Biochemical specialization within Arabidopsis RNA silencing pathways.
In plants, the RNA silencing machinery responds to numerous inputs, including viral infection, microRNAs, and endogenous siRNAs that may act both in trans and in cis. Additionally, the full spectrum of silencing outcomes has been demonstrated in plants, ranging from mRNA degradation to repression at the level of protein synthesis to chromatin remodeling. Genetic studies in Arabidopsis have indicated that individual response pathways are functionally compartmentalized. However, to date, no biochemical systems have been available to investigate the roles of specific proteins within silencing pathways or the effects of selected mutations on the biochemical activity of those components. Here, we describe the generation of Arabidopsis extracts that reproduce many aspects of RNA silencing reactions in vitro. We find that specific members of the Dicer and Argonaute families have distinct biochemical activities, which provides insight into their roles within RNA silencing pathways in Arabidopsis.
Qi
Yijun
Y
Cold Spring Harbor Laboratory, Watson School of Biological Sciences, 1 Bungtown Road, Cold Spring Harbor, New York 11724, USA.
Denli
Ahmet M
AM
Hannon
Gregory J
GJ
D000255:Adenosine Triphosphate / Q000378:metabolism; D017360:Arabidopsis / Q000235:genetics* / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism; D060565:Argonaute Proteins; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D010936:Plant Extracts / Q000378:metabolism; D034622:RNA Interference; D012330:RNA, Double-Stranded / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D034743:RNA-Induced Silencing Complex / Q000235:genetics / Q000378:metabolism; D043244:Ribonuclease III / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics
C117457:AGO1 protein, Arabidopsis; D029681:Arabidopsis Proteins; D060565:Argonaute Proteins; D018797:Cell Cycle Proteins; D035683:MicroRNAs; D010936:Plant Extracts; D012330:RNA, Double-Stranded; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D034743:RNA-Induced Silencing Complex; D000255:Adenosine Triphosphate; C402383:DCL1 protein, Arabidopsis; D043244:Ribonuclease III