SAM-synthase specific effects on metabolism and mitochondria underlie resistance to heat stress in C. elegans

S-adenosylmethionine (SAM), produced by SAM synthases, is critical for various cellular regulatory pathways and the synthesis of diverse metabolites. Studies have often equated the effects of knocking down one synthase with broader SAM-dependent outcomes such as histone methylation or phosphatidylcholine (PC) production. However, many organisms express multiple synthase genes including humans and Caenorhabditis elegans. Evidence in C. elegans, which possesses an expanded family of SAM synthases, suggest that the enzymatic source of SAM impacts its function. For instance, loss of sams-1 leads to enhanced heat shock survival and increased lifespan, whereas reducing sams-4 adversely affects heat stress survival. Here, we reveal that loss of sams-1 exerts age-dependent effects on nuclear-encoded mitochondrial gene expression, mitochondrial metabolites, and mitophagy. Notably, we find that that SAMS-1 exerts synthase-specific effects on PC production. We propose a mechanistic framework wherein the reduced SAM from SAMS-1 acts through PC to impact mitochondrial fission and mitophagy, thereby enhancing survival during heat stress. This study highlights multifaceted roles of SAM across metabolic pathways and synthase-specific SAM functions. Overall design: Lysis of young adult C. elegans was performed in 0.5% SDS, 5% ß-ME, 10 mM EDTA, 10 mM Tris-HCl ph7.4, 0.5 mg/ml Proteinase K, before purification of RNA by TRI-Reagent (Sigma). cDNA was produced with Transcriptor First-strand cDNA kits (Roche), and RT-PCR was performed using Kappa SYBR Green 2X Mastermix. RNA for sequencing was purified using RNAeasy columns (Qiagen). RNA sequencing (including library construction was performed by BGI (Hong Kong). Reads were analyzed through the NextFlow analysis platform (https://github.com/DanHUMassMed/RNA-Seq-Nextflow), using DeSeq2 to identify differentially expressed genes. Gene set enrichment was performed using WormCat (http://www.wormcat.com).

S-腺苷甲硫氨酸（S-adenosylmethionine, SAM）由SAM合酶催化合成，广泛参与多种细胞调控通路及多样代谢物的合成。过往研究常将单种合酶敲低的效应等同于更广泛的SAM依赖性功能变化，例如组蛋白甲基化或磷脂酰胆碱（phosphatidylcholine, PC）的生成。然而包括人类与秀丽隐杆线虫（Caenorhabditis elegans）在内的诸多生物，均表达多组合酶基因。在拥有扩增SAM合酶家族的秀丽隐杆线虫中已有研究证据表明，SAM的酶学来源会影响其功能：例如，敲除sams-1可提升线虫的热应激存活能力并延长寿命，而抑制sams-4则会对热应激存活产生负面影响。本研究揭示，sams-1的缺失会对核编码线粒体基因表达、线粒体代谢物及线粒体自噬产生年龄依赖性的调控效应。值得注意的是，我们发现SAMS-1对PC的生成具有合酶特异性的调控作用。我们提出了一套机制框架：由SAMS-1产生的SAM减少后，会通过PC通路影响线粒体分裂与线粒体自噬，进而提升热应激状态下的存活能力。本研究凸显了SAM在多条代谢通路中的多方面作用，以及不同合酶介导的SAM功能特异性。

整体实验设计：使用含0.5% 十二烷基硫酸钠（SDS）、5% β-巯基乙醇（β-ME）、10 mM 乙二胺四乙酸（EDTA）、10 mM Tris-HCl（pH7.4）及0.5 mg/ml蛋白酶K的裂解液对年轻成年秀丽隐杆线虫进行裂解，随后通过TRI试剂（Sigma）纯化RNA。使用Transcriptor第一链cDNA合成试剂盒（Roche）制备cDNA，以Kappa SYBR Green 2X预混液进行RT-PCR实验。用于测序的RNA通过Qiagen的RNAeasy层析柱进行纯化。RNA测序（含文库构建）由华大基因（香港）完成。测序reads通过NextFlow分析平台（https://github.com/DanHUMassMed/RNA-Seq-Nextflow）进行分析，使用DeSeq2鉴定差异表达基因。基因集富集分析采用WormCat工具（http://www.wormcat.com）完成。