Multiplexed functional genomic analysis of somatic 5' untranslated region mutations across the spectrum of human prostate cancer (RNASeq)

The functional consequences of genetic variants within 5' untranslated regions (UTRs) on a genome-wide scale are poorly understood in disease. We developed a high-throughput multi-layer massively parallel sequencing-based method called PLUMAGE (Pooled full-length UTR Multiplex Assay on Gene Expression) to quantify the molecular consequences of somatic 5' UTR mutations found across 229 prostate cancer patients with localized to metastatic disease. We show that 5' UTR mutations can control both transcript levels and mRNA translation rates through the creation of new DNA binding elements or RNA-based cis-regulatory motifs. We also discover that single point mutations can simultaneously impact both transcript levels and mRNA translation of the same gene. Using gene editing technology, we validate that a single point mutation in the oncogenic CKS2 5' UTR can increase mRNA specific translation. Turning to a molecular pathway critical for cancer, we provide evidence that functional 5' UTR mutations in the MAP kinase signaling pathway can upregulate pathway-specific gene expression and are associated with distinct clinical outcomes. Our study reveals the diverse mechanisms by which the mutational landscape of 5' UTRs can co-opt multiple levels of gene expression and demonstrates that single nucleotide alternations within leader sequences are functional in cancer. Overall design: Next-generation sequencing of metastatic castration-resistant prostate cancer (mCRPC) tumors using Illumina TruSeq Stranded mRNA Library prep.

目前，基因组范围内5'非翻译区（5' untranslated regions, UTRs）内遗传变异在疾病中的功能后果仍未被充分阐明。本研究开发了一种基于大规模并行测序的多层高通量检测方法PLUMAGE（Pooled full-length UTR Multiplex Assay on Gene Expression），用于量化229例局限性至转移性前列腺癌患者中检出的体细胞5'非翻译区突变的分子效应。本研究证实，5'非翻译区突变可通过形成新的DNA结合元件或RNA顺式调控基序，同时调控转录本水平与mRNA翻译速率。此外，本研究发现单点突变可同时影响同一基因的转录本水平与mRNA翻译过程。本研究借助基因编辑技术，验证了致癌基因CKS2的5'非翻译区单点突变可提升mRNA的特异性翻译效率。针对癌症发生的关键分子通路，本研究提供证据表明，MAP激酶信号通路（MAP kinase signaling pathway）中具有功能的5'非翻译区突变可上调通路特异性基因表达，且与独特的临床结局相关。本研究揭示了5'非翻译区突变景观通过多种机制调控基因表达多个层级的途径，并证实先导序列内的单核苷酸改变在癌症中具有功能活性。实验整体设计：采用Illumina TruSeq Stranded mRNA Library prep，对转移性去势抵抗性前列腺癌（metastatic castration-resistant prostate cancer, mCRPC）肿瘤样本进行下一代测序。