Functional significance of methionyl-tRNA synthetase and AIMP3/p18 in translational initiation

Abstract

단백질합성효소 (ARS

aminoacyl-tRNA synthetase)는 고등생물에서 9개의 ARSs와 3개의 보조인자가 multisynthetase complex (MSC)를 이루고 있으며, 그 중에서 methionyl-tRNA synthetase (MRS)는 methionine을 자신의 상보적인 initiator tRNA에 붙여주는 효소로서 암의 억제효과를 가지고 있는 보조인자인 ARS-interacting multifunctional protein 3 (AIMP3)와 결합하고 있다. 위의 두 단백질이 각각 여러 가지 기능을 가지고 있으나 단백질합성과 관련하여 왜 특이적으로 결합을 하고 있는지 그리고 어떻게 조절하는지 아직 밝혀진 바가 없었다. 본 연구에서는 이 두 단백질이 어떻게 단백질합성에 관여하여 조절하는지 규명하였다. 제 1 단원에서는 단백질합성 및 AIMP3의 암 억제 활성의 조절에 있어서 MRS의 두 가지 역할에 대하여 기술하였다. MRS와 AIMP3는 각각의 GST 유사 부위를 통하여 결합을 하며, 자외선 처리시 그 결합이 떨어지는 것을 확인하였다. 이러한 현상은 general control nonrepressed 2 (GCN2)에 의존적으로 일어나며, GCN2에 의하여 MRS가 662번째 Serine에 인산화되는 것을 볼 수 있었다. 인산화가 된 MRS는 구조변화를 유도하여 AIMP3를 complex에서 놓아주게 되며, 활성 및 전반적인 단백질합성을 줄여주게 됨을 확인할 수 있었다. 또한 자외선과 같은 DNA 손상에 반응하는 eIF2a와 함께 단백질합성 저해를 위해 서로 협력한다는 것을 증명하였다. 이와 같은 기작으로 MRS가 DNA 손상에 관련된 스트레스에 의한 단백질합성 조절 및 암 억제 활성을 조절할 것으로 판단된다. 제 2 단원에서는 AIMP3가 단백질합성 개시 complex로 charged initiator tRNA (Met-tRNAiMet)의 전달을 매개함으로써 단백질합성시작 단계를 조절한다는 것을 기술하였다. MRS와의 결합을 통하여 MSC에 속해있는 AIMP3는 MRS에 의해 charge된 Met-tRNAiMet와 결합한다. 이는 initiator tRNA에 특이적이며, initiator tRNA의 acceptor stem에 결합해 있는 methionine을 인식하여 결합한다. AIMP3와 MRS는 eIF2g와 각각 결합하며, eIF2a와 g가 MRS와 AIMP3와 함께 complex를 형성할 수 있음을 확인하였다. 또한 AIMP3는 MRS와 eIF2g의 결합을 매개하며, 활성화된 eIF2g가 AIMP3와 결합하는 것을 확인하였다. 마지막으로 AIMP3가 위와 같은 현상을 통하여 3중복합인자 (initiator ternary complex) 형성을 조절하고 이를 통하여 전체적인 단백질합성에 관여함을 밝혔다.

Aminoacyl-tRNA Synthetases (ARSs) are essential enzymes for protein synthesis which catalyze the attachment of amino acid to their cognate tRNA. In higher eukaryote, nine ARSs is forming multisynthetase complex (MSC) with three non-enzymatic cofactors, called ARS-interacting multifunctional proteins (AIMPs). The several functions of ARSs and AIMPs were reported, including signal transduction, immune response, metabolism and cancer related activities. In MSC, methionyl-tRNA sythetase (MRS) ligates methionine to initiator tRNA for translation initiation, and makes a strong interaction with AIMP3, known to be tumor suppressor. Recently, the importance of MRS in global translational regulation was suggested. Although MRS and AIMP3 have many noncanonical functions, it is unclear that why their specific interaction is needed and how the interaction is affected in translational regulation. In translational regulation, the correlation of ARSs and AIMPs is not well-known. In this study, I elucidated the role of MRS and AIMP3 in global translation. In part 1, dual roles of human MRS was described in the regulation of cytosolic translation and molecular interaction with AIMP3, coupling control of protein synthesis to UV stress. MRS and AIMP3 bound each other via GST homology domain. However MRS was dissociated with AIMP3 under UV irradiation. The dissociation of AIMP3 and MRS depended on General control nonrepressed 2 (GCN2). MRS was phosphorylated by UV-induced GCN2. The phosphorylation of MRS at Ser662 residue induced a conformational change that is propagated to the N-terminal extension, and then led to AIMP3 releasing from MSC. The modification of MRS affected in the binding ability of initiator tRNA. Therefore, global translation was reduced by phosphorylation of MRS. In addition, MRS cooperated with eukaryotic initiation factor 2 alpha subunit (eIF2a) which responses to DNA damage condition such as UV irradiation. As a result, MRS regulated global translation and tumor suppressor activity of AIMP3 from DNA damage by mechanism which was explained in this study. In part 2, I demonstrated that AIMP3 regulates translation initiation by mediating transfer of charged initiator tRNA to initiation complex. In MSC, AIMP3 associated with MRS and charged initiator tRNA (Met-tRNAiMet). AIMP3 specifically interacted with Met-tRNAiMet but not unacylated through the recognition of methionine and acceptor stem. AIMP3 and MRS interacted with eukaryotic initiation factor 2 gamma subunit (eIF2g). Furthermore, AIMP3 mediated association of MRS and active eIF2g Downregulation of AIMP3 reduced ternary complex formation and protein synthesis. As a result, AIMP3 regulated formation of initiation ternary complex, so AIMP3 may regulate the accuracy and efficiency of translation. Through parts I and II, I elucidated role of MRS and AIMP3 as a regulator of translation initiation. MRS phosphorylation was related with DNA damage response and translational inhibition under UV stress. AIMP3 was dissociated from MRS under UV irradiation, thereby inhibiting ternary complex formation

this chain of events led to reduced rate of protein synthesis. This crosstalk may be important to keep the accuracy and efficiency of protein synthesis.