Abstract
Eukaryotic ribosomes assemble by association of ribosomal RNA with ribosomal proteins into nuclear precursor particles, which undergo a complex maturation pathway coordinated by non-ribosomal assembly factors. Here, we provide functional insights into how successive structural re-arrangements in ribosomal protein S3 promote maturation of the 40S ribosomal subunit. We show that S3 dimerizes and is imported into the nucleus with its N-domain in a rotated conformation and associated with the chaperone Yar1. Initial assembly of S3 with 40S precursors occurs via its C-domain, while the N-domain protrudes from the 40S surface. Yar1 is replaced by the assembly factor Ltv1, thereby fixing the S3 N-domain in the rotated orientation and preventing its 40S association. Finally, Ltv1 release, triggered by phosphorylation, and flipping of the S3 N-domain into its final position results in the stable integration of S3. Such a stepwise assembly may represent a new paradigm for the incorporation of ribosomal proteins.
Highlights
Eukaryotic ribosomes assemble by association of ribosomal RNA with ribosomal proteins into nuclear precursor particles, which undergo a complex maturation pathway coordinated by non-ribosomal assembly factors
The crystal structure of the r-protein S3 (Rps3)/Yar1 complex showed that the concave ankyrin-repeat surface of Yar1 binds the N-terminal a-helix of Rps3, while the Rps3 C-domain is in contact with the second a-helix of the fourth ankyrin-repeat23
To address how the complex is organized in solution, thereby avoiding crystallization artefacts, and to model the N- and C-terminal extensions of Yar1 and Rps3 missing in the crystal structure, we determined the solution structure of the complex by small-angle X-ray scattering (SAXS)
Summary
Eukaryotic ribosomes assemble by association of ribosomal RNA with ribosomal proteins into nuclear precursor particles, which undergo a complex maturation pathway coordinated by non-ribosomal assembly factors. Most non-ribosomal 40S assembly factors present in the SSU processome leave the biogenesis pathway again in the nucleus, a few factors (namely Ltv, Enp, Pno, Nob, Dim, Tsr, Rio, Hrr, Fap and Rrp12) accompany the pre-ribosomal 40S particle to the cytoplasm. Beak formation coincides with the phosphorylation-dependent release of Ltv by the kinase Hrr, which is supposed to be the initial step in cytoplasmic 40S maturation7,8,15 During these remodelling steps, the initially weakly associated, salt-extractable Rps adopts its final position and becomes stably bound to the 40S particle; the mechanistic basis for this stable incorporation of Rps has remained elusive. We have unveiled the intricate mechanisms regulating the stable assembly of Rps, which promotes the final steps of cytoplasmic 40S maturation
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