Coupling of 5S RNP rotation with maturation of functional centers during large ribosomal subunit assembly

Abstract

The protein composition and structure of assembling 60S ribosomal subunits undergo numerous changes as pre-ribosomes transition from the nucleolus to the nucleoplasm. This includes stable anchoring of the Rpf2 subcomplex containing 5S rRNA, rpL5, rpL11, Rpf2 and Rrs1, which initially docks onto the flexible domain V of rRNA at earlier stages of assembly. In this work, we tested the function of the C-terminal domain (CTD) of Rpf2 during these anchoring steps, by truncating this extension and assaying effects on middle stages of subunit maturation. The rpf2Δ255-344 mutation affects proper folding of rRNA helices H68-70 during anchoring of the Rpf2 subcomplex. In addition, several assembly factors (AFs) are absent from pre-ribosomes or in altered conformations. Consequently, major remodeling events fail to occur: rotation of the 5S RNP, maturation of the peptidyl transferase center (PTC) and the nascent polypeptide exit tunnel (NPET), and export of assembling subunits to the cytoplasm.

The final step of 40S ribosomal subunit maturation is controlled by a dual key lock

“…The function of RIO1 in the release of DIM2 and NOB1 echoes the roles that other NTPases play in pre-ribosomal particle remodeling along the maturation pathway. Their nucleotide-hydrolyzing activity was shown to power the release of other RBFs, as well as their own release, contributing to the unidirectionality of the maturation ( Hedges et al, 2005 ; Kargas et al, 2019 ; Knüppel et al, 2018 ; Micic et al, 2020 ; Mitterer et al, 2019 ; Weis et al, 2015 ; Zemp et al, 2009 ). NTPases might also have additional roles than conformational switches and probe the correct conformation of functional sites.…”

Puf6 primes 60S pre-ribosome nuclear export at low temperature

“…On the 60S subunit, the H68 TL GAAA motif (rRNA domain IV) engages in an unusual tertiary contact with a kissing loop (KL) formed between H22 (rRNA domain I) and H88 (rRNA domain V) bringing together distant secondary structure elements. Cryo-EM studies suggest this contact contributes to the compaction of the flexible subunit interface (rRNA domains III–V) with the rigid solvent-exposed side (rRNA domains I, II, and VI) on the 60S pre-ribosome 8 , 54 . Using FRET reporters, we provide evidence for the reconstitution of the KL, and docking of the TL GAAA onto the KL in vitro.…”

Analysis of subunit folding contribution of three yeast large ribosomal subunit proteins required for stabilisation and processing of intermediate nuclear rRNA precursors

“…On the other hand, the non-positioned flexible (sub-)domains in the mis-assembled particles should substantially increase at a multitude of other sites the accessibility for general nuclear RNA degradation machineries. That effect is likely still increased in the subpopulations of misassembled particles for which numerous factors were not anymore detectable (Nog1TAP_L2-B, Nog1TAP_L25-A, Nog1TAP-L34B, see S1_Appendix, compare also with mutant LSU precursor populations depleted of factors in [46]). These populations might represent newly made subunits with limited access to these factors, possibly through a delay of their release from accumulating misassembled particles.…”