Arginine methylation regulates phase separation of TAF15 C-terminal LC domain

다국어 초록 (Multilingual Abstract)

Transcription is intricately regulated by RNA polymerase II (Pol II) and various transcription factors. TAF15, a component of the TFIID complex and an RNA-binding protein (RBP), utilizes low-complexity (LC) domains to mediate phase separation and participate in transcriptional regulation. While the N-terminal LC domain's function is known, the role of the C-terminal LC domain remains poorly understood. This study elucidates the effect of arginine methylation on the TAF15 C-terminal LC domain. In-cell and in vitro experiments showed that protein arginine N-methyltransferase 1 (PRMT1) catalyzes arginine methylation at R406, R408, and R411. This post-translational modification suppresses the phase separation capacity of TAF15; increasing PRMT1 concentrations led to higher methylation and the dissolution of liquid-like droplets. Conversely, both methylation-deficient and ALS-linked mutations exhibited significantly reduced droplet dissolution compared to WT, confirming methylation as a critical phase separation regulatory mechanism. When the nuclear localization signal (NLS) was intact, nuclear distribution was similar between TAF15 WT and the methylation-deficient mutation (R3K). However, in the absence of NLS, the methylation-deficient mutation formed distinct cytoplasmic granules under basal conditions, a finding mirrored by PRMT1 inhibitor (furamidine) treatment. Furthermore, recovery assays revealed that both the methylation-deficient and ALS-linked mutation in TAF15 exhibited slowed stress granule (SG) dynamics following stress, resulting in significantly more residual stress granules compared to WT. Collectively, these findings highlight the critical role of PRMT1-mediated arginine methylation in regulating both TAF15's phase separation and stress granule dynamics. This regulation is essential for normal cellular functions, and its disruption may contribute to the molecular mechanisms underlying ALS pathogenesis.

국문 초록 (Abstract)

TAF15는 FUS와 EWS와 함께 FET family를 구성하는 RNA 결합 단백질로 유전자가 발현하기 위한 전사 과정에 핵심적인 역할을 하고 있다. 이러한 TAF15는 N-terminal과 C-terminal에 단순하고 반복적인 아미노산 조성으로 이루어진 low complexity (LC) 도메인을 가지고 있다. 최근 연구들에 의하면, 이러한 LC 도메인에서는 phase separation을 통해 cross-β 구조의 가역적인 중합체 (polymer)를 형성하며, 이는 in vitro에서 hydrogel과 liquid-like droplet을 형태로 나타나는 특징이 있음이 밝혀졌다. 구조와 기능이 잘 알려진 N-terminal과 달리, TAF15의 C-terminal 도메인의 기능 및 이를 조절하는 특정 기전에 대한 이해는 제한적이다. 본 연구에서는 TAF15 C-terminal 도메인에서 일어나는 phase separation이 PRMT1 매개 arginine methylation에 의해 조절된다는 사실을 확인하였다. 특히, 이 methylation이 일어나는 잔기는 ALS 관련 TAF15 변이체 잔기 중 하나와 동일한 위치임을 확인하였다. In vitro liquid-like droplet formation assay 결과, ALS 관련 TAF15 변이체와 메틸화 결핍 TAF15 변이체가 WT과 비교하여 PRMT1 농도 증가에 따른 droplet의 형성 저해가 현저하게 감소함을 확인하였다. 또한 이러한 methylation 조절은 TAF15의 liquid-liquid phase separation뿐만 아니라 TAF15의 세포 내 localization에도 영향을 미침을 확인하였다. 세포 스트레스 회복 분석에서는 두 변이체 모두 느린 스트레스 과립 동역학을 보였으며, WT 대비 더 많은 잔존하는 세포질 puncta를 초래했다. 종합적으로, 본 연구는 PRMT1 매개 arginine methylation가 TAF15 C-terminal 도메인의 phase separation 및 스트레스 과립 동역학을 조절하는 핵심 요인이며, 이 조절의 이상이 ALS 발병 기전에 기여할 수 있음을 시사한다.

목차 (Table of Contents)

• 1. Introduction 1
• 2. Material and Methods 4
• 3. Results 9
• 3-1. Arginine methylation of the C-terminal low complexity domain of TAF15 9
• 3-2. Identification of arginine methylation sites on TAF15 10
• 3-3. Phase separation of the C-terminal LC domain of TAF15 can be regulated by arginine methylation mediated by PRMT1 12
• 3-4. PRMT1 inhibition and arginine methylation defects promote cytoplasmic granule formation of TAF15 14
• 3-5. Effects of R-to-K mutations on cytoplasmic granule localization of TAF15 15
• 3-6. Methylation deficiency of TAF15 impairs stress granule dynamics 16
• 4. Discussion 33
• 5. References 36