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被探索。由于数百个 rDNA 单元位于多个位点，因此不可能对哺乳动物细胞中的 rRNA 进行基因修饰，从而阻碍了对核糖体功能的理解。扩展片段 (ES)、在真核生物进化过程中序列和大小不同的触手状 rRNA 扩展是否可能在翻译控制中发挥功能性作用仍然难以捉摸。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)，这是一种生成适应物种的 ES 并在优先与 ES 相关的转录组中绘制特定 mRNA 区域的通用方法。VELCRO-IP RNA-seq 在哺乳动物 ES、ES9S、鉴定了大量通过 ES 选择性募集到核糖体的转录物。我们进一步表征了一组 5' UTR，它们通过 ES9S 介导的核糖体结合促进不依赖帽的翻译。因此，我们提出了一种研究核糖体神秘 ES 的技术，揭示它们在基因特异性翻译中的功能。