Characterization of intra- and intercellular ARGONAUTE localization and trafficking

• Applicant: Dr. Jochen Gohlke
• Subject Area: Plant Genetics and Genomics
• Term: from 2014 to 2016
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 254981177

Modification of chromatin is a major mechanism for regulation of gene expression. For example, accurate maintenance and modification of DNA methylation is essential for diverse regulatory processes and environmental responses. Induction of DNA methylation by ARGONAUTE (AGO)-bound siRNAs has emerged as a key concept of targeted modification of DNA. Targeted chromatin modification by DNA methylation is important for transgenerational silencing in response to external stimuli. AGO proteins confer such modifications by binding siRNAs and guiding the silencing machinery to their corresponding DNA sequences in the nucleus. SiRNAs are mobile and act systemically, but the mechanisms of siRNA transport have not been explored. Despite their systemic functions and need to pass through the cytoplasm, most of our knowledge about RNA-directed DNA methylation is limited to the nucleus. As AGO proteins are frequently localized cytoplasmically and are able to bind siRNAs, these proteins may act in siRNA reception or trafficking. To assess the role of cytoplasmic AGO in reception of mobile siRNAs, grafting experiments with different Arabidopsis ecotypes and -mutants will be conducted. Total and AGO-bound small RNAs will be isolated from rootstocks. By using high-throughput sequencing, mobile RNAs will be identified by single nucleotide polymorphisms. From the proportion of mobile RNAs in the total RNAs and the AGO-bound fraction, this experiment will allow to draw conclusions regarding AGO function in scavenging unbound mobile siRNAs or intercellular siRNA transport. Protein fusions and mutations will be used to control the nuclear and cytoplasmic accumulation of AGO to better understand the biological role of protein localization. Transient cotransformation of AGO proteins with a nuclear-localized protein will reveal inter- or intracellular AGO movement by fluorescence microscopy.To analyze AGO functions in systemic silencing, a meristem-specific GFP-silencer system in combination with grafting will be employed to evaluate systemic silencing responses of AGO mutants by fluorescence intensity. This experiment is expected to deliver information about AGO function and localization in transduction and reception of mobile silencing signals in meristems. Collectively, these experiments will provide new insights into local and systemic RNA transit and uncover new functions of components from the transcriptional silencing machinery.

• DFG Programme: Research Fellowships
• International Connection: USA
• Host: Professorin Rebecca Mosher, Ph.D.