RNA binding protein PRRC2B mediates translation of specific proteins and regulates cell cycle progression [Polysome-seq]

Accumulating evidence suggests that posttranscriptional regulation of gene expression, including regulation of RNA splicing, transport, modification, translation, and degradation, primarily relies on RNA binding proteins (RBPs). However, the functions of many RBPs remain understudied. Here, we characterized the function of a novel RBP, Proline-Rich Coiled-coil 2B (PRRC2B). Through photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) and deep sequencing, we identified transcriptome-wide CU- or GA-rich PRRC2B binding sites around the translation initiation codon on a specific cohort of mRNAs in HEK293T cells. These PRRC2B target mRNAs, including oncogenes and cell cycle regulators such as CCND2 (cyclin D2), exhibited decreased translation upon PRRC2B knockdown as revealed by sequencing polysome-associated RNA, resulting in decreased G1/S phase transition and cell proliferation. Antisense oligonucleotides (ASOs) blocking PRRC2B-CCND2 mRNA interaction decreased CCND2 translation, thus inhibited G1/S transition and cell proliferation. Mechanistically, PRRC2B interactome capture analysis revealed RNA-independent interactions with eukaryotic initiation factors eIF4G2, eIF3, and FXR1. The interaction with eIF4G2 is essential for PRRC2B function since unlike wildtype PRRC2B, eIF4G2-interacting defective mutants failed to rescue the translation deficiency caused by PRRC2B knockdown. Taken together, our findings reveal that PRRC2B, by interacting with eIF4G2, is essential for efficient translation of specific proteins required for cell cycle progression and cell proliferation. We performed polysome-seq on HEK293T cells and its derivatives (shPRRC2B). Total RNA and polysome-associated RNA extracted from HEK293T cells and its knockdown derivatives (shPRRC2B) were subjected to RNA-seq. Biological triplicates were performed. Samples were subjected to DNA removal by DNase I and polyA enrichment before library construction by NGS Library Prep following the manufacturer's protocols. Paired-end sequencing was conducted at Novogene using NovaSeq6000 S4 with a depth of 20M reads/sample.越来越多的研究证据表明，基因表达的转录后调控——涵盖RNA剪接、转运、修饰、翻译及降解等过程的调控——主要依赖于RNA结合蛋白（RNA Binding Protein, RBP）。然而目前仍有大量RNA结合蛋白的功能尚未得到充分解析。本研究对一种新型RNA结合蛋白——富含脯氨酸卷曲螺旋2B（Proline-Rich Coiled-coil 2B, PRRC2B）的功能开展了系统表征。 通过光活化核糖核苷增强交联免疫沉淀（photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation, PAR-CLIP）结合深度测序技术，我们在HEK293T细胞的特定mRNA队列中，于翻译起始密码子附近鉴定出了转录组范围内PRRC2B结合的、富含CU或GA序列的位点。 这些PRRC2B靶mRNA（包括癌基因及细胞周期调控因子，如CCND2，即细胞周期蛋白D2）在PRRC2B敲低后翻译水平显著下调，该结果通过多聚核糖体结合RNA测序得到验证，进而导致G1/S期转换及细胞增殖能力受损。 靶向阻断PRRC2B与CCND2 mRNA相互作用的反义寡核苷酸（Antisense Oligonucleotides, ASOs）可抑制CCND2的翻译过程，从而阻滞G1/S期转换并降低细胞增殖活性。 机制层面的分析显示，PRRC2B相互作用组捕获实验揭示其可与真核起始因子eIF4G2、eIF3及FXR1发生非RNA依赖的相互作用。其中与eIF4G2的相互作用对PRRC2B的功能至关重要：相较于野生型PRRC2B，无法结合eIF4G2的突变体无法挽救PRRC2B敲低所引发的翻译缺陷。 综上，本研究结果表明，PRRC2B通过与eIF4G2相互作用，对于细胞周期进程及细胞增殖所需的特定蛋白质的高效翻译不可或缺。 本研究对HEK293T细胞及其衍生的PRRC2B敲低细胞系（shPRRC2B）开展了多聚核糖体测序（polysome-seq）：从HEK293T细胞及其敲低衍生细胞中提取的总RNA与多聚核糖体结合RNA，均按照NGS文库制备试剂盒的厂商操作规程，经脱氧核糖核酸酶I（DNase I）去除基因组DNA并进行polyA富集后，构建测序文库。实验设置三次生物学重复。样本在采用NGS文库制备试剂盒（NGS Library Prep）按照厂商规程构建文库前，先经DNase I去除DNA并进行polyA富集。测序采用NovaSeq6000 S4平台，由诺禾致源（Novogene）完成双端测序，每个样本的测序深度为20M reads。