Identification of stable, high copy number, medium-sized RNA degradation intermediates that accumulate in plants under non-stress conditions.

It is becoming increasingly evident that the RNA degradome is a crucial component of the total cellular RNA pool. Here, we present an analysis of the medium-sized RNAs (midi RNAs) that form in Arabidopsis thaliana. Our analyses revealed that the midi RNA fraction contained mostly 20-70-nt-long fragments derived from various RNA species, including tRNA, rRNA, mRNA and snRNA. The majority of these fragments could be classified as stable RNA degradation intermediates (RNA degradants). Using two dimensional polyacrylamide gel electrophoresis, we demonstrated that high copy number RNA (hcn RNA) degradants appear in plant cells not only during stress, as it was earlier suggested. They are continuously produced also under physiological conditions. The data collected indicated that the accumulation pattern of the hcn RNA degradants is organ-specific and can be affected by various endogenous and exogenous factors. In addition, we demonstrated that selected degradants efficiently inhibit translation in vitro. Thus, the results of our studies suggest that hcn RNA degradants are likely to be involved in the regulation of gene expression in plants.

RNA degradome--its biogenesis and functions.

RNA degradation is among the most fundamental processes that occur in living cells. The continuous decay of RNA molecules is associated not only with nucleotide turnover, but also with transcript maturation and quality control. The efficiency of RNA decay is ensured by a broad spectrum of both specific and non-specific ribonucleases. Some of these ribonucleases participate mainly in processing primary transcripts and in RNA quality control. Others preferentially digest mature, functional RNAs to yield a variety of molecules that together constitute the RNA degradome. Recently, it has become increasingly clear that the composition of the cellular RNA degradome can be modulated by numerous endogenous and exogenous factors (e.g. by stress). In addition, instead of being hydrolyzed to single nucleotides, some intermediates of RNA degradation can accumulate and function as signalling molecules or participate in mechanisms that control gene expression. Thus, RNA degradation appears to be not only a process that contributes to the maintenance of cellular homeostasis but also an underestimated source of regulatory molecules.

2D-PAGE as an effective method of RNA degradome analysis.

The continuously growing interest in small regulatory RNA exploration is one of the important factors that have inspired the recent development of new high throughput techniques such as DNA microarrays or next generation sequencing. Each of these methods offers some significant advantages but at the same time each of them is expensive, laborious and challenging especially in terms of data analysis. Therefore, there is still a need to develop new analytical methods enabling the fast, simple and cost-effective examination of the complex RNA mixtures. Recently, increasing attention has been focused on the RNA degradome as a potential source of riboregulators. Accordingly, we attempted to employ a two-dimensional gel electrophoresis as a quick and uncomplicated method of profiling RNA degradome in plant or human cells. This technique has been successfully used in proteome analysis. However, its application in nucleic acids studies has been very limited. Here we demonstrate that two dimensional electrophoresis is a technique which allows one to quickly and cost-effectively identify and compare the profiles of 10-90 nucleotide long RNA accumulation in various cells and organs.

A new family of ferritin genes from Lupinus luteus--comparative analysis of plant ferritins, their gene structure, and evolution.

Ferritins are one of the most important elements of cellular machinery involved in iron management. Despite extensive studies conducted during the last decade, many factors regulating the expression of ferritin genes in plants remain unknown. To broaden our knowledge about the mechanisms controlling ferritin production in plant cells, we have identified and characterized a new family of ferritin genes (from yellow lupine). We have also inventoried all available plant ferritins and their genes and subjected them to a complex bioinformatic analysis. It showed that the conservative structure of ferritin genes was established much earlier than it was thought before. The first introns in ferritin genes appeared already in green algae. The number and location of introns have been finally established in mosses, over 400 million years ago, and are strictly preserved in all plants from bryophytes to dicots. Comparison of ferritin gene promoters revealed that the 14-bp-long iron-dependent regulatory sequence (IDRS), identified earlier in Arabidopsis and maize, is characteristic for all higher plants. Moreover, we found that a highly conserved IDRS can be extended (extIDRS) up to 22 bp. Phylogenetic analysis of plant ferritins showed that polypeptides of the eudicot clade can be divided into two subclasses (eudicot-1 and eudicot-2). Interestingly, we found that genes encoding proteins classified as eudicot-1 and eudicot-2 are equipped with class-specific promoters. This suggests that eudicot ferritins are structurally and perhaps functionally diverse. Based on the above observations, we were able to identify conservative elements (ELEM1--6) other than extIDRS within plant ferritin gene promoters. We also found E-boxes and iron-responsive sequence elements FeRE1 and 2, characteristically distributed within ferritin promoters. Because most of the identified conserved sequences are located within or in close proximity of extIDRS, we named this fragment of the plant ferritin gene promoter the regulatory element rich region.

The brome mosaic virus-based recombination vector triggers a limited gene silencing response depending on the orientation of the inserted sequence.

In some RNA viruses (e.g. in brome mosaic virus, BMV), the same factor (intra- or intermolecular hybridization between viral RNA molecules) is capable of inducing two different processes: RNA silencing and RNA recombination. To determine whether there is some interplay between these two phenomena, we have examined if the BMV-based recombination vector containing a plant-genome-derived sequence can function as a gene-silencing vector. Surprisingly, we found that neither dsRNA forming during the replication of the BMV-based vector nor highly structured regions of its genome were effective RNAi triggers. Only mutants carrying a sequence complementary to the target mRNA functioned as gene silencing vectors and were steadily maintained in the infected plant. The constructs containing a sense sequence or inverted repeats did not induce gene silencing but instead were eliminated from the plant cells.

Ferritins and nodulation in Lupinus luteus: iron management in indeterminate type nodules.

An ability to form symbiotic associations with rhizobia and to utilize atmospheric nitrogen makes legumes ecologically successful. High iron content in legume grains, partially relocated from root nodules, is another-nutritional-advantage of this group of plants. The ferritin complex is the major cell iron storage and detoxification unit and has been recognized as a marker of many stress-induced responses. The possible participation of ferritin in nodule formation and functioning was investigated here. Correlation of increased accumulation of both ferritin polypeptide and mRNA with actual in situ localization of ferritin allowed ferritin synthesis in the developing, indeterminate-type root nodules to be related to differentiating bacteroid tissue. This kind of tissue, in contrast to the determinate-type nodules, is present in lupin nodules at almost all stages of their development. Interestingly, it was found that, in this type of nodule, senescence starting in the decaying zones induces ferritin accumulation in younger, still active, tissues. Based on the presented data, and in correlation with previous results, some aspects of the regulation of expression of lupin ferritin genes are also discussed.