Glyphosate treatment mediates the accumulation of small discrete 5'- and 3'-terminal fragments of 18S rRNA in plant cells.

Under many kinds of stress, eukaryotic cells rapidly decrease the overall translation level of the majority of mRNAs. However, some molecular mechanisms of protein synthesis inhibition like phosphorylation of eukaryotic elongation factor 2 (eEF2), which are known to be functional in animals and yeast, are not implemented in plants. We suggest that there is an alternative mechanism for the inhibition of protein synthesis in plant cells and possibly, in other eukaryotes, which is based on the discrete fragmentation of 18S rRNA molecules within small ribosomal subunits. We identified four stress-induced small RNAs, which are 5'- and 3'-terminal fragments of 18S rRNA. In the present work, we studied the induction of 18S rRNA discrete fragmentation and phosphorylation of the α-subunit of eukaryotic initiation factor 2 (eIF2α) in germinated wheat embryos in the presence of glyphosate, which imitates the condition of amino acid starvation. Using northern and western blotting, we have shown that stress-induced 18S rRNA fragments started to accumulate in wheat embryos at glyphosate concentrations that did not evoke eIF2α phosphorylation. It was also found that cleavage of 18S rRNA near the 5'-terminus began much earlier than eIF2α phosphorylation, which became noticeable only at higher concentration (500 µM) of glyphosate. This result suggests that discrete fragmentation of 18S rRNA may constitute a regulatory mechanism of mRNA translation in response to stress and may occur in plant cells in parallel with and independently of eIF2α phosphorylation. The identified small 5'- and 3'-terminal fragments of 18S rRNA that accumulate during various stresses may serve as stress resistance markers in the breeding of economically important plant crops.

Constructing the constitutively active ribosomal protein S6 kinase 2 from Arabidopsis thaliana (AtRPS6K2) and testing its activity in vitro.

Ribosomal protein S6 (RPS6) is the only phosphorylatable protein of the eukaryotic 40S ribosomal subunit. Ribosomes with phosphorylated RPS6 can selectively translate 5'TOP-(5'-terminal oligopyrimidine)-containing mRNAs that encode most proteins of the translation apparatus. The study of translational control of 5'TOP-mRNAs, which are preferentially translated when RPS6 is phosphorylated and cease to be translated when RPS6 is de-phosphorylated, is particularly important. In Arabidopsis thaliana, AtRPS6 is phosphorylated by kinase AtRPS6K2, which should in turn be phosphorylated by upper level kinases (AtPDK1 - at serine (S) 296, AtTOR - at threonine (T) 455 and S437) for full activation. We have cloned AtRPS6K2 cDNA gene and carried out in vitro mutagenesis replacing codons encoding S296, S437 and T455 by triplets of phosphomimetic glutamic acid (E). After the expression of both natural and mutated cDNAs in Escherichia coli cells, two recombinant proteins were isolated: native AtRPS6K2 and presumably constitutively active AtRPS6K2(S296E, S437E, T455E). The activity of these variants was tested in vitro. Both kinases could phosphorylate wheat (Triticum aestivum L.) TaRPS6 as part of 40S ribosomal subunits isolated from wheat embryos, though the non-mutated variant had less activity than phosphomimetic one. The ability of recombinant non-mutated kinase to phosphorylate TaRPS6 can be explained by its phosphorylation by bacterial kinases during the expression and isolation steps. The phosphomimetically mutated AtRPS6K2(S296E, S437E, T455E) can serve as a tool to investigate preferential translation of 5'TOP-mRNAs in wheat germ cell-free system, in which most of 40S ribosomal subunits have phosphorylated TaRPS6. Besides, such an approach has a biotechnological application in producing genetically modified plants with increased biomass and productivity through stimulation of cell growth and division.

[Fragment of mRNA coding part that is complementary to region 1638-1650 of wheat 18S rRNA functions as a translational enhancer].

Possible involvement of 18S rRNA fragment 1638-1650 including basements of the helices h44 and h28 and nucleotides of the ribosomal decoding site in the cap-independent translation initiation on plant ribosomes is studied. This rRNA fragment is shown to be accessible for complementary interactions within the 40S ribosomal subunit. It is found that the sequence complementary to the 18S rRNA fragment 1638-1650 is able to enhance efficiency of a reporter mRNA translation when placed just after the initiation codon. The results obtained indicate that in the course of the cap-independent translation initiation, complementary interactions can occur between mRNA coding sequence and 18S rRNA fragment in the region of the ribosomal decoding site.

[Region 1112-1123 in the central domain of 18S rRNA in 40S subunits of plant ribosomes: accessibility for complementary interactions and the functional role].

The binding of the 18S RNA of the 40S subunits of wheat germ ribosomes to an oligodeoxyribonucleotide complementary to the 1112-1123 region of the central domain of this RNA molecule has been studied. The selective binding of this oligomer to the complementary RNA fragment and the inhibition of the translation of uncapped chimeric RNA containing enhancer sequences in the 5'-untranslated region upstream of the reporter sequence coding for beta-glucuronidase has been shown in a cell-free protein-synthesizing system. The use of a derivative of the aforementioned oligomer containing an alkylating group at the 5' end allowed for the demonstration that the 1112-1123 region of 18S RNA can form a heteroduplex with the complementary sequence of the oligomer. The data obtained show that the 1112-1123 region in loop 27 of the central domain of 18S RNA of 40S ribosomal subunits is exposed on the subunit surface and probably participates in the cap-independent binding of the subunits to mRNA due to the complementary interaction with the enhancer sequences.

ARC-1, a sequence element complementary to an internal 18S rRNA segment, enhances translation efficiency in plants when present in the leader or intercistronic region of mRNAs.

The sequences of different plant viral leaders with known translation enhancer ability show partial complementarity to the central region of 18S rRNA. Such complementarity might serve as a means to attract 40S ribosomal subunits and explain in part the translation-enhancing property of these sequences. To verify this notion, we designed beta-glucuronidase (GUS) mRNAs differing only in the nature of 10 nt inserts in the center of their 41 base leaders. These were complementary to consecutive domains of plant 18S rRNA. Sucrose gradient analysis revealed that leaders with inserts complementary to regions 1105-1114 and 1115-1124 ('ARC-1') of plant 18S rRNA bound most efficiently to the 40S ribosomal subunit after dissociation from 80S ribosomes under conditions of high ionic strength, a treatment known to remove translation initiation factors. Using wheat germ cell-free extracts, we could demonstrate that mRNAs with these leaders were translated more than three times more efficiently than a control lacking such a complementarity. Three linked copies of the insert enhanced translation of reporter mRNA to levels comparable with those directed by the natural translation enhancing leaders of tobacco mosaic virus and potato virus Y RNAs. Moreover, inserting the same leaders as intercistronic sequences in dicistronic mRNAs substantially increased translation of the second cistron, thereby revealing internal ribosome entry site activity. Thus, for plant systems, the complementary interaction between mRNA leader and the central region of 18S rRNA allows cap-independent binding of mRNA to the 43S pre-initiation complex without assistance of translation initiation factors.

[Detection of a new small RNA, induced by heat shock, in wheat seed ribosomes]. / Obnaruzhenie v ribosomakh zarodyshei pshenitsy novoi maloi RNK, indutsiruemoi teplovym shokom.

Changes in the small cytoplasmic RNA (scRNA) composition have been examined in wheat embryos exposed to heat shock conditions. A novel scRNA of about 135 nucleotides in length termed as 5.3S RNA, has been detected for the first time. With a rise in temperature from 26 degrees C up to 37 degrees C, the amount of 5.3S RNA increases 5 to 6 times. It was found that 5.3S RNA is localized predominantly on ribosomes and also in the fraction of native 40S ribosomal subunits known to contain translation preinitiation complexes. Upon dissociation of 80S monoribosomes under high ionic strength conditions (0.5 M KCl), 5.3S RNA remains to be bound to the 40S subunit, thus indicating their strong and specific interaction. 5.3S RNA is neither a precursor of the 5S rRNA nor a discrete fragment of high molecular weight ribosomal RNA. It was supposed that 5.3S RNA is transcribed by RNA polymerase III and involved in protein synthesis regulation in plant cells under heat shock conditions at the level of translation initiation.

[Release of informosomes from isolated nuclei of wheat embryos in an in vitro system]. / Osvovozhdenie informosom iz izolirovannykh iader zarodyshei. pshenitsy v sisteme in vitro.

The properties of the informosomes released from isolated nuclei of wheat embryos were studied in an in vitro system. The release of the informosomes from isolated nuclei of wheat embryos labelled with [3H]uridine is stimulated by exogeneous ATP. Optimal conditions for informosomes release from nuclei were determined. The particles obtained have heterogeneous sedimentation coefficients in sucrose gradient ranging from 10 to 60S. The particles are sensitive to low concentrations of RNase. Buoyant density in CsCl is 1.40-1.43 g/cm3. RNA extracted from particles is characterized by heterogeneous distribution in sucrose gradient between 4 and 20S. The informosomes contain two main peptides with molecular mass 36 000 and 51 000 and a number of minor peptides.