Biochemical And Structural Studies Of RNA Silencing Suppressors

RNA silencing is gene regulatory mechanisms with specificity determined by small RNAs of 21-30 nt in length and is an important means in virus defense in plants. To counter defense, viruses encode diverse types of RNA silencing suppressor (VSR). Elucidation of biochemical property and structure of each virus RNA silencing suppressor is the key to understand their mechanism of action. This work studies the biochemical property and structure of a few known VSRs, including NS3 of Rice stripe virus, P26 of Foxtail mosaic virus and P14 of Pothos latent virus. These proteins have been expressed, purified to high homogeneity and subjected to crystallization. However, no crystal has been obtained. Meanwhile, biochemical property of NS3 has been characterized. We show that NS3 is a zinc-binding protein and forms a stable dimer. NS3 recognizes a?10 base pair region in double stranded RNA with nanomolar affinity, and associates in multiple copies with dsRNAs of 20 bp or longer. A few basic residues (R50?K77?K112?R124G?K173?K174?R175?K196) likely involved in RNA binding have been identified by mutagenesis. We could not get crystal of P26 protein which likely resulted from the property protein aggregation. P14 protein always precipitated when the protein mix with precipitant we selected, though the protein concentration is low.The biogenesis of eukaryotic ribosome requires many putative RNA helicases that are thought to remodel the RNA/RNP structure formed in ribosome assembly. We characterize the structure of one such RNA helicase Dbp8 from budding yeast and its interacting protein Esf2. The two proteins were expressed, purified and subjected to crystallization. No crystal was obtained. Pull-down assay shows that Dbp8 cannot stably associate with Esf2, in contrary with what was reported in the literature.