Visualizing the Assembly Pathway of Nucleolar Pre-60S Ribosomes

Abstract

SummaryEukaryotic 60S ribosomal subunits are comprised of three rRNAs and ∼50 ribosomal proteins. The initial steps of their formation take place in the nucleolus, but, owing to a lack of structural information, this process is poorly understood. Using cryo-EM, we solved structures of early 60S biogenesis intermediates at 3.3 Å to 4.5 Å resolution, thereby providing insights into their sequential folding and assembly pathway. Besides revealing distinct immature rRNA conformations, we map 25 assembly factors in six different assembly states. Notably, the Nsa1-Rrp1-Rpf1-Mak16 module stabilizes the solvent side of the 60S subunit, and the Erb1-Ytm1-Nop7 complex organizes and connects through Erb1’s meandering N-terminal extension, eight assembly factors, three ribosomal proteins, and three 25S rRNA domains. Our structural snapshots reveal the order of integration and compaction of the six major 60S domains within early nucleolar 60S particles developing stepwise from the solvent side around the exit tunnel to the central protuberance.

Modular assembly of the nucleolar pre-60S ribosomal subunit

“…State 1 is highly similar to state A (EMD-3888) 16 . States 2 and 2A correspond closely to state B (EMD-3889) 16 , but state B lacks the Ssf1–Rrp15–Rrp14 module and Mak11. Built assembly factors that become ordered or leave in subsequent particles are indicated with arrows.…”

“…The structural data presented in this paper (states 1, 2, and 3) are complemented by recent data on pre-60S assembly: states C (EMD-3893) and E (EMD-3891) 16 . State 1 is highly similar to state A (EMD-3888) 16 .…”

“…Built assembly factors that become ordered or leave in subsequent particles are indicated with arrows. Two possible pathways are shown that result in the final incorporation of the DEAD-box helicase Spb4 (previously unidentified 16 ).…”

“… a , b , Comparative views of state 2 ( a ) and state E (PDB 6ELZ) 16 ( b ), highlighting that the binding of the Brx1 CTD to Rrp1 prevents premature incorporation of Rpl28. c , d , Comparative views of state E ( c ) and the Nog2 particle (PDB 3JCT) ( d ), highlighting that the presence of Spb1 prevents the binding of Bud20.…”

“…While this manuscript was under review, complementary structural data on the nucleolar assembly of the large ribosomal subunit was published 16 . Differences in purification conditions resulted in the isolation of distinct states, which may represent assembly stages or breakdown products.…”