The young and the restless: Isolating the dynamic mammalian preribosomes

Abstract

Despite the advances in understanding the assembly of yeast preribosomes using affinity purification and structural analysis, studies on mammalian ribosome biogenesis have lagged behind. Using an unbiased method to purify native mammalian preribosomal complexes from the nucleus, Abetov et al. now uncover two types of premature ribonucleoprotein complexes that are nutrient- and mTOR-dependent. This purification scheme, combined with genome-editing techniques, could be exploited to untangle the complexities underlying human ribosome biogenesis and ribosomopathies.

Introduction

Ribosomes are macromolecular machineries that function to synthesize proteins. As the quantity and quality of ribosomes are crucial for normal cell growth, their biogenesis and assembly occur in a highly dynamic, regulated, and ordered manner (1, 2). Ribosome formation originates in the nucleolus. A massive preribosomal complex from this nuclear compartment was termed the 90S particle in the 1970s based on its sedimentation (3, 4). Studies over the years have revealed that this complex contains the nascent pre-rRNA that associates with assembly or biogenesis factors (AFs),² ribosomal proteins, and small nucleolar RNAs. The 90S particle is subjected to processing and maturation steps that give rise to the 60S and 40S subunits, which translocate into the cytoplasm and coalesce as the 80S translation-competent ribosome. Efforts to elucidate preribosome assembly were accelerated by employing tandem affinity purification (TAP) of tagged ribosomal proteins in yeast (5). Combined with yeast genetics, proteomics, and structural analysis using cryo-EM, we now have a more comprehensive list of components and sequence of events involved in this process (reviewed in Refs. 1 and 2). However, it is not clear whether these insights in yeast directly translate to mammalian systems, and better strategies are needed to tackle the more elusive mammalian preribosomes. Abetov et al. now describe a finely tuned purification scheme designed to isolate the fleeting native mammalian preribosomal complexes (6), revealing new environmentally sensitive intermediates and changing our understanding of these immature forms.

Although it is expected that key aspects of ribosome biogenesis are conserved from yeast to humans due to broad conservation of numerous components, there remains a need to map this process in higher eukaryotes. First, methods such as large-scale computationally based screens of the mammalian proteome and more targeted approaches have revealed many conserved but also unique preribosomal components and biogenesis factors present in mammals (1, 7). Hence, a more unbiased approach using purification of native proteins could identify other nonconserved proteins that play roles in ribosome assembly and maturation. Second, whereas nutrients serve as a common regulator of ribosome biogenesis in lower and higher eukaryotes, multicellular organisms are subject to hormonal and other environmental cues to coordinate whole-body growth. Thus, these multiple inputs likely confer more complex regulatory mechanisms to mammalian ribosome biogenesis. Third, ribosomes have apparently tissue-specific functions, as revealed by human ribosomopathies (8), meaning that an improved understanding of shared and unique processes could inform therapeutic strategies for these diseases. The challenge in obtaining a more nonbiased picture of the components of preribosomal complexes in mammals is that purification methods must be harsh enough to exclude the mature cytoplasmic ribosomal complexes while being mild enough to preserve the dynamic intermediates in the nucleolus. The new approach developed by Abetov et al. relies on a meticulous isolation of intact nuclei devoid of cytoplasmic mature ribosomes by combining lysis of nuclei with mild sonication to obtain soluble nuclear lysates. Sucrose gradient fractionation of the nuclear fractions resolved two types of ribonucleoprotein complexes in various human cancer, primary, and mouse cells. A sharp peak with an 85S particle was termed the composed preribosome (CPRib), whereas multiple smaller peaks of 105S-125S were named the intermediate preribosomes (IPRibs) (Fig. 1). The authors speculate that the IPRib is a transitional complex, where the 125S complex represents the main complex that undergoes disassembly (regulated or purification-induced) to more intermediate 115S and 105S species. Mass spectrometry analysis of the CPRib and IPRib fractions identified more than 70 of 80 known ribosomal proteins. More than 30 ribosomal AFs and processing factors that play roles in rRNA modifications were also identified, indicating that rRNAs from the mammalian preribosomes undergo acetylation, methylation, or pseudouridinylation. Pulse-chase labeling experiments revealed that the IPRib precedes the formation of CPRib. Indeed, CPRib contained only mature rRNA and lacked rRNA modification factors. A ribosomal biogenesis factor, Ly1 antibody-reactive (LYAR), previously identified as a cell growth–regulating nucleolar protein (9), was selectively enriched in the CPRib. Although the precise nature of CPRib remains to be characterized, its size is reminiscent of the 90S preribosome described in earlier studies (3).

Figure 1.

Purification of mammalian preribosome complexes. Native preribosomal complexes purified from mammalian nuclear extracts reveal two main types of ribonucleoprotein complexes, the IPRib (105S-125S) and the CPRib (85S). The preribosome complex (consisting of ribosomal proteins and rRNAs) is shown associated with rRNA modification factors (MF) and AFs. The 30S and 32S rRNA precursors were observed less in the CPRib complex. The ribosomal AF, LYAR, is found only on the CPRib.

The authors next investigated whether the new intermediates were sensitive to environmental cues as in other species. Both IPRib and CPRib peaks were also sensitive to amino acid starvation or inhibition of mTOR, suggesting that the quantity and/or assembly of preribosomes is also dependent on nutrients and mTOR signaling. Interestingly, whereas mTOR inhibition increased the mature cytosolic 80S monosome peak due to polysome disintegration (an effect of decreased translation), it dampened the nuclear 85S peak of the CPRib, further indicating that the CPRib is distinct from the mature cytoplasmic ribosomes. Together, the findings reveal progressive maturation of preribosomes wherein a larger, more dynamic IPRib complex containing the premature rRNAs undergoes rRNA modification followed by formation of the smaller, more stable CPRib complex with mature rRNAs harboring distinct ribogenesis factors.

The purification scheme described by Abetov et al. provides intriguing evidence for new intermediates in ribosomal assembly. However, as the authors note, further refinement is needed to capture more fragile preribosomal complexes, such as those formed during earlier stages with nascent rRNAs as well as more mature complexes en route to the cytoplasm. Growth conditions, such as nutrient levels, and pathological conditions may also affect preribosome integrity, thus necessitating improvement of purification strategies. Given the recent amenability of mammalian cells to genomic editing, it would be feasible to conduct epitope tagging or mutation of key preribosomal components, which could allow isolation of the more unstable complexes. Improved purification methods to recover different intermediate complexes will additionally facilitate structural determination of these complexes and identification of individual components by proteomics. Characterizing component functions and modifications as well as regulatory factors at the different assembly and maturation stages would significantly advance our knowledge of mammalian ribosome biogenesis. Because ribosome biogenesis is intimately linked to nutrients and mTOR signaling (10), it would be important to delineate how metabolite levels impact modifications of pre-rRNAs, ribosomal proteins, and biogenesis factors. Given the tissue-specific defects associated with ribosomopathies, which are inherited disorders that often have predisposition to cancer (8), improved purification methods also promise to facilitate understanding of the mechanisms underlying these pathologies. Overall, the study provides new impetus to explore the native state and fascinating drama behind the assembly of these young and restless preribosomes.