[Efficient cap-dependent in vitro and in vivo translation of mammalian mRNAs with long and highly structured 5'-untranslated regions].

According to generally accepted scanning model proposed by M. Kozak, the secondary structure of 5'-untranslated regions (5'-UTR) of eukaryotic mRNAs can only cause an inhibitory effect on the translation initiation since it would counteract migration of the 40S ribosomal subunit along the mRNA polynucleotide chain. Thus, the existence of efficiently translatable mRNAs with long and highly structured 5'-UTRs is not compatible with the cap-dependent scanning mechanism. It is expected that such mRNAs should use alternative ways of translation initiation to be efficiently translated, first of all the mechanism of the internal ribosome entry mediated by special RNA structures called IRESes (for Internal Ribosome Entry Sites), which have been proposed to reside within their 5'-UTRs. In this paper, it is shown that this point of view is not correct and most probably based on experiments of mRNA translation in rabbit reticulocyte lysate. This cell free system does not reflect correctly the ratio of translation efficiencies of various mRNAs which is observed in the living cell. Using five different mRNAs of similar design which possess either relatively short leaders of cellular mRNAs (beta-globin and beta-actin mRNAs) or long and highly structured 5'-UTRs (c-myc, LINE-1, Apaf-1 mRNAs), we show that the translation activities of all tested 5'-UTRs are comparable, both in transfected cells and in a whole cytoplasmic extract of cultivated cells. This activity is strongly dependent on the presence of the cap at their 5'-ends.

[Molecular environment of the IIId subdomain of the IRES element of hepatitits C virus RNA on the human 40S ribosomal subunit].

The molecular environment of the key subdomain IIId of the internal ribosome entry site (IRES) element of hepatitis C virus (HCV) RNA in the binary complex with the human 40S ribosomal subunit was studied. To this end, HCV IRES derivatives bearing perfluorophenylazido groups activatable by mild UV at nucleotide G263 or A275 in the subdomain IIId stem were used. They were prepared by the complementarily addressed modification of the corresponding RNA transcript with alkylating oligodeoxynucleotide derivatives. None of the RNA derivatives were shown to be crosslinked to the 18S rRNA. It was found that the photoreactive groups of the IRES G263 and A275 nucleotides are crosslinked to ribosomal proteins S3a, S14, and S16. For the IRES derivative with the photoreactive group in nucleotide G263, the degree of modification of proteins S14 and S16 was greater than that of S3a, whereas the derivative containing the same photoreactive group in nucleotide A275 was mainly crosslinked to proteins S3a and S14. An analysis of the data led to the conclusion that, in the binary complex of HCV IRES elements with the small subunit of the 80S ribosome, its subdomain IIId stem is located on the outer subunit surface between the head and the body next to the "beak" near the exit of mRNA from the ribosome.

[Binding of the IRES of hepatitis C virus RNA to the 40S ribosomal subunit: role of protein p40].

Ribosomal protein p40 is a structural component of the 40S ribosomal subunit, which is partially homologuos to prokaryotic ribosomal protein S2 and has a long eukaryote-specific C-terminal region. In the present work, we have studied the binding of the Internal Ribosome Entry Site (IRES) of the hepatitis C virus (HCV) RNA to the 40S ribosomal subunit either deficient on protein p40, or saturated with the recombinant p40, or pre-bound to monoclonal antibodies (MAB) 4F6 against p40. It was shown that the apparent association constant of HCV IRES binding to 40S subunits directly depends on p40 content in the subunits. Binding of MAB 4F6 against p40 to 40S subunits prevented the HCV IRES binding by the subunits and blocked translation of the IRES-containing RNA in cell-free translation system. The data obtained point to the involvement of the ribosomal protein p40 in the binding of the HCV IRES by ribosomes and therefore in initiation of translation of RNA of this virus.

[Similar features in mechanisms of translation initiation of mRNAs in eukaryotic and prokaryotic systems].

Using as examples non-canonical features of translation initiation for some bacterial and mammalian mRNAs with unusual 5'- untranslated regions (5'-UTR) or lacking these regions (leaderless mRNAs), the authors of this review discuss similarities in mechanisms of translation initiation on prokaryotic and eukaryotic ribosomes.

[Molecular environment of the subdomain IIIe loop of the RNA IRES element of hepatitis C virus on the human 40S ribosomal subunit].

The molecular environment of the internal ribosome entry site (IRES) element of hepatitis C viral (HCV) RNA in the binary complex with the human 40S ribosomal subunit was studied. To this end, RNA derivatives bearing mild UV-reactive perfluorophenylazide groups at nucleotide G87 in IRES domain II and at nucleotide A296 in the subdomain IIIe loop were used, which were prepared by the RNA complementarily-addressed modification with alkylating oligonucleotide derivatives. None of the RNA derivatives were shown to be crosslinked to the 18S rRNA of the 40S subunit. It was found that the photoreactive group of IRES nucleotide A296 was crosslinked to the 40S subunit S2/S3a, S5, and p40 (SOA) proteins. No protein crosslinking was observed for the RNA derivative containing the same photoreactive group in nucleotide G87. It was concluded that the subdomain IIIe loop of the HCV RNA IRES element in the complex with the 40S subunit is located on the outer subunit surface between the head and the body next to the "beak" near the entrance into the mRNA-binding channel. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2006, vol. 32, no. 3; see also http://www.maik.ru.

[Minor secondary-structure variation in the 5'-untranslated region of the beta-globin mRNA changes the concentration requirements for eIF2]. / Neznachitel'nye variatsii vtorichnoi struktury 5'-netransliruemoi oblasti mRNK beta-globina izmeniaiut trebovaniia k kontsentratsii faktora initsiatsii eIF2.

Nucleotide sequence changes increasing the number of paired bases without producing stable secondary structure elements in the 5'-untranslated region (5'-UTR) of the beta-globin mRNA had a slight effect on its translation in rabbit reticulocyte lysate at its low concentration and dramatically decreased translation efficiency at a high concentration. The removal of paired regions restored translation. Addition of purified eIF2 to the lysate resulted in equal translation efficiencies of templates differing in structure of 5'-UTR. A similar effect was observed for p50, a major mRNP protein. Other mRNA-binding initiation factors, eIF4F and eIF3B, had no effect on the dependence of translation efficiency on mRNA concentration. Analysis of the assembly of the 48S initiation complex from its purified components showed that less eIF2 is required for translation initiation on the beta-globin mRNA than on its derivative containing minor secondary structure elements in 5'-UTR. According to a model proposed, eIF2 not only delivers Met-tRNA, but it also stabilizes the complex of the 40S ribosome subunit with 5'-UTR, which is of particular importance for translation initiation on templates with structured 5'-UTR.

[Approach to identifying the functionally important segments of RNA, based on complementation-addressed modification]. / Podkhod k vyiavleniiu funktsional'no vazhnykh uchastkov RNK, osnovannyi na komplementarno-adresovannoi modifikatsaii.

An approach based on complementation-addressed modification of nucleic acids by oligodeoxyribonucleotide derivatives was proposed for changing the spatial structure of particular RNA sites in order to study their role in the biological activity of the total RNA molecule. Hepatitis C virus (HCV) IRES was used as a model. Oligodeoxyribonucleotide derivatives contained a 4-[N-(2-chloroethyl)-N-methylamino]benzylamino group at the 5'-P and were complementary to various RNA sites located in regions of hairpins II, IIId, or IIIe. Covalent adducts resulting from RNA alkylation with the derivatives were isolated by denaturing PAGE and tested for binding with the 40S subunit of human ribosomes. Structural alteration of hairpin II had no effect, whereas alteration of hairpin IIIe substantially reduced the binding. The RNA with modified hairpin IIId showed virtually no binding with the 40S subunit. Hairpin IIId was assumed to play a critical role in the binding of HCV IRES with the 40S subunit.

[Study of molecular mechanisms of translation initiation in mammals using in vitro reconstruction of initiation complexes]. / Rekonstruktsiia initsiiruiushchikh kompleksov in vitro dlia issledovaniia molekuliarnykh mekhanizmov initsiatsii transliatsii u mlekopitaiushchikh.

Papers on the mechanisms of translation initiation in mammals studied by reconstruction of initiation complexes from individual components are reviewed. The author points to the constraints of this approach and to the pitfalls ignoring which one might come to erroneous conclusions and even artifacts. In addition, some methods employed in the field as well as some technical problems are discussed in the paper, together with the means of obviating them. The review could be a guidebook for newcomers into this quite labor-consuming field.

[Internal initiation of translation in eukaryotes. Chemical probing of the encephalomyocarditis virus RNA IRES-element in the 48S preinitiation complex]. / Vnutrenniaia initsiatsiia transliatsii u éukariot. Khimicheskoe zondirovanie IRES-élementa RNK virusa éntsefalomiokardita v sostave 48S preynitsiatornogo kompleksa.

Using in vitro T7 polymerase system, the transcript containing the IRES-element (nts 315-833), and the initial part of the coding sequence of encephalomyocarditis virus (EMCV) RNA (nts 834-1155) was prepared. Its complex with the 40S ribosomal subunit (48S preinitiation complex) was then isolated by sucrose gradient sedimentation from ascites carcinoma Krebs2 cell extracts after preincubation with the transcript. The complex was treated with dimethylsulphate (DMS), a common reagent for chemical probing of A and C residues in single-stranded RNA regions. The modified nucleotides were identified by primer extension inhibition analysis in reverse transcription reaction. The pattern of modification of the 48S complex was compared with that for the corresponding free mRNP. Multiple protection of A residues against DMS modification was found in the domains of the IRES-element proximal to the initiation AUG codon (nt 834-836). The mechanism of internal translational initiation of EMCV RNA and other picornaviral RNAs is discussed.

[Availability of DNP-modified 5'- and 3'-ends of the poly(U) fragment bound to the 30S ribosomal subunit for interaction with antibodies]. / Dostupnost' DNF-modifitsirovannykh 5'- i 3'-kontsov fragmenta poli(U), sviazannogo s 30 S-subchastitsei ribosomy, dlia vzaimodeistviia s antitelami.

Modification of the 5'- or 3'-terminal nucleotide residues of poly(U) by dinitrophenyl haptens was carried out. The accessibility of the modified ends of short poly(U) (20-70 nucleotide residues) bound to the 30S subunit of E. coli ribosomes for the interaction with antibodies that are specific against the dinitrophenyl group, was investigated. Some peculiarities of the structural organization of the "entry" (3'-end) and "exit" (5'-end) sites of the template polynucleotide on the 30S subunit and the possibility of the template polynucleotide folding on the ribosome are discussed.

[Modification of sulfhydryl groups in ribosomal proteins by a dinitrophenyl hapten]. / Modifikatsiia sul'fgidril'nykh grupp ribosomnykh belkov dinitrofenil'nym gaptenom.

A dinitrophenyl hapten capable of protein SH-group modification was synthesized and the specificity of its reaction with SH-groups of the E. coli ribosomal proteins was studied. The possibility of incorporation of the Dnp-modified protein into ribosomal subunits by in vitro reconstitution was demonstrated with the ribosomal protein S12. The Dnp-hapten attached to the protein S12 was found to be accessible for interaction with the Dnp-specific antibodies and therefore to be exposed on the surface of the reconstituted 30S subunit. Thus, the approach for incorporation of the antigenic groups into the protein components of supramolecular structures was proposed.