Structural Analysis Reveals Features of Ribosome Assembly Factor Nsa1/WDR74 Important for Localization and Interaction with Rix7/NVL2

Abstract

SUMMARY Ribosome assembly is a complex process that requires hundreds of essential assembly factors, including Rix7 (NVL2 in mammals) and Nsa1 (WDR74 in mammals). Rix7 is a type-II double ring, AAA-ATPase, which is closely related to the well-known Cdc48/p97. Previous studies in Saccharomyces cerevisiae suggest that Rix7 mediates the release of Nsa1 from nucleolar pre-60S particles however the underlying mechanisms of this release are unknown. Through multiple structural analyses we show that S. cerevisiae Nsa1 is composed of an N-terminal seven bladed-WD40 domain followed by a lysine rich C-terminus that extends away from the WD40 domain and is required for nucleolar localization. Co-immunoprecipitation assays with the mammalian homologues identified a well-conserved interface within WDR74 that is important for its association with NVL2. We further show that WDR74 associates with the D1 AAA domain of NVL2, which represents a novel mode of binding of a substrate with a type-II AAA-ATPase.

Visualizing the Assembly Pathway of Nucleolar Pre-60S Ribosomes

“…This solvent-side location of Nsa1 explains why failure of its removal during 60S biogenesis, due to an N-terminal deletion in the Rix7 AAA ATPase, still allows the formation of functional 80S ribosomes and polysomes ( Kressler et al., 2008 ). The loop between the fourth and fifth β-blade of Nsa1 (Loop 4D-5A) ( Figure 2 A) was described to mediate the interaction between the human homologs of Nsa1 (WDR74) and Rix7 (NVL2) ( Lo et al., 2017 ). Yet in yeast, we found that the Nsa1 loop 4D-5A is involved in interactions with ES7a and Mak16; it is therefore not readily accessible for interaction with Rix7 ( Figure 2 A).…”

Cryo-EM structure of an early precursor of large ribosomal subunit reveals a half-assembled intermediate

“…In addition to binding Mak16, Rpf1 also binds L32, Nsa1 and the base of helix ES7a. Nsa1 contains a WD domain (Lo et al, 2017 ) and is docked at H19, ES7a and Rpf1. Rrp1 folds into a super-helical structure and contacts L4, L18, the N-terminus of L7, Mak16 and RNA helix ES7a.…”

“…The unmodeled density was assigned with assistance of the BALBES-MOLREP pipeline (Brown et al, 2015 ) and CXMS data. The crystal structures of the Brx1 and Ebp2 complex determined here and the Nsa1 structure (Lo et al, 2017 ) were fitted as rigid body. Homology models of Rpf1 and Has1 were created by SWISS-MODEL (Biasini et al, 2014 ).…”

Modular assembly of the nucleolar pre-60S ribosomal subunit

“…New assembly factors Mak16, Rrp1, Nop16, Erb1-NTD, Rrp14, Rrp15 and Ebp2 and segments of rRNA were modelled de novo . Previously determined crystal structures of assembly factors Nsa1 (PDB 5SUI) 26 , Ytm1 (PDB 5CXB) and Erb1-CTD (PDB 4U7A) 27 were docked and manually adjusted. Has1, Ssf1, Brx1, Rpf1 and Mak11 were initially docked from Phyre2 models 28 , and manually built to fit the density.…”