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

RNA解旋酶通过其在结构重塑RNA和核糖核蛋白(RNP)复合体中的能量依赖性功能，对基因表达的各个方面起着关键的调节作用。这种结构重排通常是通过它们在解开RNA双链方面的经典功能实现的，但最近也发现了一系列其他分子功能，包括退火RNA链和钳制RNA，突显了这些酶的灵活性。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，死盒RNA解旋酶主要与其RNA底物的骨架相互作用，使它们固有的非特异性。因此，RNA解旋酶如何在复杂的细胞环境中识别它们的目标RNA，以及是什么优化了每个RNA解旋酶的特定功能，仍然是重要的开放问题。核糖体的产生是一个基本的细胞途径，需要过多的RNA解旋酶，这些基本概念可以用来探索。核糖体合成包括四个核糖体RNA的转录、修饰和加工，以及大约80个核糖体蛋白的组装。这一复杂的过程以严格的层级方式发生，并由一系列不可逆转的重塑步骤驱动，其中许多步骤是由RNA解旋酶催化的。到目前为止，作为核糖体生物发生的典型模型，RNA解旋酶在酵母中的各种功能已经被描述，然而，最近出现的许多核糖体生物发生辅因子在人类细胞中具有不同的或附加的功能。对这些因子在高等真核生物中的作用的详细了解的重要性随着一些遗传病的发现而突显出来，这些遗传病被称为核糖体疾病，这些疾病是由编码核糖体生物发生因子的基因突变引起的。本项目致力于研究人类细胞核糖体生物发生过程中涉及的RNA解旋酶的特征，一方面更好地了解核糖体组装过程中的关键重构事件，另一方面深入了解非常规辅助因子对解旋酶的调节机制。我们将使用CRAC方法确定选定的解旋酶在核糖体前复合体上的结合位点。这将指导这些酶在核糖体前重塑的不同方面的功能分析。同时，我们将在结构和生化水平上探索RNA解旋酶与专用辅因子蛋白的相互作用，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。

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.