TRUB1 is the predominant pseudouridine synthase acting on mammalian mRNA via a predictable and conserved code.
TRUB1 is the predominant pseudouridine synthase acting on mammalian mRNA via a predictable and conserved code.
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DOI：
10.1101/gr.207613.116
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发表时间：
2017-03
期刊：
影响因子：
7
通讯作者：
Schwartz S
中科院分区：
文献类型：
--
作者：
Safra M;Nir R;Farouq D;Vainberg Slutskin I;Schwartz S
在合成之后，RNA可以被修饰超过100种化学上不同的修饰，这可以潜在地在转录后调节RNA表达。假尿苷（Pseudouridine，PdR）是近年来发现的一种广泛存在于酵母mRNA中并受其动态调控的蛋白质，但对PdR在哺乳动物mRNA中的存在、调控和生物合成却知之甚少。在这里，我们试图描述哺乳动物mRNA上的酶景观，以确定催化酶形成的主要酶（PUSs），并了解控制其对选定靶标的特异性的因素。我们首先开发了一个框架，允许分析，评估和整合的mapping，我们将其应用于来自30个人类样本的> 25亿读取。这些图谱，加上遗传扰动，使我们能够发现TRUB1和PUS7是作用于哺乳动物mRNA的两个关键PUSs，并对控制TRUB1特异性的序列和结构元件进行计算建模，实现了对其底物的近乎完美的预测（AUC = 0.974）。然后，我们使用大规模平行报告分析验证并扩展了这些图谱和TRUB1的推断特异性，在该分析中，我们监测了数千种合成设计的序列变体的TRUB1水平，这些序列变体包含假尿苷化靶周围的序列或系统设计的干扰RNA序列和结构的突变体。我们的研究结果提供了一个广泛的和高质量的表征的转录组范围内分布的pseudouridine在人类和控制它的因素，并提供了一个重要的资源，为社会，铺平了道路的功能和机制解剖这一新兴层的转录后调控。
Following synthesis, RNA can be modified with over 100 chemically distinct modifications, which can potentially regulate RNA expression post-transcriptionally. Pseudouridine (Ψ) was recently established to be widespread and dynamically regulated on yeast mRNA, but less is known about Ψ presence, regulation, and biogenesis in mammalian mRNA. Here, we sought to characterize the Ψ landscape on mammalian mRNA, to identify the main Ψ-synthases (PUSs) catalyzing Ψ formation, and to understand the factors governing their specificity toward selected targets. We first developed a framework allowing analysis, evaluation, and integration of Ψ mappings, which we applied to >2.5 billion reads from 30 human samples. These maps, complemented with genetic perturbations, allowed us to uncover TRUB1 and PUS7 as the two key PUSs acting on mammalian mRNA and to computationally model the sequence and structural elements governing the specificity of TRUB1, achieving near-perfect prediction of its substrates (AUC = 0.974). We then validated and extended these maps and the inferred specificity of TRUB1 using massively parallel reporter assays in which we monitored Ψ levels at thousands of synthetically designed sequence variants comprising either the sequences surrounding pseudouridylation targets or systematically designed mutants perturbing RNA sequence and structure. Our findings provide an extensive and high-quality characterization of the transcriptome-wide distribution of pseudouridine in human and the factors governing it and provide an important resource for the community, paving the path toward functional and mechanistic dissection of this emerging layer of post-transcriptional regulation.