Essential Roles of Conserved Pseudouridines in Helix 69 for Ribosome Dynamics in Translation

The widespread distribution of pseudouridine (?), an isomer of the canonical uridine base, in RNA indicates its functional importance to the cell. In eukaryotes, it is estimated that around 2% of ribosomal RNA nucleotides are pseudouridines, most of which are located in functional regions of the ribosome. Defects in RNA pseudouridylation induce a range of detrimental effects from compromised cellular protein biosynthesis to disease phenotypes in humans. However, genome-wide changes to mRNA translation profiles by ribosomes lacking specific conserved pseudouridines have not been extensively studied. Here, using a new genomic method called 5PSeq and in vitro biochemistry, we investigated changes in ribosome dynamics and cellular translation profiles upon loss of ?2258 and ?2260 in helix 69, the two most conserved pseudouridines in the ribosome in yeast cells. We found that inhibiting the formation of these two pseudouridines challenges ribosomes to maintain the correct open reading frame and causes generally faster ribosome dynamics in translation. Furthermore, mutant ribosomes are more prone to pause while translating a subset of GC-rich codons, especially rare codons such as Arg (CGA) and Arg (CGG). These results demonstrate the presence of ?2258 and ?2260 contributes to the dynamics of the H69 RNA stem-loop, and helps to maintain functional interactions with the tRNAs as they move within the ribosome. The optimality of this ribosome-tRNA interaction is likely to be more critical for those limited tRNAs that decode rare codons. Consistent with the changes in ribosome dynamics, we observe that IRES-mediated translation is compromised in the mutant ribosome. These results explain the importance of ?2258 and ?2260 in H69 to maintain cellular fitness. The strong conservation of ?2258 and ?2260 in the ribosomes from bacteria to humans indicates their functional significance in modulating ribosome functions. It's likely that the identified functions of these covalent modifications are conserved across species. Overall design: 8 samples for 5Pseq experiments using various yeast strains.

假尿苷（pseudouridine）作为经典尿苷的异构体，在RNA中广泛分布，这一特征提示其对细胞具有重要的功能意义。在真核生物中，约2%的核糖体RNA（ribosomal RNA）核苷酸为假尿苷，其中大多数位于核糖体的功能区域。RNA假尿苷化修饰的缺陷会引发一系列有害影响，从受损的细胞蛋白质生物合成，到人类的疾病表型。然而，目前针对缺失特定保守假尿苷的核糖体所导致的mRNA翻译谱全基因组变化，尚未得到广泛研究。本研究借助一种名为5PSeq的新型基因组学方法结合体外生物化学实验，对酵母细胞内核糖体69号螺旋上两个最保守的假尿苷——ψ2258和ψ2260缺失后，核糖体动力学变化及细胞翻译谱的改变进行了探究。研究发现，抑制这两个假尿苷的形成会对核糖体维持正确开放阅读框（open reading frame, ORF）的能力造成挑战，同时普遍导致翻译过程中核糖体的动力学加快。此外，突变核糖体在翻译一组富含GC的密码子时更易发生暂停，尤其是精氨酸（arginine, Arg）的稀有密码子CGA和CGG。上述结果表明，ψ2258和ψ2260的存在有助于维持69号螺旋RNA茎环的动态结构，并帮助核糖体在转运RNA（transfer RNA, tRNA）于核糖体内移动时维持功能性相互作用。这种核糖体-tRNA相互作用的最优性，对于那些解码稀有密码子的有限tRNA群体而言可能更为关键。与核糖体动力学变化的观测结果一致，我们观测到突变核糖体中内部核糖体进入位点（internal ribosome entry site, IRES）介导的翻译过程受到损伤。这些结果阐释了69号螺旋上的ψ2258和ψ2260对于维持细胞适应性的重要性。从细菌到人类的核糖体中，ψ2258和ψ2260均高度保守，这表明它们在调控核糖体功能方面具有重要的功能意义。这些共价修饰的已鉴定功能很可能在跨物种间保守。总体实验设计：使用不同酵母菌株开展5PSeq实验，共设置8个样本。