We describe a novel approach to separate two ribosome populations from the same cells and use this method, and RNA-seq, to identify the mRNAs bound to S. cerevisiae ribosomes with and without Rps26, a protein linked to the pathogenesis of Diamond Blackfan Anemia (DBA). These analyses reveal that Rps26 contributes to mRNA-specific translation by recognition of the Kozak sequence in well-translated mRNAs, and that Rps26-deficient ribosomes preferentially translate mRNA from select stress response pathways. Surprisingly, exposure of yeast to these stresses leads to the formation of Rps26-deficient ribosomes and to the increased translation of their target mRNAs. These results describe a novel paradigm, the production of specialized ribosomes, which play physiological roles in augmenting the well-characterized transcriptional stress response with a heretofore unknown translational response, thereby creating a feed forward loop in gene-expression. Moreover, the simultaneous gain-of-function and loss-of-function phenotypes from Rps26-deficient ribosomes can explain the pathogenesis of DBA.

During translation initiation, 40S ribosomes scan the mRNA until they encounter the start codon, where conformational changes produce a translation-competent 80S complex. Destabilizing the scanning complex results in misinitiation at non-AUG codons, demonstrating its importance for fidelity. Here, we use a combination of biochemical and genetic analyses to demonstrate that the ability of the nascent subunit to adopt the scanning complex is tested during assembly via structural mimicry. Specifically, formation of the 80S-like assembly intermediate, which structurally resembles scanning complexes, requires the correct folding of two rRNA elements in the subunit head and the proper positioning of the universally conserved head proteins Rps3, Rps15, Rps20, and Rps29. rRNA misfolding impairs the formation of 80S-like ribosomes, and bypass of individual checkpoints using cancer-associated mutations produces ribosomes defective in accurate start-site selection. Thus, the formation of 80S-like assembly intermediates is a quality control step that ensures scanning competence of the nascent subunit.

Cell growth relies on Hrr25/CK1δ-directed phosphorylation of Ltv1, which allows its release from nascent 40S ribosomal subunits and promotes subunit maturation.

SUMMARY Late stage 40S ribosome assembly is a highly regulated, dynamic process that occurs in the cytoplasm, alongside the full translation machinery. Seven assembly factors (AFs) regulate and facilitate maturation, but the mechanisms through which they work remain undetermined. Here, we present a series of structures of the immature small subunit (pre-40S) determined by three-dimensional (3D) cryogenic electron microscopy with 3D sorting to assess the molecule’s heterogeneity. These structures demonstrate extensive structural heterogeneity of interface AFs that likely regulates subunit joining during 40S maturation. We also present structural models for the beak and the platform, two regions where the low resolution of previous studies did not allow for localization of AFs, and the rRNA, respectively. These models are supported by biochemical analyses using point variants and suggest that maturation of the 18S 3’-end is regulated by dissociation of the AF Dim1 from the subunit interface, consistent with previous biochemical analyses.

The correct assembly of ribosomes from ribosomal RNAs (rRNAs) and ribosomal proteins (RPs) is critical, as indicated by the diseases caused by RP haploinsufficiency and loss of RP stoichiometry in cancer cells. Nevertheless, how assembly of each RP is ensured remains poorly understood. We use yeast genetics, biochemistry, and structure probing to show that the assembly factor Ltv1 facilitates the incorporation of Rps3, Rps10, and Asc1/RACK1 into the small ribosomal subunit head. Ribosomes from Ltv1-deficient yeast have substoichiometric amounts of Rps10 and Asc1 and show defects in translational fidelity and ribosome-mediated RNA quality control. These defects provide a growth advantage under some conditions but sensitize the cells to oxidative stress. Intriguingly, relative to glioma cell lines, breast cancer cells have reduced levels of LTV1 and produce ribosomes lacking RPS3, RPS10, and RACK1. These data describe a mechanism to ensure RP assembly and demonstrate how cancer cells circumvent this mechanism to generate diverse ribosome populations that can promote survival under stress.