Evgeny Smirnov

Background: Ribosomal DNA (rDNA), the most abundantly expressed locus in the human genome, is represented by hundreds of units per cell. Each unit includes a 13 kb long region (47S rDNA) containing the genes of ribosomal particles, and a 30 kb long intergenic spacer (IGS). The 47S rDNA is transcribed with varying intensity in different units, some of which remain permanently silent. A key intermediator of this silencing is the promoter-associated RNA (pRNA) produced from a 2 kb long gene situated upstream of the rDNA transcription start site. Recent studies, including ours, suggest that the sequence variability, which normally occurs in mammalian cells, may account for the selective transcription of different rDNA units. The present work is based on the deep sequencing of a pRNA gene fragment and its RNA product and subsequent bioinformatic analysis. Results: We found that a certain SNV, which converts the CCC motif into CCT, as well as deletions which reduce the number of (CCCT) tandem repeats, were significantly more frequent in the DNA than in the respective transcripts. Conclusions: These findings allowed us to establish directly the inhibitory effect of DNA variants on the expression of pRNA and thus (indirectly) the promoting effect on the production of ribosomal RNA. Our results also suggest that (CCCT) _n /(GGGA) _n DNA repeats and the respective (GGGA) _n RNA repeats may form triplex structures, facilitating the function of pRNA.

Non-canonical structures (NCS) refer to the various forms of DNA that differ from the B-conformation described by Watson and Crick. It has been found that these structures are usual components of the genome, actively participating in its essential functions. The present review is focused on the nine kinds of NCS appearing or likely to appear in human ribosomal DNA (rDNA): supercoiling structures, R-loops, G-quadruplexes, i-motifs, DNA triplexes, cruciform structures, DNA bubbles, and A and Z DNA conformations. We discuss the conditions of their generation, including their sequence specificity, distribution within the locus, dynamics, and beneficial and detrimental role in the cell.

Human ribosomal DNA is represented by hundreds of repeats in each cell. Every repeat consists of two parts: a 13 kb long 47S DNA with genes encoding 18S, 5.8S, and 28S RNAs of ribosomal particles, and a 30 kb long intergenic spacer (IGS). Remarkably, transcription does not take place in all the repeats. The transcriptionally silent genes are characterized by the epigenetic marks of the inactive chromatin, including DNA hypermethylation of the promoter and adjacent areas. However, it is still unknown what causes the differentiation of the genes into active and silent. In this study, we examine whether this differentiation is related to the nucleotide sequence of IGS. We isolated ribosomal DNA from the nucleoli of human-derived HT1080 cells, and separated methylated and non-methylated DNA by chromatin immunoprecipitation. Then, we used PCR to amplify a 2 kb long region upstream of the transcription start and sequenced the product. We found that six SNVs and a series of short deletions in a region of simple repeats correlated with the DNA methylation status. These data indicate that variability of IGS sequence may initiate silencing of the ribosomal genes. Our study also suggests a number of pathways to this silencing that involve micro-RNAs and/or non-canonical DNA structures.

In human cells, each rDNA unit consists of the ~13 kb long ribosomal part and ~30 kb long intergenic spacer (IGS). The ribosomal part, transcribed by RNA polymerase I (pol I), includes genes coding for 18S, 5.8S, and 28S RNAs of the ribosomal particles, as well as their four transcribed spacers. Being highly repetitive, intensively transcribed, and abundantly methylated, rDNA is a very fragile site of the genome, with high risk of instability leading to cancer. Multiple small mutations, considerable expansion or contraction of the rDNA locus, and abnormally enhanced pol I transcription are usual symptoms of transformation. Recently it was found that both IGS and the ribosomal part of the locus contain many functional/potentially functional regions producing non-coding RNAs, which participate in the pol I activity regulation, stress reactions, and development of the malignant phenotype. Thus, there are solid reasons to believe that rDNA locus plays crucial role in carcinogenesis. In this review we discuss the data concerning the human rDNA and its closely associated factors as both targets and drivers of the pathways essential for carcinogenesis. We also examine whether variability in the structure of the locus may be blamed for the malignant transformation. Additionally, we consider the prospects of therapy focused on the activity of rDNA.

In human cells, ribosomal DNA (rDNA) is arranged in ten clusters of multiple tandem repeats. Each repeat is usually described as consisting of two parts: the 13 kb long ribosomal part, containing three genes coding for 18S, 5.8S and 28S RNAs of the ribosomal particles, and the 30 kb long intergenic spacer (IGS). However, this standard scheme is, amazingly, often altered as a result of the peculiar instability of the locus, so that the sequence of each repeat and the number of the repeats in each cluster are highly variable. In the present review, we discuss the causes and types of human rDNA instability, the methods of its detection, its distribution within the locus, the ways in which it is prevented or reversed, and its biological significance. The data of the literature suggest that the variability of the rDNA is not only a potential cause of pathology, but also an important, though still poorly understood, aspect of the normal cell physiology.

Numerous studies show that various genes in all kinds of organisms are transcribed discontinuously, i.e. in short bursts or pulses with periods of inactivity between them. But it remains unclear whether ribosomal DNA (rDNA), represented by multiple copies in every cell, is also expressed in such manner. In this work, we synchronized the pol I activity in the populations of tumour derived as well as normal human cells by cold block and release. Our experiments with 5-fluorouridine (FU) and BrUTP confirmed that the nucleolar transcription can be efficiently and reversibly arrested at +4°C. Then using special software for analysis of the microscopic images, we measured the intensity of transcription signal (incorporated FU) in the nucleoli at different time points after the release. We found that the ribosomal genes in the human cells are transcribed discontinuously with periods ranging from 45 min to 75 min. Our data indicate that the dynamics of rDNA transcription follows the undulating pattern, in which the bursts are alternated by periods of rare transcription events.

Numerous studies show that various genes in all kinds of organisms are transcribed discontinuously, i.e. in short bursts or pulses with periods of inactivity between them. But it remains unclear whether ribosomal DNA (rDNA), represented by multiple copies in every cell, is also expressed in such manner. In this work, we synchronized the pol I activity in the populations of tumour derived as well as normal human cells by cold block and release. Then using special software for analysis of the microscopic images, we measured the intensity of transcription signal revealed by incorporated 5-fluorouridine (FU) in the nucleoli at different time points after the release. We found that the ribosomal genes in the human cells are transcribed discontinuously with periods ranging from 45 min to 75 min. Our data indicate that the dynamics of rDNA transcription follows the undulating pattern, in which the bursts are alternated by periods of rare transcription events.

In human cells, the intergenic spacers (IGS), which separate ribosomal genes, are complex approximately 30 kb-long loci. Recent studies indicate that all, or almost all, parts of IGS may be transcribed, and that at least some of them are involved in the regulation of the ribosomal DNA (rDNA) transcription, maintenance of the nucleolar architecture, and response of the cell nucleus to stress. However, since each cell contains hundreds not quite identical copies of IGS, the structure and functions of this locus remain poorly understood, and the dynamics of its products has not been specially studied. In this work, we used quantitative PCR to measure the expression levels of various rDNA regions at different times after inhibition of the transcription by Actinomycin D applied in high doses. This approach allowed us to measure real or extrapolated half-life times of some IGS loci. Our study reveals characteristic dynamic patterns suggestive of various pathways of RNA utilization and decay.

Asthma is among the most common chronic disorders of airways, which affects both children and adults. Asthma being a common disease among different segments of population, it has a high mortality rate and, in the absence of appropriate care, affects the quality of life and leads to economics losses. In a view of continuing growth in the incidence of asthma, it is important to find relevant biological targets for developing new approaches to astma therapy. Recent advances in molecular immunology, genetics, and bioinformatics allowed genes involved in the pathogenesis of asthma to be identified, which provided prerequisites for the development of new types of drugs that can regulate the activity of pathogenically significant genes. To date, a number of technologies for sequence-specific gene regulation (ASO, ribozymes, DNAzymes, EGS, DNA-decoys, U 1-adapters) are available, but RNA interference is the most promising approach in both terms of efficacy and financial cost. This review focuses on the generalization and analysis of experimental data regarding the use of RNA interference technology for the treatment of astma.