rRNA Processing in the Nucleolus. a) rDNA organization. A single tripartite rDNA unit, indicating sites of rRNA cleavage and the segments that give rise to the rRNAs of the 5′-ETS, pre-40S and pre-LSU. The “processome” is also known as the “90S preribosome.” ETS: external transcribed spacer. The term “tripartite” has also been used by some authors to specify the 18S, 5.8S, and 25S segments of rRNA. b) Subunit specialization of AFs. The Venn diagram indicates the largely distinct repertoires of AFs that contribute to the biogenesis of each type of subunit. The overlap includes several DExD/H proteins (Dbp3, Has1, Prp43), snoRNP proteins, subunits of RNA polymerase A and Rrp5 that binds sequences on either side of ITS1 (internal transcribed spacer). c) Activity cycle of AFs. Each AF is thought to cycle between a latent state (when not associated with rRNPs) and an operative state. Recruitment to the operative state requires production of new copies of ribosomal proteins and is correspondingly blocked by cycloheximide. d) Coaxial organization of the nucleolus when subunits are being produced. From left-to-right: Overview of the nucleolar crescent showing the rDNA segment (black) colocalizes with rDNA-associated proteins (rDNAPs) (red) and is enclosed by an “inner” layer of AFs (for SSU assembly, green). The remaining volume of the nucleolus is largely occupied by AFs that are engaged in assembling LSUs (blue). The points at which chromosome XII enters and leaves the crescent are indicated by (*). Two cross-sections of the coaxial cable are indicated in the lower right, either in cycling cells (left) or after metaphase arrest (right). At metaphase, when the nucleolus become elongated in the mother cell, cross-sections show the layered organization of the cable and the close apposition of the surrounding nuclear envelope (NE, purple). Note: We propose that the contour of rDNA weaves repeatedly throughout the nucleolus. See Tartakoff et al. (2021) for further detail. e) Single cycle of rRNP formation. The diagram illustrates the proposed loading of subunit-specific AFs onto rRNA transcripts during a single cycle of transcription (see Table 1 and Supplementary Table 1 for nomenclature). At the extreme left, the AFs are in their latent form, not having encountered nascent rRNA. The “principal groups” of AFs (SSU-F, LSU-Ou) are in the outer layer/volume of the coaxial structure, but a limited number (“minority group”) of AFs (SSU-In, LSU-F) are in the inner layer. As transcription begins, the SSU-In group binds the nascent transcript, followed by the SSU-F group that is recruited from the outer layer. When the 18S rRNA sequences have been transcribed and the initial downstream sequences appear, they bind LSU-F AFs and the nascent transcript extends/transfers to the outer layer where the SSU precursor is released (due to cleavage) and the LSU-Ou AFs bind. When transcription has almost completed, a site near the 3′ terminus is cleaved, thereby releasing the LSU precursor. Although not indicated, addition of ribosomal proteins occurs in conjunction with AFs. f) Flux of AFs during a single cycle of transcription. This diagram summarizes the changes of localization of AFs (lift-off) indicated in (e) as well as their return (reset) when transcription is complete, as described in Tartakoff et al. (2021).