ROK1ng the Ribosome Assembly Boat: An Investigation of the DEAD-box Protein Rok1 and its Co-factor Rrp5.

Abstract

Even though DEAD-box proteins are often referred to as RNA helicases, their described biochemical activities additionally include protein displacement from RNAs, ATP-dependent RNA binding and RNA annealing. In vitro analyses of DEAD-box proteins indicate that in all cases except one, they lack substrate specificity. However, since DEAD-box proteins have non-redundant functions and therefore high specificity in vivo, it is consequently believed that co-factors may increase the specificity of DEAD-box proteins by specifically binding individual RNA sequences. Surprisingly, until now, there is no experimental evidence for this simple hypothesis. Here, I describe both in vitro and in vivo approaches to dissect the function of two assembly factors essential for 40S ribosome maturation in Saccharomyces cerevisiae. I show that Rrp5 is an RNA-binding protein that binds specifically to rRNA sequences within the intron-like segment between 18S and 5.8S rRNAs. Interestingly, this modular protein uses some of its RNA-binding motifs to interact with rRNA in a sequence-specific fashion, while others are used to provide very high affinity. Additionally, my data provide evidence for a direct interaction between Rrp5 and the DEAD-box protein Rok1. Results from assays developed to characterize Rok1 indicate that Rok1 is a unique DEAD-box protein: it preferentially binds double-stranded RNA over single-stranded RNA and has annealing but no unwinding activity. The presence of the C-terminus of Rrp5 greatly enhances this annealing activity in an RNA-sequence specific manner. This data therefore provides evidence that co-factors can enhance the sequence specificity of DEAD-box proteins. Furthermore, the preferentially annealed RNA duplex is part of an inhibitory duplex in the pre-rRNA that serves to regulate the final cleavage step in 18S rRNA maturation. These biochemical results suggest that Rok1 and Rrp5 promote formation of this inhibitory duplex during rDNA transcription; preliminary in vivo structure probing experiments support this Rok1 requirement. Moreover, additional in vivo studies indicate that Rok1’s ATPase activity is essential for Rok1 dissociation from the ribosome. These results suggest a model by which Rok1 anneals the inhibitory duplex and associates with the pre-ribosome; when the checkpoint for Rok1 removal occurs, Rok1 can then use its ATPase activity to dissociate from the pre-ribosome.