Structural insights into assembly of the ribosomal nascent polypeptide exit tunnel

Abstract

The nascent polypeptide exit tunnel (NPET) is a major functional center of 60S ribosomal subunits. However, little is known about how the NPET is constructed during ribosome assembly. We utilized molecular genetics, biochemistry, and cryo-electron microscopy (cryo-EM) to investigate the functions of two NPET-associated proteins, ribosomal protein uL4 and assembly factor Nog1, in NPET assembly. Structures of mutant pre-ribosomes lacking the tunnel domain of uL4 reveal a misassembled NPET, including an aberrantly flexible ribosomal RNA helix 74, resulting in at least three different blocks in 60S assembly. Structures of pre-ribosomes lacking the C-terminal extension of Nog1 demonstrate that this extension scaffolds the tunnel domain of uL4 in the NPET to help maintain stability in the core of pre-60S subunits. Our data reveal that uL4 and Nog1 work together in the maturation of ribosomal RNA helix 74, which is required to ensure proper construction of the NPET and 60S ribosomal subunits.

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

“…As expected, shutting down the GAL1/10 promoter in glucose containing medium prevented growth of the three conditional r-protein expression mutants, while all four strains could be cultivated in galactose containing medium ( S2 Appendix ) [ 21 , 38 ]. Consistent with previous studies, some growth delay was observed in galactose containing medium in the strains expressing the Nog1-TAP fusion protein ( S2 Appendix , compare the colony size of the untagged with the one of tagged strains in YPG) [ 34 , 39 ]. LSU particles associated with TAP tagged Nog1 were affinity purified after four hours shift to glucose containing medium.…”

“…Indeed, corresponding densities could not be detected in Nog1-TAP associated LSU populations. The observed effects on growth rate by TAP tagging of Nog1 (see above, S2 Appendix ) and previous LSU folding analyses of particles from Nog1 mutant strains [ 34 ] thus suggest that progression of downstream nuclear LSU maturation steps was delayed in this situation to some extent.…”

“…Consistently, only minor effects on the general rRNA fold were observed by RNA structure probing in yeast LSU’s devoid of the low connectivity non-essential single domain binder rpL26 (uL24 according to the r-protein nomenclature published in [ 30 ]) [ 31 , 32 ]. By contrast, previous cryo-EM studies revealed significant impact on the SSU and LSU fold by truncations of the essential r-proteins rpS20 (uS10) and rpL4 (uL4), respectively [ 33 , 34 ].…”

The Arabidopsis 2′-O-Ribose-Methylation and Pseudouridylation Landscape of rRNA in Comparison to Human and Yeast

“…Nevertheless, Arabidopsis 60S subunit seems to be closer related to the human 60S regarding the high density of modifications. In human ribosomes, helix 74 is important for the accurate structure of the nascent polypeptide exit tunnel (NPET) (Wilson et al, 2020) and helix 93 is a contact site for hydroxylated uL2, which induces structural rearrangements in the PTC of the mature ribosomes (Yanshina et al, 2015). The same could hold true for plants as well.…”

Spatially Enriched Paralog Rearrangements Argue Functionally Diverse Ribosomes Arise during Cold Acclimation in Arabidopsis

“…PET assembly occurs much earlier in the nucleolus when pre-ribosomal particles are slowly processed and crafted [46]. Thus, disrupting PET assembly in the early biogenesis steps might cause a debilitated tunnel structure [47] and cause the need for a dedicated proof reading step catalyzed by REIL later on in the cytoplasm. After testing this example, we propose that plant ribosome heterogeneity is non-random and is likely controlled by specialized mechanisms during biogenesis that preferentially assemble groups of RPs or RP paralogs in a concerted process.…”