Modified Method of rRNA Structure Analysis Reveals Novel Characteristics of Box C/D RNA Analogues.

Ribosomal RNA (rRNA) maturation is a complex process that involves chemical modifications of the bases or sugar residues of specific nucleotides. One of the most abundant types of rRNA modifications, ribose 2'-O-methylation, is guided by ribonucleoprotein complexes containing small nucleolar box C/D RNAs. Since the majority of 2'-O-methylated nucleotides are located in the most conserved regions of rRNA that comprise functionally important centers of the ribosome, an alteration in a 2'-O-methylation profile can affect ribosome assembly and function. One of the key approaches for localization of 2'-O-methylated nucleotides in long RNAs is a method based on the termination of reverse transcription. The current study presents an adaptation of this method for the use of fluorescently labeled primers and analysis of termination products by capillary gel electrophoresis on an automated genetic analyzer. The developed approach allowed us to analyze the influence of the synthetic analogues of box C/D RNAs on post-transcriptional modifications of human 28S rRNA in MCF-7 cells. It has been established that the transfection of MCF-7 cells with a box C/D RNA analogue leads to an enhanced modification level of certain native sites of 2'-O-methylation in the target rRNA. The observed effect of synthetic RNAs on the 2'-O-methylation of rRNA in human cells demonstrates a path towards targeted regulation of rRNA post-transcriptional maturation. The described approach can be applied in the development of novel diagnostic methods for detecting diseases in humans.

[The influence of recombinant nucleophosmin 1 on artificial RNA internalization into human adenocarcinoma MCF-7 cells].

In this study we obtained and characterized the recombinant analogue of multifunctional nucleolar phosphoprotein nucleophosmin 1 (NPM1) involved in crucial cellular processes such as transcription, reparation and mitosis. The influence ofnucleophosmin 1 on extrcellular RNAs accumulation in human adenocarcinoma cells MCF-7 was analyzed. It was found that incubation of AluY RNA (n > 300 nt), U24 snoRNA analogues (n ~ 80 nt) with Npm1-His6 resulted in RNA-protein non-covalent complexes formation, but not in case of the short oligoribonucleotide (n = 22 nt). It was shown that interaction of AluY RNA analogue with Npm1-His6 significantly increases transfection efficacy of the RNA into MCF-7 human cells. Altogether, these data allow us to conclude, that nucleophosmin 1 not only binds RNA with complex secondary structure, but also promotes uptake and internalization of such RNA by human cells.

Alu- and 7SL RNA Analogues Suppress MCF-7 Cell Viability through Modulating the Transcription of Endoplasmic Reticulum Stress Response Genes.

11% of the human genome is composed of Alu-retrotransposons, whose transcription by RNA polymerase III (Pol III) leads to the accumulation of several hundreds to thousands of Alu-RNA copies in the cytoplasm. Expression of Alu-RNA Pol III is significantly increased at various levels of stress, and the increase in the Alu-RNA level is accompanied by a suppression of proliferation, a decrease in viability, and induction of apoptotic processes in human cells. However, the question about the biological functions of Pol III Alu-transcripts, as well as their mechanism of action, remains open. In this work, analogues of Alu-RNA and its evolutionary ancestor, 7SL RNA, were synthesized. Transfection of human breast adenocarcinoma MCF-7 cells with the Alu-RNA and 7SL RNA analogues is accompanied by a decrease in viability and by induction of proapoptotic changes in these cells. The analysis of the combined action of these analogues and actinomycin D or tamoxifen revealed that the decreased viability of MCF-7 cells transfected with Alu-RNA and 7SL RNA was due to the modulation of transcription. A whole transcriptome analysis of gene expression revealed that increased gene expression of the transcription regulator NUPR1 (p8), as well as the transcription factor DDIT3 (CHOP), occurs under the action of both the Alu- and 7SL RNA analogues on MCF-7 cells. It has been concluded that induction of proapoptotic changes in human cells under the influence of the Alu-RNA and 7SL RNA analogues is related to the transcriptional activation of the genes of cellular stress factors, including the endoplasmic reticulum stress response factors.

Analogues of Artificial Human Box C/D Small Nucleolar RNA As Regulators of Alternative Splicing of a pre-mRNA Target.

Small nucleolar RNAs (snoRNAs) play a key role in ribosomal RNA (rRNA) biogenesis. Box C/D snoRNAs guide the site-specific 2'-O-ribose methylation of nucleotides in rRNAs and small nuclear RNAs (snRNAs). A number of box C/D snoRNAs and their fragments have recently been reported to regulate post-transcriptional modifications and the alternative splicing of pre-mRNA. Artificial analogues of U24 snoRNAs directed to nucleotides in 28S and 18S rRNAs, as well as pre-mRNAs and mature mRNAs of human heat shock cognate protein (hsc70), were designed and synthesized in this study. It was found that after the transfection of MCF-7 human cells with artificial box C/D RNAs in complex with lipofectamine, snoRNA analogues penetrated into cells and accumulated in the cytoplasm and nucleus. It was demonstrated that the transfection of cultured human cells with artificial box C/D snoRNA targeted to pre-mRNAs induce partial splicing impairments. It was found that transfection with artificial snoRNAs directed to 18S and 28S rRNA nucleotides, significant for ribosome functioning, induce a decrease in MCF-7 cell viability.

Studies of the functional topography of Escherichia coli RNA polymerase. A method for localization of the sites of affinity labelling.

A method is proposed for localization of the sites of affinity labelling of the beta subunit of Escherichia coli RNA polymerase. The principle of this method is similar to that of the methods of rapid sequencing of nucleic acids. The polypeptide bearing a radioactive affinity label at one of the amino acid residues is subjected to short-term treatment with cyanogen bromide. The conditions of this reaction are selected in such a way that less than one cleavage occurs on average per polypeptide chain. Two series of radioactive peptides are formed, one involving all the possible N-terminal peptides and the other the C-terminal peptides. The distribution of the lengths of these peptides is studied by means of gel electrophoresis and compared with the theoretical ones based on the known amino acid sequence of the beta subunit. Obviously, the affinity label resides between the C-terminus of the shortest N-terminal radioactive peptide and the N-terminus of the shortest C-terminal radioactive peptide. In order to increase reliability and resolution of the method, partial trypsinolysis may be employed. The evidence obtained suggests that lysine residues over the regions 1036-1066, 1234-1242, and histidine-1237 are situated in the nearest neighbourhood to, or directly involved in the formation of the active center of initiating substrate binding of the beta subunit of E. coli RNA polymerase.

[Isolation and various properties of nucleoside monophosphate kinases from Escherichia coli]. / Vydelenie i nekotorye svoistva nukleozidmonofosfatkinaz iz Escherichia coli.

A technique is proposed for isolation of nucleosidemonophosphate kinases--AMP-kinase (EC 2.7.4.11), GMP-kinase (EC 2.7.4.8), CMP-kinase (EC 2.7.4.14), UMP-kinase (EC 2.7.4.14) and TMP-kinase (EC 2.7.4.9)--from E. coli MRE-600. It involves cell destroying, precipitation of nucleic acids with polyethyleneimine, fractionation with ammonium sulphate followed by chromatography on different carriers (DEAE-Toyopearl-650 M, Matrex gel Blue A, Matrex gel Red A). The technique enables all the five enzymes to be obtained separately and without contaminations with nucleotide dephosphorylating enzymes. For all the enzymes the pH optimum was found to range from 6.5 to 8.0, and Mg2+ ions were found to be the best activator for all the enzymes studied. The substrate specificity was investigated with respect to acceptors and donors of the phosphate groups. The enzymes showed strict specificity to the heterocyclic base of the acceptor phosphate group. AMP-, GMP- and CMP-kinases phosphorylated the corresponding deoxynucleoside monophosphates less effectively than ribonucleoside monophosphates. ATP was found to be the most effective phosphate donor for all the enzymes under study.

[Localization of a histidine residue in the binding site for the initiating substrate of E. coli RNA-polymerase]. / Lokalizatsiia ostatka gistidina v oblasti sviazyvaniia initsiiruiushchego substrata RNK-polimerazy E. coli.

E. coli RNA polymerase was selectively labelled in the presence of promoters at a histidine residue of the beta-subunit by treatment with GDP beta-imidazolide and then with [alpha-32P]UTP (or [alpha-33P]UTP). Partial cyanogen bromide cleavage of the labelled polypeptide afforded a series of "single-hit" labelled peptides, the electrophoretic pattern of which suggested that the labelling site was His1237. This conclusion was confirmed by a similar pattern obtained with products of the cyanogen bromide cleavage of a radioactive peptide obtained by the limited trypsinolysis (C-terminal peptide consisting of 423 amino acid residues). Interpretation of our earlier results in favour of His1116 as the labelling point (Dokl. Acad. nauk SSSR, 1985, v. 281, p. 723) was incorrect due to the electrophoretic "compression" of three labelled peptide bands.

[Localization of lysine residues in the site of initiating substrate binding of E. coli RNA-polymerase]. / Lokalizatsiia ostatkov lizina v oblasti sviazyvaniia initsiiruiushchego substrata RNK-polimerazy E. coli.

Superselective affinity labelling of E. coli RNA polymerase in a complex with the promoter-containing fragment of T7 DNA by treatment with orto-formylphenyl ester of GMP followed by addition of [alpha-33P]UTP resulted in covalent binding of the residue--pGpU (p-radioactive phosphate) with one of lysine residues of the beta-subunit, Lys1048, Lys1051, Lys1057, Lys1065. The amino acid sequence of this region of the beta-subunit of E. coli RNA polymerase has a high extent of homology with that deduced for a region of tobacco chloroplast RNA polymerase on the basis of the nucleotide sequence of the chloroplast rpoB-like gene.