title: Slicer-independent mechanism drives small-RNA strand separation during human RISC assembly.
abstract: Small RNA silencing is mediated by the effector RNA-induced silencing complex (RISC) that consists of an Argonaute protein (AGOs 1-4 in humans). A fundamental step during RISC assembly involves the separation of two strands of a small RNA duplex, whereby only the guide strand is retained to form the mature RISC, a process not well understood. Despite the widely accepted view that 'slicer-dependent unwinding' via passenger-strand cleavage is a prerequisite for the assembly of a highly complementary siRNA into the AGO2-RISC, here we show by careful re-examination that 'slicer-independent unwinding' plays a more significant role in human RISC maturation than previously appreciated, not only for a miRNA duplex, but, unexpectedly, for a highly complementary siRNA as well. We discovered that 'slicer-dependency' for the unwinding was affected primarily by certain parameters such as temperature and Mg(2+). We further validate these observations in non-slicer AGOs (1, 3 and 4) that can be programmed with siRNAs at the physiological temperature of humans, suggesting that slicer-independent mechanism is likely a common feature of human AGOs. Our results now clearly explain why both miRNA and siRNA are found in all four human AGOs, which is in striking contrast to the strict small-RNA sorting system in Drosophila.
authors.affiliation: Department of Agricultural Biotechnology, Seoul National University, Seoul 151-921, Republic of Korea.
authors.forename: June Hyun
authors.initials: JH
authors.lastname: Park
authors.affiliation: Department of Agricultural Biotechnology, Seoul National University, Seoul 151-921, Republic of Korea Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul 151-921, Republic of Korea Plant Genomics and Breeding Institute, Seoul National University, Seoul 151-921, Republic of Korea cshin@snu.ac.kr.
authors.forename: Chanseok
authors.initials: C
authors.lastname: Shin
mesh_terms: D000818:Animals; D060565:Argonaute Proteins / Q000737:chemistry / Q000378:metabolism*; D002460:Cell Line; D004330:Drosophila / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008274:Magnesium / Q000502:physiology; D017434:Protein Structure, Tertiary; D034741:RNA, Small Interfering / Q000378:metabolism; D058727:RNA, Small Untranslated / Q000378:metabolism*; D034743:RNA-Induced Silencing Complex / Q000378:metabolism*; D013696:Temperature
chemical_list: C466422:AGO2 protein, human; D060565:Argonaute Proteins; D034741:RNA, Small Interfering; D058727:RNA, Small Untranslated; D034743:RNA-Induced Silencing Complex; D008274:Magnesium