparations of recombinant squamous-cell carcinoma antigens (serpins B3 and B4) and to investigate their interactions with different monoclonal antibodies using hydrogel-based microarrays (biochips). Two genetic constructs encoding full-length serpin B3 and serpin B4 molecules were created to produce recombinant SPB3 and SPB4 proteins carrying a N-terminal His6-tag. Monoclonal antibodies against serpin B3 (H3, C5, H5, H81, and G9) were also obtained. An experimental gel-based biological microchip was designed to contain gel elements that carry immobilized antibodies against SPB3, immobilized commercial monoclonal SCC107 and SCC140 antibodies against squamous-cell carcinoma antigen (SCCA), and gel elements with immobilized SPB3 or SPB4. Judging by the specificity of recombinant SPB3 and SPB4, which bind to monoclonal antibodies against SCCA and, according to the manufacturer's data, can recognize conformational epitopes of both SPB3 and SPB4, it was concluded that the obtained recombinant serpins had the correct tertiary structure. A biochip-based direct immunoassay showed that SPB4 could bind effectively only to SCC107 and SCC140 antibodies, while SPB3 interacted specifically not only with these antibodies, but also with H3 and C5 monoclonal antibodies. Using biochip-based sandwich immunoassay, a pair of monoclonal antibodies SCC107/C5 that interacted specifically with serpin B3 but did not interact with serpin B4 was identified. Thus, it has been demonstrated that serpin B3 can be selectively determined in the presence of highly homologous serpin B4 using a biochip-based assay.

Prophylactic Admission of an In Vitro Reconstructed Complexes of Human Recombinant Heat Shock Proteins and Melanoma Antigenic Peptides Activates Anti-Melanoma Responses in Mice.

Tumor-derived autologous antigenic peptides when bound to endogenous 70 kDa family heat shock proteins (HSP70) are able to induce effective T-cell responses against tumors. However, efficacy of HSPbased vaccines in clinical practical stand point still has a number of certain limitations including an activation of immune responses against alien non-human HSPs. In this study we reconstructed the complexes of human recombinant HSPs70 (human recombinant HSP70A1B and HSC70 mixture; hrHSPs70) with antigenic lowweight peptides derived from mice B16F10 melanoma cell lysate (PepMCL) in vitro and investigated the prophylactic potential of these complexes to activate anti-tumor immunity in melanoma mouse model. Our results demonstrate that the developed prophylactic vaccine elicits melanoma-specific immune responses and anti-tumor effects against melanoma. These results suggest that hrHSPs70 has capability to reconstitute complexes with peptides obtained from tumor cells lysates in vitro and, therefore, can be used for delivery of multiple antigenic peptides into antigen-presenting cells (APCs) to activate effectors cells. Designed in such a way hrHSPs70-based prophylactic vaccines induce immune responses resulting in a significant efficient prevention of tumor growth and metastases.

Cats with inflammatory bowel disease and intestinal small cell lymphoma have low serum concentrations of 25-hydroxyvitamin D.

BACKGROUND: Inflammatory bowel disease (IBD) and intestinal small cell lymphoma (ISCL) are common diseases in cats. The prevalence of alterations in the serum concentrations of fat soluble vitamins, such as vitamin D, in cats with IBD and ISCL is unknown. HYPOTHESIS/OBJECTIVES: The objective of this study was to measure serum 25 hydroxyvitamin D (25[OH]D) concentrations in cats with IBD or ISCL. Serum 25(OH)D also was measured in healthy cats, and in hospitalized ill cats with nongastrointestinal diseases. ANIMALS: Eighty-four cats were included in the study: 23 in the healthy group, 41 in the hospitalized ill group, and 20 in the IBD/ISCL group. METHODS: Retrospective study. Serum samples for vitamin D analysis were frozen at -20°C until serum 25(OH)D was measured by high-performance liquid chromatography (HPLC). RESULTS: Although there was overlap in serum 25(OH)D concentrations among the 3 groups, serum 25(OH)D concentrations were significantly lower in the cats with IBD or ISCL compared to healthy cats (P < .0001) and hospitalized ill cats (P = .014). In the IBD/ISCL group, there was a significant moderate positive correlation between serum albumin and 25(OH)D concentrations (r = 0.58, P = .018). CONCLUSION AND CLINICAL IMPORTANCE: The median serum concentration of 25(OH)D was significantly lower in cats with IBD/ISCL than in healthy cats and in hospitalized ill cats. Additional studies are required to elucidate the mechanism of hypovitaminosis D in cats with gastrointestinal diseases, to define the best management strategy to treat this complication, and to investigate its potential prognostic implications.

Upstream open reading frames regulate translation of the long isoform of SLAMF1 mRNA that encodes costimulatory receptor CD150.

More than 40% of human genes contain upstream open reading frames (uORF) in their 5'-untranslated regions (5'-UTRs) and at the same time express at least one truncated mRNA isoform containing no uORF. We studied translational regulation by four uORFs found in the 5'-UTR of full-length mRNA for SLAMF1, the gene encoding CD150 membrane protein. CD150 is a member of the CD2 superfamily, a costimulatory lymphocyte receptor, a receptor for measles virus, and a microbial sensor on macrophages. The SLAMF1 gene produces at least two mRNA isoforms that differ in their 5'-UTRs. In the long isoform of the SLAMF1 mRNA that harbors four uORFs in the 5'-UTR, the stop codon of uORF4 overlaps with the AUG codon of the main ORF forming a potential termination-reinitiation site UGAUG, while uORF2 and uORF3 start codons flank a sequence identical to Motif 1 from the TURBS regulatory element. TURBS was shown to be required for a coupled termination-reinitiation event during translation of polycistronic RNAs of some viruses. In a model cell system, reporter mRNA based on the 5'-UTR of SLAMF1 short isoform, which lacks any uORF, is translated 5-6 times more efficiently than the mRNA with 5'-UTR from the long isoform. Nucleotide substitutions disrupting start codons in either uORF2-4 result in significant increase in translation efficiency, while substitution of two nucleotides in TURBS Motif 1 leads to a 2-fold decrease in activity. These data suggest that TURBS-like elements can serve for translation control of certain cellular mRNAs containing uORFs.

A new viral vector exploiting RNA polymerase I-mediated transcription.

We have developed a new viral vector system exploiting RNA-polymerase I transcription. The vector is based on the crucifer-infecting tobacco mosaic virus (crTMV) cDNA inserted into the rRNA transcriptional cassette (promoter and terminator). To visualize reproduction of the vector, the coat protein gene was replaced with the gene encoding green fluorescent protein (GFP) resulting in a Pr(rRNA)-crTMV-GFP construct. Our results showed that agroinjection of Nicotiana benthamiana leaves with this vector results in GFP production from uncapped crTMV-GFP RNA because RNA polymerase I mediates synthesis of rRNA lacking a cap. Coexpression of the crTMV 122 kDa capping protein gene and the silencing suppressor encoded by the tomato bushy stunt virus p19 gene stimulated virus-directed GFP production more than 100-fold. We conclude that the Pol I promoter can be used to drive transcription in a transient expression system.

New viral vector for efficient production of target proteins in plants.

A new potato virus X (PVX)-based viral vector for superproduction of target proteins in plants has been constructed. The triple gene block and coat protein gene of PVX were substituted by green fluorescent protein. This reduced viral vector was delivered into plant cells by agroinjection (injection of Agrobacterium tumefaciens cells, carrying viral vector cDNA within T-DNA, into plant leaves), and this approach allowed to dramatically reduce the size of the vector genome. The novel vector can be used for production of different proteins including pharmaceuticals in plants.

Stability of plant mRNAs depends on the length of the 3'-untranslated region.

Eukaryotic mRNAs that prematurely terminate translation are recognized and degraded by nonsense mediated decay (NMD). This degradation pathway is well studied in animal and yeast cells. The data available imply that NMD also takes place in plants. However, the molecular mechanism of recognition and degradation of plant RNAs containing premature terminator codon (PTC) is not known. Here we report that in plant cells this mechanism involves the recognition of the sizes of the 3'-untranslated regions (3'UTR). Plant 3'UTRs longer than 300 nucleotides induce mRNA instability. Contrary to mammalian and yeast cells, this destabilization does not depend on the presence of any specific sequences downstream of the terminator codon. Unlike nuclear-produced mRNAs, plant virus vector long 3'UTR-containing RNAs, which are synthesized directly in the cytoplasm, are stable and translated efficiently. This shows that RNAs produced in the cytoplasm by viral RNA-dependent RNA polymerase are able to avoid the proposed mechanism.