The Npa1p complex chaperones the assembly of the earliest eukaryotic large ribosomal subunit precursor

Abstract

The early steps of the production of the large ribosomal subunit are probably the least understood stages of eukaryotic ribosome biogenesis. The first specific precursor to the yeast large ribosomal subunit, the first pre-60S particle, contains 30 assembly factors (AFs), including 8 RNA helicases. These helicases, presumed to drive conformational rearrangements, usually lack substrate specificity in vitro. The mechanisms by which they are targeted to their correct substrate within pre-ribosomal particles and their precise molecular roles remain largely unknown. We demonstrate that the Dbp6p helicase, essential for the normal accumulation of the first pre-60S pre-ribosomal particle in S. cerevisiae, associates with a complex of four AFs, namely Npa1p, Npa2p, Nop8p and Rsa3p, prior to their incorporation into the 90S pre-ribosomal particles. By tandem affinity purifications using yeast extracts depleted of one component of the complex, we show that Npa1p forms the backbone of the complex. We provide evidence that Npa1p and Npa2p directly bind Dbp6p and we demonstrate that Npa1p is essential for the insertion of the Dbp6p helicase within 90S pre-ribosomal particles. In addition, by an in vivo cross-linking analysis (CRAC), we map Npa1p rRNA binding sites on 25S rRNA adjacent to the root helices of the first and last secondary structure domains of 25S rRNA. This finding supports the notion that Npa1p and Dbp6p function in the formation and/or clustering of root helices of large subunit rRNAs which creates the core of the large ribosomal subunit RNA structure. Npa1p also crosslinks to snoRNAs involved in decoding center and peptidyl transferase center modifications and in the immediate vicinity of the binding sites of these snoRNAs on 25S rRNA. Our data suggest that the Dbp6p helicase and the Npa1p complex play key roles in the compaction of the central core of 25S rRNA and the control of snoRNA-pre-rRNA interactions.

Assembly and early maturation of large subunit precursors

“…Domain V leads to the appearance of Nop13, a known nucleolar pre-60S factor and three members of the Npa/Urb subcomplex: Urb1, Nop8, and Rsa3. These proteins form a subcomplex, which also includes Urb2 and the helicase Dbp6, involved in the earliest steps of large subunit biogenesis (Rosado et al 2007;Joret et al 2018). While its role in large subunit assembly is still poorly understood, these data suggest that this complex associates with domain V.…”

The nucleolus as a polarized coaxial cable in which the rDNA axis is surrounded by dynamic subunit-specific phases

“…Consistent with such a role, they form a complex that can be recovered in an early precursor of LSUs. 60,61 Figure 4. Differential relocation of subunit-specific AFs (A) Three ovals at the left: in cells that are making subunits, both SSU-In and SSU-F AFs localize to the inner layer (first oval).…”

Assembly and early maturation of large subunit precursors

“…Domain V leads to the appearance of Nop13, a known nucleolar pre-60S factor and 3 members of the Npa/Urb subcomplex: Urb1, Nop8 and Rsa3. These proteins form a subcomplex, which also includes Urb2 and the helicase Dbp6, involved in the earliest steps of large subunit biogenesis (Rosado et al 2007;Joret et al 2018). While its role in large subunit assembly is still poorly understood, this data suggests that this complex associates with domain V.…”