Cryo-electron microscopy and crosslinking data revealed the position of Ltv1 and Enp1 on the pre-40S solvent side 9, 21. Rps3 forms a ternary complex with the assembly factors Ltv1 and Enp1, which is salt-extractable from pre-ribosomal particles 8.

Cytoplasmic 40S maturation events include the final rRNA processing step of 20S pre-rRNA into mature 18S rRNA by the endonuclease Nob1 (refs 16, 17, 18), which takes place during a translation-like cycle in 80S-like ribosomes after the transient joining of mature 60S subunits 10, 19, 20.Ī crucial structural reorganization within the 40S head domain, occurring before 20S pre-rRNA cleavage, involves the r-protein S3 (Rps3) and leads to the formation of the characteristic beak structure, shaped by protrusion of 18S rRNA helix 33 (ref. After fulfilling their distinct tasks, they are progressively released and recycled back to the nucleus in a hierarchical order that is not entirely resolved so far 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15. Although most non-ribosomal 40S assembly factors present in the SSU processome leave the biogenesis pathway again in the nucleus, a few factors (namely Ltv1, Enp1, Pno1, Nob1, Dim1, Tsr1, Rio2, Hrr25, Fap7 and Rrp12) accompany the pre-ribosomal 40S particle to the cytoplasm. Most of the ∼80 r-proteins are assembled co-transcriptionally with a precursor rRNA, forming the large 90S particle (also termed SSU processome) that is subsequently separated into precursors of the large 60S (LSU) and the small 40S ribosomal subunit (SSU reviewed in refs 1, 2, 3, 4). The assembly of ribosomal RNA (rRNA) and ribosomal proteins (r-proteins) into eukaryotic ribosomes is a highly complex, multi-step process, which requires the coordinated action of over 200 assembly factors and takes successively place in the nucleolus, nucleoplasm and cytoplasm. Such a stepwise assembly may represent a new paradigm for the incorporation of ribosomal proteins. Finally, Ltv1 release, triggered by phosphorylation, and flipping of the S3 N-domain into its final position results in the stable integration of S3. Yar1 is replaced by the assembly factor Ltv1, thereby fixing the S3 N-domain in the rotated orientation and preventing its 40S association. Initial assembly of S3 with 40S precursors occurs via its C-domain, while the N-domain protrudes from the 40S surface. 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. Here, we provide functional insights into how successive structural re-arrangements in ribosomal protein S3 promote maturation of the 40S ribosomal subunit. 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.

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