Research Projects

Pre-mRNA splicing is an essential process carried out by the spliceosome, a dynamic ribonucleoprotein complex composed of hundreds of proteins and RNA. Proper and efficient functioning of spliceosomes is associated with massive conformational and compositional changes throughout the different stages of the pre-mRNA splicing cycle. While spliceosomal components and their regulation have been extensively studied in yeast, plants and mammals, the role of molecular chaperones, particularly J-domain proteins (JDPs) in spliceosome remodeling in plants remains unexplored. This project focuses on AtDjC37, the Arabidopsis thaliana ortholog of the essential yeast spliceosomal disassembly factor Cwc23, which harbors both a J-domain and a conserved RNA recognition motif (RRM), unlike its budding yeast counterpart. We hypothesize that AtDjC37 plays a crucial role in spliceosome remodeling, with both the J-domain and RRM contributing to its functional relevance in pre-mRNA splicing, thereby affecting plant growth, development, and stress adaptation. The objectives are to 1. Elucidate the functional relevance of AtDjC37 in Arabidopsis thaliana. 2. Assess the role of AtDjC37 in pre-mRNA splicing. To address these aims, the project will involve comprehensive phenotypic characterization of AtDjC37 loss-of-function mutants (knock-down mutants) to identify developmental or stress associated defects. This will be complemented by full-length and domain-specific complementation assays using constructs lacking either the J-domain or RRM, to dissect the functional contribution of these domains on AtDjC37 functionality. Promoter::reporter assays will also be employed to examine spatio-temporal gene expression of AtDjC37 gene. RNA sequencing will be used to identify global splicing defects arising from AtDjC37 disruption. To elucidate the underlying molecular mechanisms, protein-protein interaction studies using yeast two-hybrid (Y2H) and immunoprecipitation followed by mass spectrometry (IP-MS) will be conducted. Additionally, sub-cellular localization and co-localization of AtDjC37 with known spliceosomal markers will be studied through confocal microscopy. The significance of this research lies in uncovering a previously uncharacterized spliceosomal chaperone in plants, bridging the fields of RNA biology and molecular chaperone function. Outcomes might reveal novel mechanisms of RNA processing and potential targets for enhancing stress tolerance in crops, thus contributing both to fundamental understanding and to translational plant biotechnology.