Argonaute 단백질에서의 마이크로RNA 성숙 및 불안정화 조절 메커니즘에 대한 연구

학위논문 (박사)-- 서울대학교 대학원 : 농생명공학부, 2017. 2. 신찬석.Small RNA silencing is mediated by the effector RNA-induced silencing complex (RISC) that consists of an Argonaute protein (Ago 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 I 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. I showed that in human cells, the elevated physiological temperature and the functional L1-PAZ domain of the Ago proteins, drive small RNA strand separation, even when slicer-assisted pathway is absent. In contrast to the previous models, I found that slicer-deficient Ago proteins can also be programmed with highly complementary siRNAs at the physiological temperature of humans, which now clearly explains why both miRNA and siRNA are found in all four human Ago proteins. Little is known about the regulation of miRNA stability. Mature miRNAs are stabilized by binding to Ago proteins, the core components of the RISC. Recent studies suggest that interactions between miRNAs and their highly complementary target RNAs promote release of miRNAs from Ago proteins, and this in turn can lead to destabilization of miRNAs. However, the physiological triggers of miRNA destabilization with molecular mechanisms remain largely unknown. Here, using an in vitro system that consists of a minimal human Ago2-RISC in HEK293T cell lysates, I sought to understand how miRNAs are destabilized by their targets. Strikingly, I showed that miRNA destabilization is dramatically enhanced by an interaction with seedless, non-canonical targets. I then showed that this process entails not only unloading of miRNAs from Ago, but also 3ʹ end destabilization of miRNAs occurred within Ago. Furthermore, mutation analysis suggests that conformational changes in the hinge region of the Ago PAZ domain are likely to be the main driving force of the miRNA destabilization. These collective results suggest that non-canonical targets may provide a stability control mechanism in the regulation of miRNAs in humans and also highlight the remarkable flexibility of human Ago proteins that experience dynamic conformational changes during their catalytic cycle. In sum, my findings reflect another layer onto the mutual regulatory circuits between Ago proteins, miRNAs and their targets, which may provide a means to fine-tune, refine and diversify miRNAs in cells.GENERAL INTRODUCTION 1 MicroRNA biogenesis and mechanism of action in mammals 1 Structural features of Argonaute proteins 3 Argonaute as a slicer 6 Experimental validation of miRNA targets 7 The aims and scope of this dissertation 12 Chapter I. Slicer-independent Mechanism Drives Small-RNA Strand Separation during Human RISC Assembly 13 I-1. Introduction 14 I-2. Materials and Methods 17 Cell culture and siRNA transfection 17 Cell lysate preparation 17 General methods for in vitro RNAi 18 Target RNA cleavage assay 18 in vitro RISC assembly assay 18 Passenger-strand cleavage assay 19 Immunopurification of Ago2 complex and unwinding assay 19 in vitro deadenylation assay 19 Native gel mobility-shift assay 20 Northern blotting 20 Western blotting 21 Expression and tandem affinity purification of recombinant human Ago2 21 I-3. Results 23 Small RNA maturation is highly dependent on the ambient temperature both in vitro and in cells 23 Slicer-dependent unwinding increases with Mg2+ concentration and decreases with increasing temperature 29 High temperature drives duplex unwinding largely independently of slicer activity 30 Slicer-deficient Ago proteins are capable of unwinding the siRNA duplex in a temperature-dependent manner 36 Spontaneous unwinding does not occur before duplexes are loaded into the Ago protein 41 Slicer-independent unwinding depends on the thermodynamic stability of the duplex structure 47 Slicer-independent unwinding requires the functional domains of the Ago protein 53 Slicer-independent unwinding is a general mechanism for human RISC maturation 57 Slicer-independent unwinding is a conserved mechanism 60 I-4. Discussion 64 Small RNA sorting and the Mg2+ level in slicer-dependent unwinding 64 Slicer-independent unwinding in human RISCs 66 A thermodynamic perspective of RISC maturation 67 Chapter II. Non-canonical Targets Destabilize MicroRNAs in Human Argonautes 71 II-1. Introduction 72 II-2. Materials and Methods 75 Cell culture and transfection 75 Cell lysate preparation 75 In vitro target RNA-directed miRNA destabilization assay 75 Target-directed miRNA destabilization in cells 76 Northern hybridization 77 Unloading assay 77 Ago2 cleavage assay 78 In vitro RISC assembly assay 79 Antibodies 79 II-3. Results 83 miRNAs are stable in Argonaute but they are destabilized upon non-canonical target binding 83 miRNA seed pairing is important for miRNA stability 91 Non-canonical targets trigger the 3 end destabilization of miRNAs 94 Human Ago2-RISC binds seedless, non-canonical targets with extensive 3 pairing 95 A large fraction of miRNAs is still in Ago2 following target-directed destabilization 100 3ʹ complementarity confers specificity for target-directed destabilization 104 Dynamic conformational changes of the PAZ domain drive 3 end destabilization 112 Non-canonical target and anti-miR possibly employ distinct mechanisms for miRNA destabilization 118 Non-canonical target-directed mechanism is likely to operate in living cells 122 II-4. Discussion 126 CONCLUSION AND PROSPECT 132 REFERENCES 136 ABSTRACT IN KOREAN 148 PUBLICATION 150Docto