Regulation of key steps in human ribosome biogenesis by DEAD-box RNA helicases and cofactors

DEAD-box RNA 解旋酶和辅因子对人类核糖体生物发生关键步骤的调节

• 批准号: 192916677
• 负责人: Professor Dr. Markus T. Bohnsack
• 金额: --
• 依托单位: Institut für Molekularbiologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2011
• 资助国家: 德国
• 起止时间: 2010-12-31 至 2023-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/192916677?language=en
• 关键词: Regulation; key; steps; human; ribosome

RNA helicases are critical regulators of all aspects of gene expression through their energy-dependent functions in structurally remodelling RNAs and ribonucleoprotein (RNP) complexes. Such structural rearrangements are generally achieved through their classical functions in unwinding RNA duplexes but recently, a range of other molecular functions including annealing RNA strands and clamping RNAs have also been discovered, highlighting the flexibility of these enzymes. Interestingly, despite the fact that many cellular pathways involve a multitude of RNA helicases, structural studies have revealed that DEAD-box RNA helicases primarily interact with backbone of their RNA substrates, making them inherently non-specific. Therefore, how RNA helicases recognise their target RNAs within the complex cellular environment and what optimises each RNA helicase for its particular function remain important open questions. The production of ribosomes is an essential cellular pathway that requires a plethora of RNA helicases, with which these fundamental concepts can be explored. Ribosome synthesis involves the transcription, modification and processing of the four ribosomal RNAs, and assembly of approximately 80 ribosomal proteins. This complex process takes places in a strictly hierarchal manner and is driven by a series of irreversible remodelling steps, many of which are catalysed by RNA helicases. So far, a diverse range of functions of RNA helicases have been described in yeast, the prototypical model for ribosome biogenesis, however, it has recently emerged that many ribosome biogenesis cofactors have different or additional functions in human cells. The importance of a detailed understanding of the roles of these factors in higher eukaryotes is highlighted by the discovery of a number of genetic diseases, termed “ribosomopathies”, which are caused by mutations in genes coding for ribosome biogenesis factors. This project addresses the characterisation of RNA helicases involved in ribosome biogenesis in human cells to, on the one hand, achieve a better understanding of key remodelling events during ribosome assembly and, on the other hand, gain insight into the mechanisms of helicase regulation by non-conventional cofactors. We will determine the binding sites of selected helicases on pre-ribosomal complexes using the crosslinking and analysis of cDNA (CRAC) approach. This will direct functional analysis of these enzymes in different aspects of pre-ribosome remodelling. In parallel, we will explore the interactions of RNA helicases with dedicated cofactor proteins on the structural and biochemical levels to identify the critical determinants of specificity and elucidate the different mechanisms by which these proteins regulate RNA helicase activity in vivo.

RNA解旋酶通过其在结构重塑RNA和核糖核蛋白（RNP）复合物中的能量依赖性功能，是基因表达各个方面的关键调节因子。这种结构重排通常通过它们在解旋RNA双链体中的经典功能来实现，但最近，还发现了一系列其他分子功能，包括退火RNA链和夹持RNA，突出了这些酶的灵活性。有趣的是，尽管许多细胞途径涉及大量的RNA解旋酶，但结构研究表明，DEAD盒RNA解旋酶主要与其RNA底物的骨架相互作用，使其具有固有的非特异性。因此，RNA解旋酶如何在复杂的细胞环境中识别其靶RNA以及如何优化每个RNA解旋酶的特定功能仍然是重要的悬而未决的问题。核糖体的产生是一个重要的细胞途径，需要大量的RNA解旋酶，这些基本概念可以探索。核糖体合成涉及四种核糖体RNA的转录、修饰和加工，以及大约80种核糖体蛋白的组装。这个复杂的过程以严格的等级方式发生，并由一系列不可逆的重塑步骤驱动，其中许多步骤由RNA解旋酶催化。到目前为止，RNA解旋酶的各种功能已经在酵母中被描述，酵母是核糖体生物合成的原型模型，然而，最近出现的是，许多核糖体生物合成辅因子在人类细胞中具有不同或额外的功能。详细了解这些因子在高等真核生物中的作用的重要性通过发现许多遗传疾病而突出，这些遗传疾病被称为“核糖体病”，其由编码核糖体生物合成因子的基因突变引起。该项目解决了RNA解旋酶参与在人类细胞中的核糖体生物合成的表征，一方面，实现在核糖体组装过程中的关键重塑事件的更好的理解，另一方面，深入了解解旋酶的非常规辅因子的调节机制。我们将使用cDNA的交联和分析（CRAC）方法来确定所选解旋酶在前核糖体复合物上的结合位点。这将指导这些酶在前核糖体重塑的不同方面的功能分析。与此同时，我们将探讨RNA解旋酶与专用辅因子蛋白在结构和生化水平上的相互作用，以确定特异性的关键决定因素，并阐明这些蛋白质在体内调节RNA解旋酶活性的不同机制。