Song Zhuoyi, Bae Bongmin, Schnabl Simon, Yuan Fei, De Zoysa Thareendra, Akinyi Maureen V, Le Roux Charlotte A, Choquet Karine, Whipple Amanda J, Van Nostrand Eric L
Therapeutic Innovation Center & the Verna Marrs McLean Department of Biochemistry & Molecular Pharmacology, Baylor College of Medicine, Houston, TX, USA.
Department of Molecular & Cellular Biology, Harvard University, Cambridge, MA, USA.
Genome Biol. 2025 Feb 25;26(1):39. doi: 10.1186/s13059-025-03508-7.
Small nucleolar RNAs (snoRNAs) are non-coding RNAs that function in ribosome and spliceosome biogenesis, primarily by guiding modifying enzymes to specific sites on ribosomal RNA (rRNA) and spliceosomal RNA (snRNA). However, many orphan snoRNAs remain uncharacterized, with unidentified or unvalidated targets, and studies on additional snoRNA-associated proteins are limited.
We adapted an enhanced chimeric eCLIP approach to comprehensively profile snoRNA-target RNA interactions using both core and accessory snoRNA-binding proteins as baits. Using core snoRNA-binding proteins, we confirmed most annotated snoRNA-rRNA and snoRNA-snRNA interactions in mouse and human cell lines and called novel, high-confidence interactions for orphan snoRNAs. While some of these interactions result in chemical modification, others may have modification-independent functions. We showed that snoRNA ribonucleoprotein complexes containing certain accessory proteins, like WDR43 and NOLC1, enriched for specific subsets of snoRNA-target RNA interactions with distinct roles in ribosome and spliceosome biogenesis. Notably, we discovered that SNORD89 guides 2'-O-methylation at two neighboring sites in U2 snRNA that fine-tune splice site recognition.
Chimeric eCLIP of snoRNA-associating proteins enables a comprehensive framework for studying snoRNA-target interactions in an RNA-binding protein-dependent manner, revealing novel interactions and regulatory roles in RNA biogenesis.
小核仁RNA（snoRNA）是非编码RNA，主要通过引导修饰酶作用于核糖体RNA（rRNA）和剪接体RNA（snRNA）的特定位点，在核糖体和剪接体生物合成中发挥作用。然而，许多孤儿snoRNA仍未得到表征，其靶标未被鉴定或验证，并且对其他snoRNA相关蛋白的研究也很有限。
我们采用了一种改进的嵌合eCLIP方法，以核心和辅助snoRNA结合蛋白为诱饵，全面分析snoRNA与靶标RNA的相互作用。利用核心snoRNA结合蛋白，我们在小鼠和人类细胞系中证实了大多数已注释的snoRNA-rRNA和snoRNA-snRNA相互作用，并确定了孤儿snoRNA的新型高可信度相互作用。其中一些相互作用导致化学修饰，而其他相互作用可能具有与修饰无关的功能。我们发现，含有某些辅助蛋白（如WDR43和NOLC1）的snoRNA核糖核蛋白复合物，富集了在核糖体和剪接体生物合成中具有不同作用的特定snoRNA-靶标RNA相互作用子集。值得注意的是，我们发现SNORD89在U2 snRNA的两个相邻位点引导2'-O-甲基化，从而微调剪接位点识别。
snoRNA相关蛋白的嵌合eCLIP为以RNA结合蛋白依赖的方式研究snoRNA-靶标相互作用提供了一个全面的框架，揭示了RNA生物合成中的新型相互作用和调控作用。
