Role of RNA Binding Proteins and Post-Transcriptional Regulation in Response to Environmental Changes in Arabidopsis thaliana
Light and temperature are two of the most important factors that regulate plant growth and development. The adaptation to alterations in light and temperature involves programed changes in gene expression that are necessary for physiological and morphological adaptations. Transcript abundance is frequently used to monitor changes in gene expression in response to sub-optimal growth conditions. However, transcript accumulation may not accurately mirror gene expression due to extensive post-transcriptional and post-translational regulation. In this dissertation, the significance in post-transcriptional regulation in response to unanticipated alterations in light availability was evaluated in seedlings of Arabidopsis thaliana. Early darkness resulted in translational inhibition and sequestration of a subset of cellular mRNAs. The translationally regulated mRNAs were enriched in transcripts encoding chloroplastic and protein synthesis machinery. The reduced engagement of the majority of these transcripts with ribosomes was rapidly reversed upon re-illumination. These results suggest that regulation of the translational status of chloroplastic and protein synthesis mRNAs may aid in energy conservation during unanticipated darkness. Towards the elucidation of RNA binding proteins that control selective mRNA translation, the complexes of Arabidopsis Cold Shock Proteins (CSPs 1-4) were characterized. Transgenic lines over-expressing epitope-tagged CSPs were established and used for cellular fractionation and mass spectrometric protein identification of immunopurified CSP complexes. Fluorescently-tagged CSPs and associated proteins were localized in transiently transformed cells by confocal microscopy. Together, the results of this survey indicate Arabidopsis CSPs are involved in multiple processes of post-transcriptional regulation, including pre-mRNA/rRNA processing and mRNA translation. Of the four CSPs, CSP1 co-fractionated with ribosome. A mild RNase A treatment of ribosome complexes combined with sucrose gradient fractionation confirmed that CSP1 is a polysome-associated RNA binding protein. CSP1 accumulated under normal growth condition and was induced by low-temperature. A polyclonal antibody prepared to specifically recognize CSP1 protein was used to co-immunopurify native CSP1 complexes. DNA microarray hybridization was used to compare total (transcriptome), polysomal (translatome), and CSP1-associated mRNAs from plants grown under normal or low-temperature conditions. The results demonstrate that CSP1 preferentially associated with mRNAs involved in RNA processing and protein synthesis. Many of the CSP1-associated mRNA also have high 5'-UTR with a high G+C content. The transcriptome and translatome adjustments during low-temperature stress were highly correlated, in contrast to the findings with early darkness. This work provides new perspectives on post-transcriptional gene regulation in response to environmental cues inArabidopsis, as well as a foundation for future in-depth characterization of mRNA-RNP control networks in plants.