Ribosomal protein eL39 is important for maturation of the nascent polypeptide exit tunnel and proper protein folding during translation

Abstract

During translation, nascent polypeptide chains travel from the peptidyl transferase center through the nascent polypeptide exit tunnel (NPET) to emerge from 60S subunits. The NPET includes portions of five of the six 25S/5.8S rRNA domains and ribosomal proteins uL4, uL22, and eL39. Internal loops of uL4 and uL22 form the constriction sites of the NPET and are important for both assembly and function of ribosomes. Here, we investigated the roles of eL39 in tunnel construction, 60S biogenesis, and protein synthesis. We show that eL39 is important for proper protein folding during translation. Consistent with a delay in processing of 27S and 7S pre-rRNAs, eL39 functions in pre-60S assembly during middle nucleolar stages. Our biochemical assays suggest the presence of eL39 in particles at these stages, although it is not visualized in them by cryo-electron microscopy. This indicates that eL39 takes part in assembly even when it is not fully accommodated into the body of pre-60S particles. eL39 is also important for later steps of assembly, rotation of the 5S ribonucleoprotein complex, likely through long range rRNA interactions. Finally, our data strongly suggest the presence of alternative pathways of ribosome assembly, previously observed in the biogenesis of bacterial ribosomal subunits.

Ribosomal protein RPL39L is an efficiency factor in the cotranslational folding of proteins with alpha helical domains

“…Thus, to determine how RPL39/RPL39L impact the NPET structure we overexpressed the mouse RPL39 and RPL39L in RPL39 KO yeast strains. The RPL39 KO yeast lines are sensitive to cold, paromomycin and AZT, phenotypes that have been described before 21,22 . These phenotypes are rescued by both mouse RPL39 and mouse RPL39L, which indicates that RPL39L can perform the basic functions of RPL39.…”

“…6B and S5). This indicates that both mRPL39 and mRPL39L occupy the place of yRPL39 in the NPET and have a similar capacity to ensure the translation accuracy 21 and promote co-translational folding 22 in yeast. The results also suggest RPL39L shares the ancestral function of RPL39, though it may have acquired additional functions following its emergence in mammalian species.…”

“…Increased rate of translation error reduces the growth of RPL39 KO (rpl39Δ) yeast strains at cold temperatures and sensitizes the cells to translation-interfering drugs like paromomycin 21 and azetidine-2-carboxylic acid (AZT) 22 . We replicated these phenotypes in two distinct yeast RPL39 KO clones and further showed that they are rescued by the introduction of yRPL39 (rpl39Δ-ScRPL39 clones), mouse RPL39 (rpl39Δ-MmRPL39 clones) and mouse RPL39L (rpl39Δ-MmRPL39L clones) (Fig.…”

Remodelled Ribosomes Synthesise a Specific Proteome in Proliferating Plant Tissue during Cold

“…In Arabidopsis, cold heterogeneous translating ribosomes exhibit rProtein substoichiometry around the polypeptide exit tunnel (PET), with many of the rProteins being relatively removed during cold ( Martinez-Seidel et al, 2021a ). Here, we report that barley ribosomes also exhibit, on average, subtractive heterogeneity ( Briggs and Dinman, 2017 ) in the cold-ribosomal population around protein uL4 and uL29, the former being essential for PET assembly during ribosome biogenesis ( Lawrence et al, 2016 ; Gamalinda and Woolford, 2014 ; Stelter et al, 2015 ; Pillet et al, 2015 ), as its internal loops form the constriction sites of the nascent PET ( Micic et al, 2022 ) along uL29. The PET and its assembly are likely to be particularly critical during cold acclimation because both yeast ( Hung and Johnson, 2006 ) and plants ( Schmidt et al, 2013 ) have a 60S maturation factor that when knocked out, leads to cold sensitivity, namely Rei-1 in yeast and its homolog REIL in plants.…”

“…The CCT binds and promotes protein folding of newly synthesised polypeptides ( Lopez et al, 2015 ; Yébenes et al, 2011 ) or promotes their aggregation and thus protein degradation to maintain proteostasis ( Lopez et al, 2015 ; Hartl et al, 2011 ; Spiess et al, 2004 ). We have shown that during cold in plants, ribosome remodeling, i.e., subtractive heterogeneity, occurs in the proteome surrounding the PET, and the signature of a defective tunnel is protein misfolding ( Micic et al, 2022 ; Peterson et al, 2010 ; Wruck et al, 2021 ). Thus, it is conceivable that an altered tunnel structure leads to increased protein misfolding and this forces the plant cell to produce more CCT complexes to properly fold or aggregate misfolded proteins in order to fix or degrade them, respectively.…”