Crosstalk between Translational Regulation and Metabolic Remodeling in Beige Adipocyte Differentiation = 베이지 지방세포 분화 중 번역 조절 및 대사 재편성 간의 상호작용

다국어 초록 (Multilingual Abstract)

The precise contribution of regulatory events at the translational level to the shaping of distinct cellular and metabolic features in thermogenic adipocytes remains largely uncharacterized. This study elucidates two previously unidentified pathways that mediate the cross-talk between metabolic and translational regulation within beige adipocytes.
First, while the mitochondrial proteome expands significantly during the differentiation of precursors into beige adipocytes, we identified a selective translational attenuation of mRNAs encoding oxidative phosphorylation (OXPHOS) components. This attenuation was specifically restricted to OXPHOS Complexes I, III, IV, and V, whereas Complex II, which also functions as a component of the tricarboxylic acid (TCA) cycle, exhibited robust translation. The simultaneous translational upregulation of TCA cycle enzymes, including OXPHOS complex II, and the translational downregulation of other OXPHOS complexes establishes a distinct stoichiometry between OXPHOS and TCA cycle components within the mitochondria of thermogenic adipocytes, thereby acting as a key mechanism that shapes their associated metabolic activities.
Furthermore, a high-resolution analysis of ribosome positioning revealed a significant potentiation of ribosome pausing at glutamate codons in beige adipocyte differentiation. This increased stalling is attributable to the remodeling of glutamate metabolism during pan-adipocyte differentiation, which leads to a decreased availability of glutamate for tRNA charging. The glutamate codon-specific ribosome pausing reduces protein synthesis from specific genes enriched with glutamate codons, such as those associated with the actin cytoskeleton organization. Given that the disassembly of the actin cytoskeleton is a prerequisite for successful adipogenesis, our findings demonstrate that metabolic alterations during differentiation can orchestrate the developmental program by modulating translational regulation.

국문 초록 (Abstract)

열 생성 지방세포의 고유한 세포 및 대사적 특징을 형성하는 데있어, 번역 수준의 조절 기전이 미치는 기여에 대해서는 아직 명확히 규명되지 않았다. 본 연구에서는 베이지색 지방세포 내 대사 조절과 번역조절 간의 상호작용에 관여하는,이전에 알려지지 않았던 두 가지 핵심경로를 규명하였다.
첫째, 전구세포가 베이지색 지방세포로 분화하는 과정에서 미토콘드리아 단백질체는 유의미하게 확장되지만, 산화적 인산화 구성요소를 암호화하는 mRNA 에서는 선택적인 번역 약화현상이 확인되었다. 이러한 약화는 산화적 인산화 복합체 I, III, IV, V 에 국한되어 나타난 반면, TCA 회로의 구성 요소로도 기능하는 복합체 II 는 활발한 번역을 유지하였다. OXPHOS 복합체 II 를 포함한 TCA 회로 효소들의 동시적인 번역 증가와 여타 산화적 인산화 복합체들의 번역 감소는 열생성 지방세포의 미토콘드리아 내에서 OXPHOS 와 TCA 회로 구성 요소 간의 독특한 화학양론을 확립하며,이는 세포의 대사 활성을 결정짓는 핵심 기전으로 작용한다.
둘째, 리보솜 위치에 대한 고해상도 분석을 통해 베이지색 지방세포 분화 과정에서 글루탐산 코돈에서의 리보솜 정체가 유의하게 강화됨을 밝혀냈다. 이러한 정체 현상의 증가는 지방세포 분화 과정 중 일어나는 글루탐산 대사의 재편에 기인하며, 이는 tRNA 충전을 위한 글루탐산 가용성의 감소로 이어진다. 글루탐산 코돈 특이적인 리보솜 정체는 액틴 세포골격 구성과 관련된 유전자들과 같이 글루탐산 코돈이 풍부한 특정 유전자들의 단백질 합성을 감소시켰다. 액틴 세포골격의 해체가 성공적인 지방세포 형성을 위한 필수 전제 조건임을 고려할 때, 본 연구의 결과는 분화 과정 중의 대사적 변화가 번역 조절을 매개함으로써 세포의 발달 프로그램을 조율할 수 있음을 입증한다.

목차 (Table of Contents)

• ABSTRACT xii
• 1. INTRODUCTION 1
• 1.1 Beige adipocyte 1
• 1.2 Necessity of translational regulation research 7
• 1.3 Crosstalk between metabolism and gene regulation 10
• 2. MATERIALS AND METHODS 12
• 2.1 Animal model 12
• 2.2 Isolation of adipocyte precursor cells 12
• 2.3 Cell culture and chemical treatment 12
• 2.4 Ribo-seq and RNA-seq 14
• 2.4.1 Preparation of lysates for transcriptomes, translatomes, and proteomes 14
• 2.4.2 Pre-processing and alignment for Ribo-seq and RNA-seq libraries 14
• 2.4.3 Quantification of gene expression levels in Ribo-seq and RNA-Seq libraries 15
• 2.5 Proteomics 16
• 2.5.1 Sample preparation for mass spectrometry 16
• 2.5.2 High pH reversed-phase liquid chromatography for peptide fractionation 16
• 2.5.3 Liquid chromatography TMT mass spectrometry 17
• 2.5.4 Protein identification and quantitation 18
• 2.6 Oil Red O staining 19
• 2.7 Western blotting 19
• 2.8 Mitochondria isolation 20
• 2.9 Mitochondrial OXPHOS complex activity assay 20
• 2.10 Extracellular flux analysis 21
• 2.11 Glutamine/Glutamate measurement 21
• 2.12 Quantitative real-time PCR 22
• 2.13 Transfection 22
• 2.14 tRNA aminoacylation assay 23
• 2.15 Reporter assay with flow cytometry 24
• 2.16 Reporter assay with live cell image quantification 25
• 2.17 Immunocytochemistry 25
• 2.18 Bioinformatics 26
• 2.18.1 Reproducibility check of transcriptomes, translatomes, and proteomes by the principal component analysis 26
• 2.18.2 Gene set enrichment analysis with ribosome density 26
• 2.18.3 Gene list enrichment analysis 27
• 2.18.4 Determination of P-site and A-site codons of ribosomeprotected fragments (RPFs) 27
• 2.18.5 Calculation of subsequence (ribosomal A-site) abundance shift 27
• 2.18.6 Calculation of stalling score 27
• 2.18.7 Identification of ribosome stalling sites dependent upon beige adipocyte differentiation 28
• 2.18.8 Evaluation of ribosome occupancy around each codon 28
• 2.18.9 Re-analysis of publicly available Ribo-seq and RNA-seq libraries constructed using adipocyte lineage cells and adipose tissues 29
• 2.18.10 Re-analysis of publicly available mass spectrometry libraries constructed using adipocyte lineage cells 29
• 2.18.11 Re-analysis of mouse white adipose tissue single-nucleus transcriptome 30
• 2.19 Statistics 31
• 3. RESULTS 34
• Part 1. Multi-omics profiling of gene expression regulation during beige adipogenesis 34
• 3.1.1 Establishment of the beige adipocyte differentiation model and acquisition of multi-omics data 34
• 3.1.2 Identification of gene sets specifically regulated at the translational level 42
• Part 2. Attenuated mitochondrial translation selectively represses OXPHOS components to enable thermogenic adipogenesis 46
• 3.2.1 Selective translational attenuation of genes encoding OXPHOS complexes 46
• 3.2.2 Differential translational regulation between OXPHOS and TCA cycle component 58
• Part 3. Metabolic reprogramming of glutamate drives transla tional suppression of Glu-rich proteins by targeting glutamate codons during adipocyte differentiation 69
• 3.3.1 Ribosome stalling at glutamate (Glu) codons during beige adipocyte differentiation 69
• 3.3.2 Glutamate metabolic reprogramming limits Glu-tRNA charging to drive ribosome stalling 76
• 3.3.3 Glu-codon specific ribosome stalling drives actin cytoskeleton disassembly to promote adipogenesis 88
• 4. DISCUSSION 100
• REFERENCE 104
• ABBREVIATIONS 112
• DECLARATION 114
• ACKNOWLEDGEMENTS 113
• 국문요약 118