Ribosomes are sophisticated cellular organelles that catalyze translation of the mRNAs into proteins 1, 2. In all organisms, ribosomes are ribonucleoprotein complexes that consist of two ribosomal subunits (r-subunits), the large one (LSU) being about twice the size of the small one (SSU) [3]. Structural work has confirmed that, although the core of ribosomes is highly conserved in all kingdoms of life, eukaryotic cytoplasmic ribosomes are significantly larger and more complex than bacterial and archaeal ribosomes 4, 5.

The yeast Saccharomyces cerevisiae (hereafter, yeast) has been proven to be a wonderful model organism to study the structure, function and biogenesis of eukaryotic ribosomes (for reviews see, 4, 6, 7, 8, 9). In yeast, the SSU contains the 18S rRNA and 33 different ribosomal proteins (r-proteins), and it is organized into three distinct structural subdomains: body, platform and head. The LSU contains the 5S, 5.8S and 25S rRNA and 46 r-proteins, and it forms a fundamentally monolithic structure where the rRNAs are more intertwined forming six rRNA domains and harboring few notable structural features (see Figure S1), such as the central protuberance and the uL1- and P/uL10-stalks [4]. In yeast, as well as in all other studied eukaryotes, the synthesis of cytoplasmic ribosomes is a compartmentalized process that takes place largely in the nucleolus, although later steps occur in the nucleoplasm, where the pre-ribosomal particles gain export competence, and even in the cytoplasm, where the maturation of both r-subunits is finalized 6, 8, 10. In the yeast nucleolus, RNA polymerase I transcribes the mature 18S, 5.8S and 25S rDNAs as a single large precursor rRNA (pre-rRNA), whereas the pre-5S rRNA is independently synthesized by RNA polymerase III. These precursors are then processed by a complex and well characterized sequence of endo- and exonucleolytic reactions that release the mature rRNAs from the transcribed spacers (see Figure S2) 11, 12. Concomitantly, selected nucleotides within the pre-rRNAs undergo covalent modification 13, 14, 15. Neither pre-rRNA processing nor modification take place on “naked” pre-rRNAs; instead, many of the r-proteins and a plethora of protein trans-acting factors, also known as ribosome assembly factors, associate with the nascent transcripts to form the earliest pre-ribosomal particles that require further maturation 6, 10, 16. In vivo affinity-purification approaches have permitted the isolation and characterization of distinct pre-ribosomal particles that could be spatially and chronologically ordered along the ribosome assembly pathway. Assembly factors are recruited to these pre-ribosomal particles at specific cellular regions (nucleolus, nucleoplasm or cytoplasm), within delimited time windows and in a hierarchical manner; thus, enabling the sequential assembly of the r-proteins and the many conformational rearrangements that must dynamically occur to convert immature pre-ribosomal particles into translation-competent r-subunits 6, 8, 10, 16, 17, 18. Most recently, cryogenic electron microscopy (cryo-EM) has allowed the visualization of different pre-ribosomal particles on the road of ribosome maturation at atomic or near-atomic resolution, providing snapshots for the course of the maturation process (see Discussion) 6, 16, 17, 18.

It has also long been clear that r-proteins are essential components with respect to the structure and function of ribosomes 19, 20, 21, but they are also critical elements for the ribosome assembly process 7, 22, 23. In the late 70s, the Planta group deduced from smart in vivo experiments in yeast what had previously been suggested by in vitro reconstitution studies with bacterial ribosomes: distinct r-proteins, the so-called primary binding proteins, directly bind to the nascent pre-rRNAs, while secondary and tertiary binding proteins require the previous binding of one or more r-proteins [24]. It has been proposed that the initial interactions of r-proteins with nascent pre-rRNAs are weak but become reinforced as assembly proceeds 25, 26. Moreover, the contribution of most r-proteins to pre-rRNA processing and nucleo-cytoplasmic transport of pre-ribosomal particles has been well described by both individual and systematic works (e.g. 23, 27, 28, 29, 30, 31, 32, 33, 34). In the case of LSU proteins, their role during LSU assembly has been studied by functional analyses upon depletion of selected r-proteins (e.g. 28, 31, 35, 36, 37), deletion of non-essential genes (e.g. 38, 39, 40), truncation of internal loops and N- or C-terminal extensions of r-proteins (e.g. 41, 42, 43, 44) and point-mutations (e.g.29, 30, 45, 46, 47). The assembly properties of a broad number of LSU proteins has also been analyzed by assessing changes in the protein composition of pre-ribosomal intermediates through semi-quantitative proteomic analysis of affinity-purified pre-ribosomal particles upon depletion of the corresponding r-proteins or expression of different mutant variants of these r-proteins (e.g. 26, 36, 40, 44, 48, 49, 50). These latter studies have allowed us to understand the interdependences between selected r-proteins for their assembly on pre-ribosomal particles and their influence on the recruitment or release of distinct ribosome assembly factors during LSU formation. More recently, structural studies by cryo-EM analyses performed upon blockage of the assembly of specific r-proteins in yeast cells, either by depleting or expressing truncated variants of these r-proteins, have allowed a better understanding of how r-proteins contribute to the remodeling events occurring during the construction of the different LSU regions (e.g. 50, 51). Altogether, these studies helped to establish that r-proteins organize in partitioned clusters, whose members are normally neighbors or bind to the same rRNA domain in mature r-subunits. Loss-of-function of members of the same cluster leads to alike pre-rRNA processing phenotypes and has a similar effect on the progression of early, middle, late or cytoplasmic LSU assembly (see also Discussion) 7, 49, 52.

There are still a few LSU proteins that have not yet been investigated or have only been scarcely studied; thus, leaving a gap in our knowledge of the ribosome biogenesis process. In this study, we have undertaken the functional analysis of the otherwise poorly characterized yeast eL15 in ribosome synthesis. Yeast eL15, together with its neighboring r-proteins eL8 and eL36 are main interactors of 25S/5.8S rRNA domain I in mature 60S r-subunits (see later Figure 6); thus, examining eL15 is crucial to understand folding of this rRNA domain, which occurs during the earliest assembly stages of the large r-subunit in all studied prokaryotes and eukaryotes (e.g. 17, 53, 54 and references therein). In this work, we show that depletion of eL15 causes a shortage of LSUs, which is due to defective production and rapid turnover of early pre-60S ribosomal particles (r-particles). Consistently, pulse-chase experiments show impaired production of mature 25S and 5.8S rRNAs, analysis of pre-rRNA intermediates by northern blotting and primer extension reveals defective processing of 27S pre-rRNAs, most notably in the conversion of 27SA3 to 27SBS pre-rRNAs, and fluorescence microscopy suggests an impairment of nucleo-cytoplasmic export of pre-60S r-particles. We have also studied the impact of eL15 depletion on the composition of early pre-60S r-particles, which were affinity-purified using TAP-tagged Noc2 as bait. As a result, we show that eL15 is required for assembly of neighboring LSU r-proteins, including eL8, eL36 and eL13, and for the association of different ribosome assembly factors, which belong to the groups of so-called A3- and B-factors due to their roles during 27SA3 and 27SB pre-rRNA processing, respectively. We obtained similar results upon depletion of r-protein eL36, which, in addition, resemble those previously obtained upon depletion of r-protein eL8 36, 44. We conclude that eL8, eL15 and eL36 constitute a protein cluster on domain I of 25S/5.8S rRNA, which is crucial to fold this domain both by their direct binding to rRNA sequences within the domain and by their relevance for the recruitment of most of the A3-factors, which in turn facilitates 5.8S rRNA folding.