Charakterisierung des intra- und interzellulären Transports sowie der Lokalisierung von ARGONAUTE-Proteinen
Zusammenfassung der Projektergebnisse
In addition to its nuclear localization that is associated with functions in RNA-directed DNA methylation, a high proportion of AGO4 was found to be cytoplasmic. It has been proposed that AGO4 is exported to the cytoplasm for siRNA binding, but information about the possible functions of cytoplasmic AGO4 is scarce. In order to be able to distinguish between nuclear and cytoplasmic AGO4 functions, A. thaliana lines expressing AGO4 fused to a strong nuclear localization signal (SV40 - NLS) were generated. Fractionation experiments revealed that adding a strong NLS not only increases the ratio of nuclear to cytoplasmic AGO4, but also reduces total protein levels. Despite this reduction in total protein, SV40 AGO4 lines still complement the ago4-3 mutation. In line with the current model of siRNA binding, SV40-AGO4 is most likely still transported into the cytoplasm due to its internal nuclear export signals, allowing siRNA binding and methylation to function. Surprisingly, a number of independent single insertion SV40 AGO lines displayed defects in early development. Considering that cytoplasmic and nuclear accumulation of AGO4 in these lines is drastically different from the wild type situation, we hypothesize that changes in the binding pattern of AGO4 to siRNAs or its interaction partners might impact developmental processes. When analyzing cell-to-cell mobility of AGO4, there was no evidence that AGO4 itself is mobile between cells. Due to the lack of evidence for intercellular AGO4 mobility but strong effects of altering intercellular AGO4 transport, analysis of AGO4 function in siRNA reception and mobility by grafting experiments will be focused on effects of shifts in the nucleocytoplasmic AGO4 distribution. Small RNA analysis of grafted Arabidopsis C24 lines in combination with Arabidopsis mutant lines harboring reduced cytoplasmic AGO4 might reveal functions of cytoplasmic AGO4 in small RNA reception or transport and explain developmental phenotypes in SV40 AGO4 lines.
