The DEAD-box Protein Rok1 Orchestrates 40S and 60S Ribosome Assembly by Promoting the Release of Rrp5 from Pre-40S Ribosomes to Allow for 60S Maturation

Abstract

DEAD-box proteins are ubiquitous regulators of RNA biology. While commonly dubbed “helicases,” their activities also include duplex annealing, adenosine triphosphate (ATP)-dependent RNA binding, and RNA-protein complex remodeling. Rok1, an essential DEAD-box protein, and its cofactor Rrp5 are required for ribosome assembly. Here, we use in vivo and in vitro biochemical analyses to demonstrate that ATP-bound Rok1, but not adenosine diphosphate (ADP)-bound Rok1, stabilizes Rrp5 binding to 40S ribosomes. Interconversion between these two forms by ATP hydrolysis is required for release of Rrp5 from pre-40S ribosomes in vivo, thereby allowing Rrp5 to carry out its role in 60S subunit assembly. Furthermore, our data also strongly suggest that the previously described accumulation of snR30 upon Rok1 inactivation arises because Rrp5 release is blocked and implicate a previously undescribed interaction between Rrp5 and the DEAD-box protein Has1 in mediating snR30 accumulation when Rrp5 release from pre-40S subunits is blocked.

The complete structure of the small subunit processome

“…Similarly, the tetratricopeptide repeat (TPR) of Rrp5 (ref. 18), which is necessary for pre-18S processing 19,20 , is positioned in proximity to the UtpC complex 21 , Krr1 (ref. 22) and the central domain ( Supplementary Fig.…”

Covalent Protein Painting Reveals Structural Changes in the Proteome in Alzheimer Disease

“…The molecular pathways associated with these proteins are ribosome assembly (PDCD11), oxidative stress response (GSR), and protein translation (GLMN). PDCD11 supports maturation of ribosomal subunits 40S and 80S 21 and processing of 47S rRNA 22 which transiently subsides during prolonged heat shock 23 . GLMN binds to RBX1 and prevents E2 ligase recruitment and therewith Cul1 E3 ligase-mediated ubiquitinylation of substrates 24 .…”

Structural and interaction analysis of the Rrp5 C‐terminal region

“…Altogether, this list comprises Rok1, Noc1-Noc2 and Has1 as direct binders [11,12,14], and Utp10, Utp20, Utp21, Kre33, Rrp36 and Nop58 as potentially direct/indirect partners of Rrp5 [9]. However, their precise binding sites onto Rrp5 have only recently been determined [12]. Experimentally measured electron density maps based on cryo-EM preparation of pre-40S particles allowed the positioning of the Rrp5 TPR domain in close proximity to the Utp22/Rrp7 complex [17].…”

“…Rrp5 is known to be involved in a variety of interactions within the ribosome biogenesis pathway including protein and RNA binding partners [10,11,12,18]. Given that Rrp5 is conserved from yeast to mammals, it is likely that conserved surface areas correspond to the functional regions.…”