Mechanism of 5S RNP recruitment and helicase‐surveilled rRNA maturation during pre‐60S biogenesis

Abstract

Ribosome biogenesis proceeds along a multifaceted pathway from the nucleolus to the cytoplasm that is extensively coupled to several quality control mechanisms. However, the mode by which 5S ribosomal RNA is incorporated into the developing pre‐60S ribosome, which in humans links ribosome biogenesis to cell proliferation by surveillance by factors such as p53–MDM2, is poorly understood. Here, we report nine nucleolar pre‐60S cryo‐EM structures from Chaetomium thermophilum, one of which clarifies the mechanism of 5S RNP incorporation into the early pre‐60S. Successive assembly states then represent how helicases Dbp10 and Spb4, and the Pumilio domain factor Puf6 act in series to surveil the gradual folding of the nearby 25S rRNA domain IV. Finally, the methyltransferase Spb1 methylates a universally conserved guanine nucleotide in the A‐loop of the peptidyl transferase center, thereby licensing further maturation. Our findings provide insight into the hierarchical action of helicases in safeguarding rRNA tertiary structure folding and coupling to surveillance mechanisms that culminate in local RNA modification.

Structural insights into coordinating 5S RNP rotation with ITS2 pre‐RNA processing during ribosome formation

“…Next, the split‐tag PT‐rsa4 E117D NOP7‐Flag strain was subjected to tandem affinity‐purification (Cheng et al , 2019), which showed a co‐enrichment of more than 20 conserved pre‐60S factors in the final Flag eluate, among several GTPases (Nog1, Nog2, Nug1), all members of the Rix1 complex (Rix1‐Ipi3‐Ipi1), Nop53 (a recruitment factor for the exosome co‐factor Mtr4 helicase), Utp30 (a Cic1/Nsa3 homolog), and the endonuclease Las1 with its co‐factor, the polynucleotide kinase Grc3 (Gasse et al , 2015) (Fig 1B). We did not detect Rlp7 (Ribosomal‐like protein 7) in our preparation, consistent with the previous finding that Rlp7 does not exist in C. thermophilum but is replaced by the homologous Rpl7, which is the authentic ribosomal protein L7/uL30 (Lau et al , 2023). Arx1, which is another characteristic pre‐60S factor in yeast, was also absent from the Flag eluate, although it exists in C. thermophilum , but also the human homolog Ebp1 was not found on nucleoplasmic pre‐60S particles (Liang et al , 2020; Bhaskar et al , 2021).…”

“…As ITS2 and the herein C 2 cleavage site are embedded into the pre‐60S foot structure, we first had a closer look into this hallmark structure within our ct pre‐60S particles. As previously found for nucleolar ct pre‐60S particles (Lau et al , 2023), the Rsa4 E117D derived nascent 60S also exhibits this typical foot structure, containing the Rpl7/uL30 instead of the classical but homologous yeast foot factor Rlp7 (see above), and the 90S factor Utp30 instead of the homologous yeast foot factor Cic1/Nsa3 (Fig EV2C and D). Like Cic1/Nsa3 in yeast, ct Utp30 interacts with the highly conserved assembly factor ct Nop15, and in addition has direct contact to ITS2 RNA helices H1 and H2 (Fig EV2C and E).…”

“…In the past, we were able to purify and structurally characterize several 90S pre-ribosomal particles as part of the 40S biogenesis pathway from C. thermophilum (Kornprobst et al, 2016;Cheng et al, 2017Cheng et al, , 2019, but failed to isolate pre-60S particles by affinitypurification of epitope-tagged assembly factors. Recently, however, we circumvented this problem by affinity-purification of a mutant pre-60S bait, ct Ytm1E88D, which provided us with a number of nucleolar pre-60S intermediates (Lau et al, 2023).…”

“…Noteworthy, after endo‐nucleolytic cleavage at site A 2 in the ITS1, the maturation pathways of the small and large subunit proceed independently from each other, while the pre‐ribosomes travel through the nucleolus and nucleoplasm (Udem & Warner, 1972; Woolford & Baserga, 2013; Bassler & Hurt, 2019; Klinge & Woolford, 2019). In addition to the 5.8S and the 25S rRNA, the 60S large subunit contains the 5S rRNA, which is transcribed separately by RNA polymerase III and incorporated as 5S RNP early during pre‐60S maturation together with the ribosomal proteins uL18 (rpL5), uL5 (rpL11), and assembly factors Rrs1 and Rpf2 (Zhang et al , 2007; Asano et al , 2015; Kharde et al , 2015; Madru et al , 2015; Castillo Duque de Estrada et al , 2023; Lau et al , 2023). After export through the nuclear pore complexes, a final quality control checkpoint probes the integrity and reliability of the nascent ribosomal subunits in the cytoplasm (Bassler & Hurt, 2019; Klinge & Woolford, 2019).…”

“…Early nucleolar assembly intermediates of the pre‐60S maturation pathway are characterized by the presence of 27SA2 rRNA and the trimmed form 27SB pre‐rRNA. These early intermediates could be elucidated by cryo‐EM and revealed the initial folding of rRNA domains, and association of first assembly factors and ribosomal proteins (Tsuno et al , 2000; Granneman et al , 2011; Kater et al , 2017; Sanghai et al , 2018; Zhou et al , 2019; Lau et al , 2023). Later, one decisive step in nucleolar pre‐60S biogenesis is the release of the Erb1‐Ytm1 heterodimer by the AAA‐ATPase Rea1, which triggers the dissociation of interdependent assembly factors, associated with Erb1's N‐terminus (Mitterer et al , 2023).…”