Concurrent remodelling of nucleolar 60S subunit precursors by the Rea1 ATPase and Spb4 RNA helicase

Abstract

Biogenesis intermediates of nucleolar ribosomal 60S precursor particles undergo a number of structural maturation steps before they transit to the nucleoplasm and are finally exported into the cytoplasm. The AAA+-ATPase Rea1 participates in the nucleolar exit by releasing the Ytm1-Erb1 heterodimer from the evolving pre-60S particle. Here, we show that the DEAD-box RNA helicase Spb4 with its interacting partner Rrp17 is further integrated into this maturation event. Spb4 binds to a specific class of late nucleolar pre-60S intermediates, whose cryo-EM structure revealed how its helicase activity facilitates melting and restructuring of 25S rRNA helices H62 and H63/H63a prior to Ytm1-Erb1 release. In vitro maturation of such Spb4-enriched pre-60S particles, incubated with purified Rea1 and its associated pentameric Rix1-complex in the presence of ATP, combined with cryo-EM analysis depicted the details of the Rea1-dependent large-scale pre-ribosomal remodelling. Our structural insights unveil how the Rea1 ATPase and Spb4 helicase remodel late nucleolar pre-60S particles by rRNA restructuring and dismantling of a network of several ribosomal assembly factors.

The DEAD-box ATPase Dbp10/DDX54 initiates peptidyl transferase center formation during 60S ribosome biogenesis

“…2a, b ). In this intermediate, Ssf1, Rrp14 and Rrp15 have been released from the pre-60S and replaced with a new set of RBFs; the Noc2/Noc3 heterodimeric complex bridges the L1 stalk and the newly docked rRNA domain III, stabilized by the extensive interaction network of the hub RBF Spb1 34 , 35 (Figs. 2 e and 5a and Supplementary Fig.…”

“…6f ). Spb1 is a long, modular protein that acts as a hub protein to organize multiple RBFs during late nucleolar maturation 34 , 35 . Together, these sequential steps establish a chronology of how the remodeling function of Dbp10 is directly coupled to the changes in RBF composition during late nucleolar 60S maturation.…”

The DEAD-box ATPase Dbp10/DDX54 initiates peptidyl transferase center formation during 60S ribosome biogenesis

“…5l). Spb1 is a long, modular protein that acts as a hub protein to organize multiple RBFs during late nucleolar maturation 35,36 . Together, these sequential steps establish a chronology of how the remodeling function of Dbp10 is directly coupled to the changes in RBF composition during late nucleolar 60S maturation.…”

“…In turn, Rrp14 release removes a steric block to allow the organization of the C-terminal anchoring domain of Spb1. In addition to its C-terminal methyltransferase domain, Spb1 contains multiple modules that interact with 15 other pre-60S components, including the Noc2/Noc3 heterodimer, acting as a critical hub to assemble rRNA domain III 20,21,31,35,36 . These sequential events therefore directly link PTC maturation and domain III organization, revealing how a non-equilibrium, ATP-dependent step is directly connected to subsequent structural changes in the pre-60S.…”

“…These sequential events therefore directly link PTC maturation and domain III organization, revealing how a non-equilibrium, ATP-dependent step is directly connected to subsequent structural changes in the pre-60S. After rRNA domain III is assembled, the pre-60S is acted on by another DEAD-box ATPase, Spb4, which catalyzes the organization of the H61/H62/H63/H64 junction, directly linking sequential ATP-dependent steps 35,36 .…”