Full text
| |
| Author(s): |
Hugo Libonati de Araujo
Total Authors: 1
|
| Document type: | Doctoral Thesis |
| Press: | São Paulo. |
| Institution: | Universidade de São Paulo (USP). Instituto de Ciências Biomédicas (ICB/SDI) |
| Defense date: | 2023-02-02 |
| Examining board members: |
Marilis do Valle Marques;
Andrea Balan Fernandes;
Rogerio Ferreira Lourenço;
Carla Columbano de Oliveira
|
| Advisor: | Marilis do Valle Marques |
| Abstract | |
Caulobacter crescentus is an environmental alphaproteobacterium with a complex cell cycle, making it an excellent model for the study of gene regulation in this group. The stress caused by temperature downfall leads to an adaptative response known as Cold-Shock Response, characterized by alterations in several cellular components and metabolic pathways. One of those changes is the tendency of RNA molecules to form stabilized secondary structures, impairing translation and turnover. The DEAD-box RNA helicases are proteins with the ability to unwind RNA structures and RNA-proteins complexes. The aim of this work was to define the physiological roles of the DEAD-box RNA helicases RhlB and RhlE in C. crescentus, particularly at low temperature, as well as investigate the interaction of RhlB with the RNA degradosome. Growth assays at low temperature and in freezing recovery were carried out, and genes differentially expressed after cold-shock and in the rhlB and rhlE mutant strains at different growth temperatures were identified. The interaction between RhlE and the ribosome was assessed, and strains carrying FLAG-RhlB with different C-terminal regions were used to determine its binding RNAs and to map the sites involved in the interaction with the degradosome. RNA sequencing results allowed us to characterize the C. crescentus response to cold-shock, unveiling important alterations in iron metabolism, transport and detoxification systems and cation homeostasis. RhlE and Rho seem to participate in the regulation of the kdp operon at low temperature by stabilizing the transcript and/or via transcription attenuation. Although we could not conclude the effect of RhlE on the genetic expression of C. crescentus, the transcriptomic analysis of the rhlE mutant indicates that its cold shock response seems to be the same as the one observed in the wild type strain, and the experiments to identify ribosome association were inconclusive. The rhlB mutant presented a downregulation of most of the identified genes by sequencing, with an expressive increase in the number of genes after cold-shock. Among these, the main affected categories were inorganic ions transport and metabolism, translation, ribosome structure and biogenesis, and post-translational modifications. Also, a severe decrease in the number of viable cells after freezing with pre-incubation at low temperature was observed in the rhlB mutant, indicating a possible defect in the assembly of an efficient cryoprotectant response. The complete removal of RhlB C-terminus leads to a worse phenotype than a partial deletion, indicating the lack of interaction with RNase E and/or a potential reduction of its helicase activity. (AU) | |
| FAPESP's process: | 18/17309-8 - Study of the role of the DEAD-box RNA helicase RhlB in the RNA stability and gene expression in Caulobacter crescentus |
| Grantee: | Hugo Libonati de Araújo |
| Support Opportunities: | Scholarships in Brazil - Doctorate |