Collaborative Research: Role of Arabidopsis CTD-Phosphatase-Like 1 in Gene Silencing Pathways

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Intellectual meritPlant gene expression is a dynamic process that involves positive and negative regulation at multiple levels. Recent studies emphasize importance of RNA/gene-silencing mechanisms mediated by small RNA molecules, which can trigger both transcriptional and posttranscriptional gene silencing not only in plants but also in other eukaryotes. Central to the small RNA production are proteins containing double-stranded (ds) RNA binding motif (DRM). Among eukaryotes, plants contain unique DRM-containing proteins. Carboxyl-terminal domain (CTD) phosphatase-like 1 (CPL1) proteins contain an N-terminal phosphatase domain and tandemly repeated C-terminal DRM domains. The primary hypothesis to be tested in this project is that a plant-specific CPL protein fine-tunes the level of gene silencing mediated by small RNA. Since CTD phosphorylation can regulate the activity of the pol II complex, our results imply that plants evolved an additional fine-tuning mechanism of gene expression, which uses components of transcription elongation for gene silencing. This project will dissect the interaction between transcription elongation machineries and gene silencing by determining roles of CPL1 in gene silencing. The specific aims of this project are: 1) to establish CPL1 structure essential for in vivo function: essential domains of CPL1 protein will be determined by genetic complementation assays using modified CPL1 genes, 2) to establish interaction between CPL1 and HYL1; the mode of CPL1-HYL1 interaction will be determined and its relationship with CPL1 function will be analyzed, 3) to determine the RNA/gene silencing mechanism of CPL1; effect of cpl1 mutation in DNA methylation, histone modifications and small RNA accumulation/action will be analyzed using molecular genetic analysis as well as transient RNAi analysis. The body of information to be generated in this project therefore will reveal novel mechanism of plant gene regulation that will also have implications in other organisms.Broader impactsThis project will be conducted by a two-institute team consisting of two post-doctoral associates and a graduate student with support from undergraduate student(s). The participants will be trained for understanding and effective use of plant genome information and various genetic resources as well as molecular biochemical skills to validate the information. To train future scientists from diverse backgrounds, a graduate-level laboratory module for the course Physiol. Mol. Basis of Plant Stress Responses, in which students will have hands-on experience, will be taught in TAMU, some component of which will be shared in the similar course in UM. This course has been taken by numerous students in the field of field-oriented agriculture, therefore, should provide basic knowledge for sustainable agricultural practices. Undergraduate students will participate in the research program using REU supplements. An international exchange program has been initiated and is being developed with Gyeongsang Natl Univ, S. Korea, where visiting scholars and students can participate in mutual research projects. This is an excellent opportunity for students to be exposed to multi-disciplinary and multiculture research environment.