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

[摘要] 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 vivostructure 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.

[发布机构] University of Michigan

[效力级别] Chemistry

[关键词] DEAD-box Protein in Ribosome Assembly;Chemistry;Science;Chemistry