Department of Developmental Biology, Stanford University, Stanford, CA 94305, USA; Department of Genetics, Stanford University, Stanford, CA 94305, USA.
Department of Developmental Biology, Stanford University, Stanford, CA 94305, USA; Department of Genetics, Stanford University, Stanford, CA 94305, USA.
Cell Rep. 2021 Jan 19;34(3):108629. doi: 10.1016/j.celrep.2020.108629.
Roles for ribosomal RNA (rRNA) in gene regulation remain largely unexplored. With hundreds of rDNA units positioned across multiple loci, it is not possible to genetically modify rRNA in mammalian cells, hindering understanding of ribosome function. It remains elusive whether expansion segments (ESs), tentacle-like rRNA extensions that vary in sequence and size across eukaryotic evolution, may have functional roles in translation control. Here, we develop variable expansion segment-ligand chimeric ribosome immunoprecipitation RNA sequencing (VELCRO-IP RNA-seq), a versatile methodology to generate species-adapted ESs and to map specific mRNA regions across the transcriptome that preferentially associate with ESs. Application of VELCRO-IP RNA-seq to a mammalian ES, ES9S, identified a large array of transcripts that are selectively recruited to ribosomes via an ES. We further characterize a set of 5' UTRs that facilitate cap-independent translation through ES9S-mediated ribosome binding. Thus, we present a technology for studying the enigmatic ESs of the ribosome, revealing their function in gene-specific translation.
核糖体 RNA（rRNA）在基因调控中的作用在很大程度上仍未得到探索。由于数百个 rDNA 单位分布在多个基因座上，因此无法在哺乳动物细胞中遗传修饰 rRNA，这阻碍了对核糖体功能的理解。扩展片段（ESs），即类似于触手的 rRNA 延伸物，在真核生物进化过程中序列和大小都有所不同，它们是否在翻译控制中具有功能作用仍然难以捉摸。在这里，我们开发了可变扩展片段-配体嵌合核糖体免疫沉淀 RNA 测序（VELCRO-IP RNA-seq），这是一种通用的方法，可以生成适应物种的 ESs，并绘制跨越整个转录组的特定 mRNA 区域图谱，这些区域与 ESs 优先结合。将 VELCRO-IP RNA-seq 应用于哺乳动物 ES9S，鉴定出了一大组通过 ES 选择性招募到核糖体的转录本。我们进一步研究了一组 5'UTR，它们通过 ES9S 介导的核糖体结合促进帽非依赖性翻译。因此，我们提出了一种研究核糖体神秘 ESs 的技术，揭示了它们在基因特异性翻译中的功能。
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