The SRPome reveals SRP partners and non canonical functions

The signal recognition particle (SRP) is a ribonucleoprotein complex that assembles in a spatially and temporally regulated manner. Although SRP plays an essential role in co-translational targeting of proteins, new functions beyond the SRP cycle have started to emerge, including gene expression, apoptosis and stress response. This study presents the first comprehensive analysis of the eukaryotic SRP-binding partners. Two layers of the SRP interactome (SRPome), the SRP proteome and SRP transcriptome, were analysed by LC-MS/MS and RIP-seq, respectively. The majority of the newly identified protein and RNA partners of the SRP are involved in ribonucleoprotein particles (RNPs) formation, RNA processing and protein transport. SRP components have a dynamic cellular localization during the cell cycle. SRP proteins were shown to interact and co-localize with PRMT1 and PRMT5, while SRP54 was also shown to co-localize with the paraspeckle component NONO. Together these data provide a rich resource for identifying novel SRP partners and reveal several noncanonical functions of SRP that are previously unknown. Sample preparation: NTERA-2 cells were grown in DMEM media (Gibco) to 80% confluence in 15 cm dishes and crosslinked with 1% (v/v) formaldehyde for 10 minutes at RT. The cross-linking reaction was quenched by incubating for 5 minutes with glycine to a final concentration of 125 mM, followed by two washing steps with ice-cold PBS. Lysates were clarified from sonicated nuclei and SRP-RNA complexes were isolated with antibody. Libraries were prepared according to Diagenode's instructions, using CATS-RNA seq kit C05010041. Briefly, 10 ng of single stranded RNA were first chemically fragmented at 94 °C for 2 minutes, end-repaired and polyadenylated at the 3' end. Subsequently, a cDNA strand synthesis was performed in the presence of the anchored poly(dT) oligonucleotides containing terminal P7 Illumina adaptor sequence. During PCR pre-amplification (10 cycles) of the first cDNA strand, Illumina adapters carrying P5 and P7 terminal sequences as well as index sequences are incorporated into the library. The library was clean up twice with 0.9X AMPure XP beads (Beckman Coulter Life Sciences) to remove small DNA fragments and eluted in 20 µl of 1X TE buffer. Analysis: Trimming - 1 stage Trimmomatic 0.36 Reads were trimmed with Trimmomatic 0.36 with parameters (ILLUMINACLIP:TruSeq3-PE.fa:2:30:5 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:18 HEADCROP:8) Trimming - 2 stage Cutadapt 1.18 Adapters and polyA sequences were removed by Cutadapt 1.18 Alignement STAR 2.4.0.1 Reads were aligned to hg38 genome using a 2-pass mode basic procedure Peak-calling RIPSeeker 3.8

信号识别颗粒（signal recognition particle, SRP）是一类以时空调控方式组装的核糖核蛋白复合物。尽管SRP在蛋白质共翻译靶向过程中发挥不可或缺的作用，但近年来其超出SRP循环的新功能逐渐被揭示，包括基因表达、细胞凋亡与应激反应。本研究首次对真核生物SRP的结合伴侣开展全面分析：分别通过液相色谱-串联质谱（LC-MS/MS）与RNA免疫沉淀测序（RIP-seq），解析了SRP相互作用组（SRPome）的两个维度——SRP蛋白质组与SRP转录组。新鉴定的绝大多数SRP蛋白与RNA伴侣均参与核糖核蛋白颗粒（RNPs）形成、RNA加工及蛋白质转运过程。SRP组分在细胞周期中呈现动态细胞定位特征。研究表明，SRP蛋白可与精氨酸甲基转移酶PRMT1、PRMT5相互作用并共定位，而SRP54还可与核旁斑组分NONO发生共定位。上述数据为新型SRP伴侣的鉴定提供了丰富资源，并揭示了此前未被报道的SRP若干非经典功能。

样本制备：将NTERA-2细胞置于含Gibco DMEM培养基的15cm培养皿中培养至汇合度80%，于室温下用1%（体积比）甲醛交联10分钟。随后加入终浓度为125mM的甘氨酸孵育5分钟以终止交联反应，再用预冷PBS洗涤两次。对超声破碎的细胞核裂解液进行澄清处理，利用抗体分离SRP-RNA复合物。文库构建遵循Diagenode官方操作指南，使用CATS-RNA测序试剂盒C05010041完成。简要步骤如下：首先取10ng单链RNA，于94℃下化学裂解2分钟，随后进行末端修复并在3'端添加poly(A)尾；接着使用带有末端P7 Illumina接头序列的锚定poly(dT)寡核苷酸进行cDNA第一链合成。在第一链cDNA的10个循环PCR预扩增过程中，将携带P5、P7末端序列及索引序列的Illumina接头整合至文库中。使用0.9X AMPure XP磁珠（Beckman Coulter Life Sciences）对文库进行两次纯化以去除小片段DNA，最终用20μl 1X TE缓冲液洗脱文库。

数据分析：
1. 序列修剪——第一阶段：采用Trimmomatic 0.36进行序列修剪，参数为ILLUMINACLIP:TruSeq3-PE.fa:2:30:5 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:18 HEADCROP:8
2. 序列修剪——第二阶段：使用Cutadapt 1.18去除接头序列与polyA序列
3. 序列比对：采用STAR 2.4.0.1，以双流程基础步骤将读段比对至hg38参考基因组
4. 峰调用：采用RIPSeeker 3.8进行峰调用分析