Final maturation of eukaryotic ribosomes occurs in the cytoplasm and requires the sequential removal of associated assembly factors and processing of the immature 20S pre-RNA Using cryo-electron microscopy (cryo-EM), we have determined the structure of a yeast cytoplasmic pre-40S particle in complex with Enp1, Ltv1, Rio2, Tsr1, and Pno1 assembly factors poised to initiate final maturation. The structure reveals that the pre-rRNA adopts a highly distorted conformation of its 3' major and 3' minor domains stabilized by the binding of the assembly factors. This observation is consistent with a mechanism that involves concerted release of the assembly factors orchestrated by the folding of the rRNA in the head of the pre-40S subunit during the final stages of maturation. Our results provide a structural framework for the coordination of the final maturation events that drive a pre-40S particle toward the mature form capable of engaging in translation.

Eukaryotic ribosome biogenesis depends on several hundred assembly factors to produce functional 40S and 60S ribosomal subunits. The final phase of 60S subunit biogenesis is cytoplasmic maturation, which includes the proofreading of functional centers of the 60S subunit and the release of several ribosome biogenesis factors. We report the cryo-electron microscopy (cryo-EM) structure of the yeast 60S subunit in complex with the biogenesis factors Rei1, Arx1, and Alb1 at 3.4 Å resolution. In addition to the network of interactions formed by Alb1, the structure reveals a mechanism for ensuring the integrity of the ribosomal polypeptide exit tunnel. Arx1 probes the entire set of inner-ring proteins surrounding the tunnel exit, and the C terminus of Rei1 is deeply inserted into the ribosomal tunnel, where it forms specific contacts along almost its entire length. We provide genetic and biochemical evidence that failure to insert the C terminus of Rei1 precludes subsequent steps of 60S maturation.

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The ribosome is responsible for the final step of decoding genetic information into proteins. Therefore, correct assembly of ribosomes is a fundamental task for all living cells. In eukaryotes, the construction of the ribosome which begins in the nucleolus requires coordinated efforts of >350 specialized factors that associate with pre-ribosomal particles at distinct stages to perform specific assembly steps. On their way through the nucleus, diverse energy-consuming enzymes are thought to release assembly factors from maturing pre-ribosomal particles after accomplishing their task(s). Subsequently, recruitment of export factors prepares pre-ribosomal particles for transport through nuclear pore complexes. Pre-ribosomes are exported into the cytoplasm in a functionally inactive state, where they undergo final maturation before initiating translation. Accumulating evidence indicates a tight coupling between nuclear export, cytoplasmic maturation, and final proofreading of the ribosome. In this review, we summarize our current understanding of nuclear export of pre-ribosomal subunits and cytoplasmic maturation steps that render pre-ribosomal subunits translation-competent.