Ribosome biogenesis factors-from names to functions

Abstract

The assembly of ribosomal subunits is a highly orchestrated process that involves a huge cohort of accessory factors. Most eukaryotic ribosome biogenesis factors were first identified by genetic screens and proteomic approaches of pre-ribosomal particles in Saccharomyces cerevisiae. Later, research on human ribosome synthesis not only demonstrated that the requirement for many of these factors is conserved in evolution, but also revealed the involvement of additional players, reflecting a more complex assembly pathway in mammalian cells. Yet, it remained a challenge for the field to assign a function to many of the identified factors and to reveal their molecular mode of action. Over the past decade, structural, biochemical, and cellular studies have largely filled this gap in knowledge and led to a detailed understanding of the molecular role that many of the players have during the stepwise process of ribosome maturation. Such detailed knowledge of the function of ribosome biogenesis factors will be key to further understand and better treat diseases linked to disturbed ribosome assembly, including ribosomopathies, as well as different types of cancer.

Keywords: pre-rRNA processing; pre-ribosomal particle; ribosomal subunit; ribosome biogenesis factor; ribosome synthesis.

Figure 1. Overview of eukaryotic ribosome biogenesis

In the nucleolus, a rRNA precursor is transcribed by RNAPI and co‐transcriptionally joined by RPs and RBFs, giving rise to a 90S pre‐ribosomal particle. After pre‐rRNA cleavage (site 2 in human, A₂ in yeast), the pre‐40S and pre‐60S particles further mature independently in the nucleolus and the nucleoplasm. After export through the nuclear pore complex, final maturation steps occur in the cytoplasm, yielding 40S and 60S subunits competent for mRNA translation. RNAPIII transcribes the 5S rRNA (nucleolar in human cells, nucleoplasmic in yeast), which joins pre‐60S particles in the nucleolus as part of the 5S RNP complex. RNAPII transcribes mRNAs of RP and RBF genes, which are translated by the 80S ribosome in the cytoplasm and then imported into the nucleus. Structural snapshots of maturing 90S (PDB ID: 6ZQA, 6ZQC), pre‐40S (PDB ID: 6G4W, 6G4S, 6ZQF), and pre‐60S particles (PDB ID: 6EM3, 6C0F, 6ELZ, 3JCT, 5JCS, 6LU8, 6LSR) as well as mature subunits (PDB ID: 6G5H, 3J7P) are shown. RBFs are displayed in orange, Rps in green, Rpl in blue, rRNA in gray. Structures solved in yeast are shown with reduced opacity, currently no structures of the corresponding human maturation stages are available.

Figure 2. Organization of nucleoli, the rDNA locus and promoter architecture in yeast and human cells

(A) Schematic representation of a yeast nucleolus composed of fibrillar strands (FS) and granules (G) and human nucleoli consisting of three subcompartments: fibrillar center (FC), dense fibrillar component (DFC), and granular component (GC). (B) Schematic representation of rDNA architecture in S. cerevisiae and human cells. (C) Comparison of 35S/47S rDNA promoter region with associated pre‐initiation complexes in yeast and human cells. Yeast promoters contain the upstream activation sequence (UAS) bound by the upstream activating factor (UAF) complex and the central element (CE) bound by the core factor (CF) complex. Human promoters also contain two elements; the upstream core element (UCE) bound by a UBF dimer and the central element (CE) bound by selectivity factor 1 (SL1) complex (Knutson & Hahn, ; Engel et al, ; Sadian et al, ; Pilsl & Engel, ; Baudin et al, ; Girbig et al, 2022). (D) Structural model of yeast RNAPI in complex with Rrn3, the CF (PDB ID: 7OBA), and UAF complexes, bound to Tbp and promoter DNA (PDB ID: 7Z0O). RNAPI subunits are in shadows of gray, factors are color‐coded, and DNA is shown in light blue.

Figure 3. Pre‐rRNA maturation in yeast and human cells

Simplified processing pathway of the 35S pre‐rRNA in yeast and 47S pre‐rRNA in human cells, indicating processing sites of endo‐ and exonucleases in orange and blue, respectively. Several alternative processing pathways exist, which are reviewed elsewhere (Tomecki et al, ; Aubert et al, 2018).

Figure 4. Structures of the yeast and human SSU processome

Cryo‐EM structures of the yeast (PDB ID: 6ZQB, 7AJT, and 7AJU) and human (PDB ID: 7MQ8, 7MQ9, 7MQA) SSU processome particles in the pre‐A1, pre‐A1* and post‐A1 cleavage states, showing a comparison of their overall architecture. The conserved RNA components and subcomplexes are color‐coded. The pre‐rRNA (white) and individual sub‐complexes such as UTP‐A (pink), UTP‐B (light blue), UTP‐C (violet), Emg1 (green), ANN (light green) complex, and additional RBFs, are shown as surfaces. KRR1/Krr1, C1orf131/Faf1, and the NAT10/Kre33 module are indicated as pre‐A1‐specific RBFs (sand). They are released after A1 cleavage. The exosome complex (yellow) is bound to the pre‐A1* yeast particle and it is associated only with the human post‐A1 structure. In the post‐A1 structures, DHX37/Dhr1 and DIM1/Dim1 are displayed in orange.

Figure 5. Overview of late maturation steps of the small ribosomal subunit

Front and back views of human pre‐40S particles at different stages of cytoplasmic maturation as derived from cryo‐EM analyses (PDB ID: 6G4S, 6G18, 6ZXE, 6ZXF, 6ZXH). Factors involved in these steps are color‐coded and pre‐rRNA is shown in white. After RRP12 release from the state B particle, RACK1 occupies its place and the pre‐rRNA is rearranged for head formation. PNO1 directly interacts with NOB1 and keeps it in an inactive state (from state B to F2). Association of EIF1AD, concomitant with rearrangements of RIOK1, triggers PNO1 dissociation, RPS26/eS26 incorporation, and final pre‐rRNA processing. LRRC47 association prevents 60S joining until the mature decoding region is formed.

Figure 6. Overview of key nuclear maturation events of the large ribosomal subunit in yeast

Cryo‐EM structures of yeast pre‐60S particles at different stages of maturation (PDB ID: 6ELZ, 3JCT, 6YLG, 6N8J). RBFs involved in these steps are color‐coded, as well as the pre‐rRNA (white), L1 stalk (red), and 5S rRNA (orange). The nucleolar pre‐60S in state E shows a displacement of the L1 stalk from its position in the mature subunit. The successive nucleoplasmic Arx/Nog2 particle is a result of a stepwise release and binding of the indicated RBFs and the 5S RNP. The Rix‐Rea1 remodeling machinery initiates the formation of the central protuberance (CP) and rotation of the 5S RNP to its mature conformation, visible in the successive late nuclear (LN) particle.

Figure 7. Cytoplasmic maturation of 60S subunit in yeast

Cryo‐EM structures of pre‐60S particles illustrating snapshots of cytoplasmic maturation events (PDB ID: 6N8L, 7Z34, 6N8M, 6N8N, 6RZZ, 6QTZ). RBFs are color‐coded, as well as the pre‐rRNA (white), L1 stalk (red), and 5S rRNA (orange). After nuclear export, the ATPase Drg1 dissociates Rpl24 from early cytoplasmic pre‐60S particles. The release of multiple factors allows the association of the GTPase Lsg1, which in turn dissociates Nmd3 upon the incorporation of Rpl10/uL16. Following Reh1 dissociation, the GTPase Efl1 together with Sdo1 triggers the release of Tif6, resulting in mature 60S subunits.