Agata Tyczewska

Atypical DNA methylation of genes encoding cysteine-rich peptides in Arabidopsis thaliana

BackgroundIn plants, transposons and non-protein-coding repeats are epigenetically silenced by CG and non-CG methylation. This pattern of methylation is mediated in part by small RNAs and two specialized RNA polymerases, termed Pol IV and Pol V, in a process called RNA-directed DNA methylation. By contrast, many protein-coding genes transcribed by Pol II contain in their gene bodies exclusively CG methylation that is independent of small RNAs and Pol IV/Pol V activities. It is unclear how the different methylation machineries distinguish between transposons and genes. Here we report on a group of atypical genes that display in their coding region a transposon-like methylation pattern, which is associated with gene silencing in sporophytic tissues.ResultsWe performed a methylation-sensitive amplification polymorphism analysis to search for targets of RNA-directed DNA methylation in Arabidopsis thaliana and identified several members of a gene family encoding cysteine-rich peptides (CRPs). In leaves, the CRP genes are silent and their coding regions contain dense, transposon-like methylation in CG, CHG and CHH contexts, which depends partly on the Pol IV/Pol V pathway and small RNAs. Methylation in the coding region is reduced, however, in the synergid cells of the female gametophyte, where the CRP genes are specifically expressed. Further demonstrating that expressed CRP genes lack gene body methylation, a CRP4-GFP fusion gene under the control of the constitutive 35 S promoter remains unmethylated in leaves and is transcribed to produce a translatable mRNA. By contrast, a CRP4-GFP fusion gene under the control of a CRP4 promoter fragment acquires CG and non-CG methylation in the CRP coding region in leaves similar to the silent endogenous CRP4 gene.ConclusionsUnlike CG methylation in gene bodies, which does not dramatically affect Pol II transcription, combined CG and non-CG methylation in CRP coding regions is likely to contribute to gene silencing in leaves because loss of this methylation in synergid cells is associated with CRP gene expression. We discuss this unusual methylation pattern and its alteration in synergid cells as well as the possible retrogene origin and evolutionary significance of CRP genes that are methylated like transposons.

A New Short Oligonucleotide-Based Strategy for the Precursor-Specific Regulation of microRNA Processing by Dicer

The precise regulation of microRNA (miRNA) biogenesis seems to be critically important for the proper functioning of all eukaryotic organisms. Even small changes in the levels of specific miRNAs can initiate pathological processes, including carcinogenesis. Accordingly, there is a great need to develop effective methods for the regulation of miRNA biogenesis and activity. In this study, we focused on the final step of miRNA biogenesis; i.e., miRNA processing by Dicer. To test our hypothesis that RNA molecules can function not only as Dicer substrates but also as Dicer regulators, we previously identified by SELEX a pool of RNA oligomers that bind to human Dicer. We found that certain of these RNA oligomers could selectively inhibit the formation of specific miRNAs. Here, we show that these specific inhibitors can simultaneously bind both Dicer and pre-miRNAs. These bifunctional riboregulators interfere with miRNA maturation by affecting pre-miRNA structure and sequestering Dicer. Based on these observations, we designed a set of short oligomers (12 nucleotides long) that were capable of influencing pre-miRNA processing in vitro, both in reactions involving recombinant human Dicer and in cytosolic extracts. We propose that the same strategy may be used to develop effective and selective regulators to control the production of any miRNA. Overall, our findings indicate that the interactions between pre-miRNAs and other RNAs may form very complex regulatory networks that modulate miRNA biogenesis and consequently gene expression.

Selection of RNA Oligonucleotides That Can Modulate Human Dicer Activity In Vitro

Human ribonuclease Dicer is an enzyme that excises small regulatory RNAs from perfectly or partially double-stranded RNA precursors. Although Dicer substrates and products have already been quite well characterized, our knowledge about cellular factors regulating the activity of this enzyme is still limited. To learn more about this problem, we attempted to determine whether RNA could function not only as a Dicer substrate but also as its regulator. To this end, we applied an in vitro selection method. We identified 120 RNA oligomers binding human Dicer. Sixteen of them were subjected to more detailed in vitro studies. We found that 6 out of 16 oligomers affected Dicer ability to digest pre-microRNAs (miRNAs), although most of them were cleaved by this enzyme. For the 6 most active oligomers the putative mechanism of Dicer inhibition was determined. Three oligomers were classified as typical competitive inhibitors and one as an allosteric inhibitor. The remaining 2 oligomers acted as selective inhibitors. They affected the production of 1 miRNA, whereas the formation of other miRNAs was hardly influenced. In general, the data obtained suggest that one can modulate the generation of specific miRNAs by using RNA oligomers. Moreover, we found that sequences similar to those of the selected oligomers can be found within the molecules composing human transcriptome.

Deciphering the soybean molecular stress response via high-throughput approaches.

As a result of thousands of years of agriculture, humans had created many crop varieties that became the basis of our daily diet, animal feed and also carry industrial application. Soybean is one of the most important crops worldwide and because of its high economic value the demand for soybean products is constantly growing. In Europe, due to unfavorable climate conditions, soybean cultivation is restricted and we are forced to rely on imported plant material. The development of agriculture requires continuous improvements in quality and yield of crop varieties under changing or adverse conditions, namely stresses. To achieve this goal we need to recognize and understand the molecular dependencies underlying plant stress responses. With the advent of new technologies in studies of plant transcriptomes and proteomes, now we have the tools necessary for fast and precise elucidation of desirable crop traits. Here, we present an overview of high-throughput techniques used to analyze soybean responses to different abiotic (drought, flooding, cold stress, salinity, phosphate deficiency) and biotic (infections by F. oxysporum, cyst nematode, SMV) stress conditions at the level of the transcriptome (mRNAs and miRNAs) and the proteome.

Stress Responsive Non-protein Coding RNAs

Environmental or growth conditions are constantly influencing the development and ma‐ turation of organisms. To ensure precise control of gene expression under these normal as well as stress conditions, organisms developed very accurate molecular regulatory mechanisms at transcriptional and posttranscriptional levels. Key roles of small noncod‐ ing RNAs (miRNAs and siRNAs) in regulation of gene expression are no longer astonish‐ ing us nor are constantly emerging new classes of non-protein coding RNAs (tRNAderived small RNAs, snoRNA-derived small RNAs, mRNA-derived small RNAs, and long non-coding RNAs). Although there is evidence of involvement of those various classes of noncoding RNAs in response to stress conditions, in most cases, we are still lacking basic information on their targets and interplay between different regulatory pathways. In this review, we focus on “canonical” and “noncanonical” noncoding RNAs and their involvement in abiotic stress responses.

Towards Food Security: Current State and Future Prospects of Agrobiotechnology

The consistent increase in the global population, estimated to reach 9 billion people by 2050, poses a serious challenge for the achievement of global food security. Therefore, the need to feed an increasing world population and to respond adequately to the effects of climate change must be urgently considered. Progress may be achieved by applying knowledge of molecular and genetic mechanisms to create and/or improve agricultural and industrial processes. We highlight the importance of crops (wheat, maize, rice, rapeseed, and soybean) to the development of sustainable agriculture and agrobiotechnology in the EU and discuss possible solutions for ensuring food security, while also considering their social acceptance.