The AAA-ATPase Rea1 Drives Removal of Biogenesis Factors during Multiple Stages of 60S Ribosome Assembly

Abstract

The AAA(+)-ATPase Rea1 removes the ribosome biogenesis factor Rsa4 from pre-60S ribosomal subunits in the nucleoplasm to drive nuclear export of the subunit. To do this, Rea1 utilizes a MIDAS domain to bind a conserved motif in Rsa4. Here, we show that the Rea1 MIDAS domain binds another pre-60S factor, Ytm1, via a related motif. In vivo Rea1 contacts Ytm1 before it contacts Rsa4, and its interaction with Ytm1 coincides with the exit of early pre-60S particles from the nucleolus to the nucleoplasm. In vitro, Rea1's ATPase activity triggers removal of the conserved nucleolar Ytm1-Erb1-Nop7 subcomplex from isolated early pre-60S particle. We suggest that the Rea1 AAA(+)-ATPase functions at successive maturation steps to remove ribosomal factors at critical transition points, first driving the exit of early pre-60S particles from the nucleolus and then driving late pre-60S particles from the nucleus.

CRM1 and its ribosome export adaptor NMD3 localize to the nucleolus and affect rRNA synthesis

“…28,30 The maturation of pre-ribosome particles depends on successive, but transient interactions of several non-ribosomal proteins. 7,8 Stoichiometric interaction of NMD3 with 60S subunit and its binding interface Expression plasmids. Myc-NMD3 expression plasmid (RC203044) was purchased from Origene.…”

“…This process is mediated by over 150 participant proteins, occurs sequentially through dynamic protein exchange, and takes place in discrete nucleolar subdomains. [7][8][9][10] The nucleolus consists of three subdomains, the fibrillar center (FC), the dense fibrillar component (DFC) and the granular component (GC). 2 Various proteins are specifically associated with these domains and used to mark them.…”

Ribosome Biogenesis in the YeastSaccharomyces cerevisiae

“…During the transition from early preribosomes to intermediate pre-rRNPs, the AAA ATPases Rix7 and Rea1 catalyze removal of Nsa1 and Ytm1 plus Rsa4, respectively (Kressler et al 2008;Ulbrich et al 2009;Bassler et al 2010). Amino-terminal extensions of these ATPases are thought to direct them to their protein substrates, and ATP-dependent conformational switches are thought to disrupt pre-rRNP architecture, to release target factors from preribosomes.…”

The N-terminal extension of yeast ribosomal protein L8 is involved in two major remodeling events during late nuclear stages of 60S ribosomal subunit assembly

“…Subsequently, the Rea1 ATPase catalyzes removal of Rsa4 and Nog2 ( Fig. 7A; Bassler et al 2010;Kressler et al 2012;Baßler et al 2014). When the N-terminal extension of L8 was truncated, we observed decreased amounts of Sda1, the Rix1-subcomplex, and Rea1 in Nog2-containing pre-ribosomes.…”

“…These nucleotides are part of the rRNA binding site of assembly factor Erb1 (Granneman et al 2011;Thoms et al 2015). Thus, it is tempting to speculate that this stabilization mediated by the L35 extension may be involved in Erb1 removal, which is coupled with cleavage of ITS2 (Thomson and Tollervey 2005;Bassler et al 2010).…”