Human ribosomal protein S13 regulates expression of its own gene at the splicing step by a feedback mechanism

The expression of ribosomal protein (rp) genes is regulated at multiple levels. In yeast, two genes are autoregulated by feedback effects of the protein on pre-mRNA splicing. Here, we have investigated whether similar mechanisms occur in eukaryotes with more complicated and highly regulated splicing patterns. Comparisons of the sequences of ribosomal protein S13 gene (RPS13) among mammals and birds revealed that intron 1 is more conserved than the other introns. Transfection of HEK 293 cells with a minigene-expressing ribosomal protein S13 showed that the presence of intron 1 reduced expression by a factor of four. Ribosomal protein S13 was found to inhibit excision of intron 1 from rpS13 pre-mRNA fragment in vitro. This protein was shown to be able to specifically bind the fragment and to confer protection against ribonuclease cleavage at sequences near the 5′ and 3′ splice sites. The results suggest that overproduction of rpS13 in mammalian cells interferes with splicing of its own pre-mRNA by a feedback mechanism

Design principles for bifunctional targeted oligonucleotide enhancers of splicing

Controlling the patterns of splicing of specific genes is an important goal in the development of new therapies. We have shown that the splicing of a refractory exon, SMN2 exon 7, could be increased in fibroblasts derived from patients with spinal muscular atrophy by using bifunctional targeted oligonucleotide enhancers of splicing (TOES) oligonucleotides that anneal to the exon and contain a ‘tail’ of enhancer sequences that recruit activating proteins. We show here that there are striking agreements between the effects of oligonucleotides on splicing in vitro and on both splicing and SMN2 protein expression in patient-derived fibroblasts, indicating that the effects on splicing are the major determinant of success. Increased exon inclusion depends on the number, sequence and chemistry of the motifs that bind the activator protein SRSF1, but it is not improved by increasing the strength of annealing to the target site. The optimal oligonucleotide increases protein levels in transfected fibroblasts by a mean value of 2.6-fold (maximum 4.6-fold), and after two rounds of transfection the effect lasted for a month. Oligonucleotides targeted to the upstream exon (exon 6 in SMN) are also effective. We conclude that TOES oligonucleotides are highly effective reagents for restoring the splicing of refractory exons and can act across long introns

Diethyl [2,2,2-trifluoro-1-phenyl­sulfonyl­amino-1-(trifluoro­meth­yl)eth­yl]phospho­nate

The title compound, C13H16F6NO5PS, is of inter­est with respect to inhibition of serine hydro­lases. Its structure contains a 1.8797 (13) Å P—C bond and two inter­molecular N—H⋯O=P hydrogen bonds, resulting in centrosymmetric dimers. An intra­molecular N—H⋯O=P hydrogen bond is also present

Many Faces of Next-Generation Sequencing in Gene Expression Studies

Gene expression is a fundamental cellular process that ensures the transfer of information encoded in a gene into the final functional product [...

Many Faces of Next-Generation Sequencing in Gene Expression Studies

Gene expression is a fundamental cellular process that ensures the transfer of information encoded in a gene into the final functional product [...

S20, S18 and S16 that contact rRNA present

in the eukaryotic ribosomal proteins S25

FORMATION OF STUDENTS’ READINESS TO WORK IN THE SYSTEM OF DISTANCE LEARNING FOR DISABLED CHILDREN

Abstract. The aim of the research is to reveal pedagogical conditions of students’ readiness to work in the system of distance learning for disabled children.Methods. The methods involve theoretical methods (analysis, synthesis, specification, generalization, modeling) and empirical methods (diagnostical questionnaires, monitoring of students’ educational activity, methods of statistical data processing).Results and scientific novelty. The components of readiness of future teachers for work in system of distance learning are structured; qualitative characteristics of each of these components are made. The levels of the specified readiness are determined; the pedagogical conditions of its development are designated. The model of formation of knowledge, skills of students in the field of distance education of pupils with special needs is offered.Practical significance. The course «Distance Educational Technologies in the Work with Disabled Children» is worked out and implemented in the university educational process

Knockdown of the Ribosomal Protein eL38 in HEK293 Cells Changes the Translational Efficiency of Specific Genes

The protein eL38 is one of the smallest proteins of the mammalian ribosome, which is a component of its large (60S) subunit. The haploinsufficiency of eL38 in mice leads to the Tail-short mutant phenotype characterized by defects in the development of the axial skeleton caused by the poor translation of mRNA subsets of Hox genes. Using the ribosome profiling assay applied to HEK293 cells knocked down of eL38, we examined the effects of the lack of eL38 in 60S subunits on gene expression at the level of translation. A four-fold decrease in the cell content of eL38 was shown to result in significant changes in the translational efficiencies of 150 genes. Among the genes, whose expression at the level of translation was enhanced, there were mainly those associated with basic metabolic processes; namely, translation, protein folding, chromosome organization, splicing, and others. The set of genes with reduced translation efficiencies contained those that are mostly involved in the processes related to the regulation of transcription, including the activation of Hox genes. Thus, we demonstrated that eL38 insufficiency significantly affects the expression of certain genes at the translational level. Our findings facilitate understanding the possible causes of some anomalies in eL38-deficient animals

Reorganization of the Landscape of Translated mRNAs in NSUN2-Deficient Cells and Specific Features of NSUN2 Target mRNAs

The RNA cytosine C5 methyltransferase NSUN2 has a variety of RNA substrates and plays an important role in mRNA metabolism. NSUN2 binds to specific sequences enriched in exosomal mRNAs, suggesting its possible involvement in the sorting of mRNAs into exosomes. We applied the photoactivatable.4-thiouridine-enhanced cross-linking and immunoprecipitation assay involving high-throughput RNA sequencing (RNA-seq) to HEK293T cells to determine NSUN2 mRNA targets. NSUN2 cross-linking sites were found in more than one hundred relatively abundant mRNAs with a high GC content and a pronounced secondary structure. Then, utilizing RNA-seq for the total and polysome-associated mRNA from HEK293T cells with and without the knockdown of NSUN2, we identified differentially expressed genes, as well as genes with altered translational efficiency (GATEs). It turned out that the up-regulated GATE mRNAs were much shorter on average than the down-regulated ones, and their GC content was higher; moreover, they contained motifs with C residues located in GC-rich environments. Our findings reveal the specific features of mRNAs that make them potential targets for NSUN2 and expand our understanding of the role of NSUN2 in controlling translation and, possibly, in mRNA sorting into exosomes implemented through the methylation of cytosine residues