Arabidopsis HOT3/eIF5B1 constrains rRNA RNAi by facilitating 18S rRNA maturation during translation initiation (18S END-Seq). Arabidopsis HOT3/eIF5B1 constrains rRNA RNAi by facilitating 18S rRNA maturation during translation initiation (18S END-Seq)

Ribosome biogenesis is essential for protein synthesis in gene expression. Yeast eIF5B has been shown biochemically to facilitate 18S rRNA 3’ end maturation during late-40S ribosomal subunit assembly and gate the transition from translation initiation to elongation. But the effects of eIF5B have not been studied at the genome-wide level in any organism, and 18S rRNA 3’ end maturation is poorly understood in plants. Arabidopsis HOT3/eIF5B1 was found to promote development and heat-stress acclimation by translational regulation, but its molecular function remained unknown. Here, we show that HOT3 is a late-stage ribosome biogenesis factor that facilitates 18S rRNA 3’ end processing and is a translation initiation factor that globally impacts the transition from initiation to elongation. By developing and implementing 18S-ENDseq, we revealed previously unknown events in 18S rRNA 3’ end maturation or metabolism. We quantitatively defined new processing hotspots and identified adenylation as the prevalent non-templated RNA modification at the 3’ ends of pre-18S rRNAs. Aberrant 18S rRNA maturation in hot3 further activated RNAi to generate RDR1- and DCL2/4-dependent risiRNAs mainly from a 3’ portion of 18S rRNA. We further showed that risiRNAs in hot3 were predominantly localized in ribosome-free fractions not responsible for the 18S rRNA maturation or translation initiation defects in hot3. Our study uncovered the molecular function of HOT3/eIF5B1 in 18S rRNA maturation at the late-40S assembly stage and revealed the regulatory crosstalk among ribosome biogenesis, mRNA translation initiation, and siRNA biogenesis in plants. Overall design: Qualification and quantitation analysis of 18S rRNA 3' end for 18-day-old seedlings of WT, hot3-2, hot3-2 dcl2-1 dcl4-2, and dcl2-1 dcl4-2

核糖体生物发生对于基因表达中的蛋白质合成至关重要。酵母真核翻译起始因子5B（eIF5B）已通过生化实验证实，可在晚期40S核糖体亚基组装过程中促进18S核糖体RNA（18S rRNA）的3'端成熟，并调控翻译起始向延伸的转变过程。但目前尚未在任何生物的全基因组水平上研究eIF5B的功能，且植物中18S rRNA的3'端成熟机制仍知之甚少。拟南芥HOT3/eIF5B1已被发现通过翻译调控促进植物发育与热应激适应，但其分子功能始终未被阐明。

本研究证实，HOT3是一类晚期核糖体生物发生因子，可促进18S rRNA的3'端加工；同时也是一类翻译起始因子，能够全局性影响翻译起始向延伸的转变过程。通过开发并应用18S-ENDseq技术，我们揭示了此前未被发现的18S rRNA 3'端成熟或代谢事件。我们定量界定了全新的加工热点位点，并鉴定出腺苷酸化是前体18S rRNA（pre-18S rRNA）3'端普遍存在的非模板RNA修饰。hot3突变体中异常的18S rRNA成熟过程进一步激活了RNA干扰（RNAi）途径，主要以18S rRNA的3'区段为模板生成依赖于RDR1和DCL2/4的核糖体来源小干扰RNA（ribosome-derived small interfering RNAs, risiRNAs）。我们进一步证实，hot3中的risiRNAs主要定位于无核糖体组分，而该组分与hot3中18S rRNA成熟缺陷或翻译起始缺陷并无关联。本研究阐明了HOT3/eIF5B1在晚期40S核糖体亚基组装阶段参与18S rRNA成熟的分子功能，并揭示了植物中核糖体生物发生、mRNA翻译起始与小干扰RNA生物发生之间的调控串扰。

实验整体设计：对野生型（WT）、hot3-2、hot3-2 dcl2-1 dcl4-2以及dcl2-1 dcl4-2四个基因型的18日龄幼苗，开展18S rRNA 3'端的定性与定量分析。