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

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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.