Ph.D. thesis: The cellular epitranscriptome: analysis of N6-methyladenosine (m6A) in Arabidopsis thaliana RNAs upon acclimation.
Several chemical modifications in cellular RNAs have been identified to date. The most common internal modification of eukaryotic RNAs is known as m 6 A. This modification is able to configure the outcome of gene expression by adjusting RNA decay, translation efficiency, RNA structure and alternative splicing. Although thousands of m6A sites were found in RNAs of several plant species, nearly nothing is known about the impact of m6A on plant acclimation. Here, we showed that mRNAs encoding writers, erasers and readers - proteins capable of installing, removing and recognizing/interpreting RNA modifications, respectively, - are responsive to cold, heat and high light conditions in Arabidopsis thaliana. Quantification of m6A by mass spectrometric analysis showed that m6A-modified poly(A)-enriched mRNAs are more abundant under cold acclimation. Under this condition, knockdown lines for components of the major m6A writer complex showed a decrease in photosynthetic performance, especially in the fip37-4 mutant. Immunological analysis of photosynthetic proteins and blue native gels has shown that under cold specific proteins and supercomplexes are downregulated in fip37-4. Interestingly, fip37-4 has a further role in thylakoid organization under cold as revealed by chloroplast ultrastructure analysis. In addition, ROS formation was increased while the expression of cold acclimation-related genes was reduced in fip37-4 at low temperatures. Plant development regarding leaf area, weight, chlorophyll/anthocyanin content and root development was impaired under control, but a much stronger deficiency especially in photosynthesis was clear under cold acclimation. Altogether, these results indicate that FIP37-based RNA methylations play crucial roles in plastid functions under cold and several other aspects of plant development.
Main techniques: DNA cloning, immunoblotting techniques, Northern blots/qRT-PCR, EMSAs (Electrophoretic Mobility Shift Assay), m6A quantification (colorimetric /ELISA-like assays and HPLC+LC-MS), IMAGING-PAM Chlorophyll Fluorescence System, Blue Native gels.
Master dissertation: Study of DEAD-box RNA helicase encoded by the gene rhlE in Caulobacter crescentus
Caulobacter crescentus is a Gram-negative bacterium, non-pathogenic and free-living, present mainly in aquatic environments. This species has been used as an important model system for the study of the cell cycle, due to the capability of their cells to divide into two distinct daughter cells. Its complex life cycle, combined with the gene control developed by this species in response to its environment, makes it an excellent model for studies of genes related to mechanisms of environmental stress response. At low temperature, the formation of secondary structures in the RNA is energetically favorable; this process can, however, jeopardize its functions and, consequently, the cellular processes in which they participate. At this point, it is essential the expression of specialized proteins, such as cold shock proteins, RNA helicases and RNA chaperones, in order to help the cell to adjust to the new environmental condition. In this context, the RNA helicases can perform a remarkable role, since mutants show reduced fitness at 30°C and high sensitivity to cold, suggesting an important cellular role, which still remains unknown. This project proposes to construct a strain in which the protein will be fusioned to an epitope recognized by a commercial antibody, allowing study its expression during the cell cycle and at different stress conditions. Also its interaction with specific RNAs will be investigated by coimmunoprecipitation and identification of RNAs by large scale sequencing (High-throughput sequencing of RNA isolated by cross-linking immunoprecipitation - HITS-CLIP). Therefore, the understanding of its expression regulatory signals and interaction with RNAs will allow the elucidation of its cellular role.
Main techniques: DNA cloning, immunoblotting techniques, co-immunoprecipitation assays, beta galactosidase assay, RNAseq and HITS-CLIP.