Sultan Ch. Agalarov

The methylation of adenosine in the N⁶ position (m⁶A) is a widely used modification of eukaryotic mRNAs. Its importance for the regulation of mRNA translation was put forward recently, essentially due to the ability of methylated mRNA to be translated in conditions of inhibited cap-dependent translation initiation, e.g., under stress. However, the peculiarities of translation initiation on m⁶A-modified mRNAs are not fully known. In this study, we used toeprinting and translation in a cell-free system to confirm that m⁶A-modified mRNAs can be translated in conditions of suppressed cap-dependent translation. We show for the first time that m⁶A-modified mRNAs display not only decreased elongation, but also a lower efficiency of translation initiation. Additionally, we report relative resistance of m⁶A-mRNA translation initiation in the absence of ATP and inhibited eIF4A activity. Our novel findings indicate that the scanning of m⁶A-modified leader sequences is performed by a noncanonical mechanism.

The formation of ribosomal 48S initiation complexes at the start codon of the mRNA leader sequence that encodes obelin has been studied using the method of primer extension inhibition (toeprinting). Experiments have been performed in a system composed of purified individual components required to initiate translation. The influence of the dominant negative mutant of factor eIF4A (R362Q) on translation initiation has been studied. It has been shown that the presence of the mutant in reaction mixture has no effect on efficiency of formation of the 48S complexes at start codon of the template studied.

The translation of uncapped mRNAs encoding luciferase and green fluorescent protein in a cellfree translation system based on wheat germ extract has been studied. It turned out that two simultaneously translated (in one tube) different templates in a certain range of concentrations not only do not compete, but mutually enhance each other's translation. It has been shown that the synthesis of luciferase in the presence of mRNA that encodes green fluorescent protein is much more effective than in the translation of only luciferase mRNA at the same concentration. Similarly, the efficiency of the synthesis of green fluorescent protein increases in the presence of the template that encodes luciferase. It follows that the total effect of the concurrent translation of two different mRNAs exceeds the sum of the effects of the translation of each mRNA separately.

The formation of ribosomal 48S initiation complexes at the start codon of the mRNA leader sequence that encodes obelin has been studied using the method of primer extension inhibition (toeprinting)....

Recently we described a novel phenomenon observed during eukaryotic translation in a cell-free system: the coupling of initiation and termination on different mRNA molecules. Here we show that the phenomenon does not depend on a special mode of initiation. The mRNAs with certain leader sequences known to require different determinants for successful initiation were examined. Even in a case of using the intergenic internal ribosome entry site (IRES) of cricket paralysis virus RNA as the leader sequence, while no initiation factors are required, the effect of coupling is well expressed, including trials in the presence of hippuristanol as an inhibitor of eIF4A. Thus, the effect persists in the absence of scanning and does not depend on initiator tRNA and eIF2. The results suggest that the initiation factors are not involved in the coupling mechanism.

The formation of ribosomal 48S initiation complexes at the start AUG codon of uncapped mRNA leader sequences was studied using the methodology of primer extension inhibition (toe-printing). The experiments were performed in the system composed of purified individual components required for translation initiation. The formation of ribosomal 48S initiation complexes at the initiation codon was tested depending on the presence of the initiation factors eIF4F, eIF4A, and eIF4B. Several mRNAs containing short leader sequences lacking the extended secondary structure were studied. It was found that 48S ribosomal complexes at mRNAs with such leaders were not formed in the absence of eIF4F. In contrast, the removal of either eIF4A or eIF4B from the experimental system was found to be dispensable for the formation of the 48S complex.

In vitro reconstitution systems of ribosomal subunits from free ribosomal RNA and ribosomal proteins are helpful tool for studies on the structure, function and assembly of ribosome. Using this system mutant or modified ribosomal proteins or ribosomal RNA can be incorporated into ribosomal subunits for studying ribosome structure and function. Developing the protocol for reconstitution of 30S subunits from an extreme thermophilic bacterium Thermus thermophilus can be beneficial especially for structural studies, as proteins and nucleic acids from this organism are very stable and crystallize easier than those from mesophilic organisms.

The objective of the present work was to determine whether it is possible to use a nonhydrolyzable analog of ATP (AMP-PNP) as an inhibitor of ATP-dependent scanning of the leader sequence of eukaryotic mRNA in translation initiation. The formation of ribosomal 48S initiation complexes at the start codon of the capped mRNA leader sequence of rabbit β-globin mRNA was studied. The study was carried out in a system composed of individual components of translation initiation. The dependences of the efficiency of formation of 48S initiation complexes on ATP concentration and incubation time were obtained in the absence and presence of AMP-PNP. It was found that AMP-PNP did not affect the efficiency of formation of 48S initiation complexes in all cases under study. We conclude that the uncleavable analog of ATP, AMP-PNP, is not an inhibitor of translation initiation in eukaryotes.

The recombinant mRNAs with 5[prime]-untranslated region, called omega leader, of tobacco mosaic virus RNA are known to be well translated in eukaryotic cell-free systems, even if deprived of cap structure. Using the method of primer extension inhibition (toe-printing), the ribosomal particles were shown to initiate translation at uncapped omega leader when its 5[prime]-end was blocked by a stable RNA-DNA double helix, thus providing evidence for internal initiation. The scanning of the leader sequence and the formation of ribosomal 48S initiation complexes at the initiation AUG codon occurred in the absence of ATP-dependent initiation factor eIF4F, as well as without ATP. The latter results implied the ability of ribosomal initiation complexes for ATP-independent, diffusional wandering (also called bi-directional movement) along the leader sequence during scanning.

The task of the present work was to answer the question: is the free 5′-end needed for effective translation of a model polyribonucleotide template — polyuridylic acid — in a bacterial (E. coli) cell-free system? For this purpose, the template activities of the original polyuridylic acid with its free 5′-end and the polyuridylic acid with blocked 5′-end were compared in the bacterial cell-free translation system. To block the 5′-end, the cytidylic oligodeoxyribonucleotide with fluorescein residue at its 5′-end and uridylic oligoribonucleotide sequence at its 3′-end, schematically described as FAM(dC)₁₀(rU)₅₀, was covalently attached (ligated) to the 5′-end of the template polyuridylic acid. It was shown that the efficiency of polyphenylalanine synthesis on the 5′-blocked template and on the polyuridylic acid with free 5′-end was virtually the same. It was concluded that bacterial ribosomes are capable of effectively initiating translation at the polyuridylic sequence independently of the 5′-end of template polyribonucleotide, i.e. via an internal initiation mechanism, in the absence of a Shine-Dalgarno sequence and AUG start codon.