Publications
RNA-binding proteins (RBPs) are key post-transcriptional regulators that play a substantial role during stress adaptation. Recent proteome-wide surveys have uncovered a large number of new and “unconventional” RBPs such as metabolic enzymes, yet little is known about the reconfiguration of the RNA-binding proteome (RBPome) and RNA-enzyme interactions in response to cellular stress. Here, we applied RNA-interactome capture to monitor the dynamics of the mRBPome upon mild oxidative stress in the yeast Saccharomyces cerevisiae. Among the 257 proteins that significantly changed RNA associations, we observed the coordinated remodeling of RNA-binding enzymes — particularly of the central carbon metabolism — that complemented known metabolic responses. Furthermore, we recognized the propensity for paralogous specific alterations of enzyme-RNA interactions. Our results suggest coordinated cross talk between RNA-enzyme interactions and intermediary metabolism to maintain the physiological and molecular balance upon oxidative stress, perhaps through specialization of paralogous during evolution.
In vivo characterization of RNA-protein interactions is the key for understanding RNA regulatory mechanisms. Herein, we describe a protocol for detection of proteins interacting with polyadenylated RNAs in the yeast Saccharomyces cerevisiae. Proteins are crosslinked to nucleic acids in vivo by ultraviolet (UV) irradiation of cells, and poly(A)-containing RNAs with bound proteins are isolated from cell lysates using oligo[dT]25 beads. RBPs can be detected by immunoblot analysis or with mass spectrometry to define the mRNA-binding proteome (mRBPome) and its changes under stress.
Despite the advances in treatment using chemotherapy or targeted therapies, due to static survival rates, non-small cell lung cancer (NSCLC) is the major cause of cancer-related deaths worldwide. Epigenetic-based therapies have been developed for NSCLC by targeting DNA methyltransferases (DNMTs) and histone-modifying enzymes. However, treatment using single epigenetic agents on solid tumours has been inadequate; whereas, treatment with a combination of DNMTs inhibitors with chemotherapy and immunotherapy has shown great promise. Dietary sources of phytochemicals could also inhibit DNMTs and cancer stem cells, representing a novel and promising way to prevent and treat cancer. Herein, we will discuss the different DNMTs, DNA methylation profiling in NSCLC as well as current demethylating agents in ongoing clinical trials. Therefore, providing a concise overview of future developments in the field of epigenetic therapy in NSCLC.
How stable and temperature-dependent variations in DNA methylation and nucleosome occupancy influence alternative splicing (AS) remains poorly understood in plants. To answer this, we generated transcriptome, whole-genome bisulfite, and MNase sequencing data for an epigenetic Recombinant Inbred Line (epiRIL) of A. thaliana at normal and cold temperature. For comparative analysis, the same data sets for the parental ecotype Columbia (Col-0) were also generated, whereas for DNA methylation, previously published high confidence methylation profiles of Col-0 were used. Significant epigenetic differences in an identical genetic background were observed between Col-0 and epiRIL lines under normal and cold temperatures. Our transcriptome data revealed that differential DNA methylation and nucleosome occupancy modulate expression levels of many genes and AS in response to cold. Collectively, DNA methylation and nucleosome levels exhibit characteristic patterns around intron-exon boundaries at normal and cold conditions, and any perturbation in them, in an identical genetic background is sufficient to modulate AS in Arabidopsis.
Alternative splicing (AS) is a major gene regulatory mechanism in plants. Recent evidence supports co-transcriptional splicing in plants, hence the chromatin state can impact AS. However, how dynamic changes in the chromatin state such as nucleosome occupancy influence the cold-induced AS remains poorly understood. Here, we generated transcriptome (RNA-Seq) and nucleosome positioning (MNase-Seq) data for Arabidopsis thaliana to understand how nucleosome positioning modulates cold-induced AS. Our results show that characteristic nucleosome occupancy levels are strongly associated with the type and abundance of various AS events under normal and cold temperature conditions in Arabidopsis. Intriguingly, exitrons, alternatively spliced internal regions of protein-coding exons, exhibit distinctive nucleosome positioning pattern compared to other alternatively spliced regions. Likewise, nucleosome patterns differ between exitrons and retained introns, pointing to their distinct regulation. Collectively, our data show that characteristic changes in nucleosome positioning modulate AS in plants in response to cold.
Plants use complex gene regulatory mechanisms to overcome diverse environmental challenges. For instance, cold stress induces rapid and massive transcriptome changes via alternative splicing (AS) to confer cold tolerance in plants. In mammals, mounting evidence suggests chromatin structure can regulate co-transcriptional AS. Recent evidence also supports co-transcriptional regulation of AS in plants, but how dynamic changes in DNA methylation and the chromatin structure influence the AS process upon cold stress remains poorly understood. In this study, we used the DNA methylation inhibitor 5-Aza-2'-Deoxycytidine (5-aza-dC) to investigate the role of stochastic variations in DNA methylation and nucleosome occupancy in modulating cold-induced AS, in (Arabidopsis). Our results demonstrate that 5-aza-dC derived stochastic hypomethylation modulates nucleosome occupancy and AS profiles of genes implicated in RNA metabolism, plant hormone signal transduction, and of cold-related genes in response to cold stress. We also demonstrate that cold-induced remodelling of DNA methylation regulates genes involved in amino acid metabolism. Collectively, we demonstrate that sudden changes in DNA methylation via drug treatment can influence nucleosome occupancy levels and modulate AS in a temperature-dependent manner to regulate plant metabolism and physiological stress adaptation.