Pre-ribosomal WDR74 module coordinates the early and late pre-rRNA processing stages for the NVL2-mediated regulation of 60S ribosome biogenesis.

WD repeat domain 74 (WDR74) is a nucleolar protein involved in the early stages of pre-60S maturation in the ribosome biogenesis pathway. In later stages, WDR74 interacts with MTR4, an RNA helicase that functions with the exosome nuclease complex, and is dissociated upon ATP hydrolysis by the chaperone-like nuclear VCP-like 2 (NVL2) AAA-ATPase. We previously reported that ATP hydrolysis-defective NVL2 causes aberrant accumulation of WDR74 on the MTR4-exosome complex at the nucleolar periphery and in the nucleoplasm and that this nuclear redistribution of WDR74 leads to the unusual cleavage of the early rRNA precursor within the internal transcribed spacer 1 sequence. However, the precise mechanisms underlying this NVL2-mediated regulation is largely obscure. In this study, co-immunoprecipitation combined with mass spectrometry revealed that WDR74 functions as part of a pre-ribosomal subcomplex, termed the WDR74 module, consisting of eukaryotic conserved WDR74, RPF1, MAK16, and RRP1. Each component of the WDR74 module was mutually essential for the interaction of other members with MTR4, and all components were required for the accurate cleavage of pre-rRNA during 60S ribosome biogenesis. Moreover, impaired release of WDR74 from the MTR4-exosome complex caused by NVL2 dysfunction prevented MTR4 from recruiting PICT1, an MTR4 adaptor protein required for the 3'-end maturation of 5.8S rRNA. Our results highlight the key role of the WDR74 module in coordinating the early pre-rRNA cleavage and late processing of pre-5.8S rRNAs.