Analysis of the In Vivo Assembly Pathway of Eukaryotic 40S Ribosomal Proteins

Abstract

In eukaryotes, in vivo formation of the two ribosomal subunits from four ribosomal RNAs (rRNAs) and approximately 80 ribosomal proteins (r-proteins) involves more than 150 nonribosomal proteins and around 100 small noncoding RNAs. It is temporally and spatially organized within different cellular compartments: the nucleolus, the nucleoplasm, and the cytoplasm. Here, we present a way to analyze how eukaryotic r-proteins of the small ribosomal subunit (SSU) assemble in vivo with rRNA. Our results show that key aspects of the assembly of eukaryotic r-proteins into distinct structural parts of the SSU are similar to the in vitro assembly pathway of their prokaryotic counterparts. We observe that the establishment of a stable assembly intermediate of the eukaryotic SSU body, but not of the SSU head, is closely linked to early rRNA processing events. The formation of assembly intermediates of the head controls efficient nuclear export of the SSU and cytoplasmic pre-rRNA maturation steps.

A hierarchical model for assembly of eukaryotic 60S ribosomal subunit domains

“…These scattered interactions of members of early-, intermediate-, and late-acting RPLs with rRNA do not immediately suggest a 59-39 directionality of assembly. However, some contacts might be preferentially established earlier, while some interactions occur later, explaining the progressive stabilization of RPL binding (Ferreira-Cerca et al 2007;Sahasranaman et al 2011;Ohmayer et al 2013). Interestingly, with the exception of L3 in domain VI, early-acting RPLs have more interactions with the 59 half of 25S rRNA in domains I and II, while late-acting RPLs have more interactions with the 39 half of 25S rRNA in domains IV and V (Supplemental Table 1).…”

“…Furthermore, we also know that most RPLs are found in the earliest pre-60S particles, yet their association with preribosomes is thought to become more stable as assembly proceeds, by creating more contacts with pre-rRNA (Ferreira-Cerca et al 2007;Adilakshmi et al 2008;Ohmayer et al 2013). Thus, analyzing the RPL content of preribosomes depleted of early-, middle-, and late-acting RPLs enabled us to arrive at a conclusion that assembly of the 60S ribosomal subunit is hierarchical.…”

“…Knowing these final endpoints of RP localization provides a strategic platform to investigate the connection between spatial and temporal aspects of ribosome biogenesis. Most RPs assemble with preribosomes early in the biogenesis pathway (Kruiswijk et al 1978;Ferreira-Cerca et al 2007;Babiano and de la Cruz 2010;Babiano et al 2012;Gamalinda et al 2013;Ohmayer et al 2013). As the nascent subunits mature, the association of RPs with pre-rRNA is strengthened by dynamic rearrangement of initial protein-RNA encounter complexes (Ferreira-Cerca et al 2007;Adilakshmi et al 2008;Ohmayer et al 2013).…”

“…However, phenotypic analyses of these mutants were primarily limited to assaying effects on pre-rRNA processing and nucleocytoplasmic export of preribosomes. Nevertheless, investigations of small subunit RPs (RPSs) suggested that the two discernable structural domains of the 40S subunit are sequentially assembled (Ferreira-Cerca et al 2007; O' Donohue et al 2010). The ''body'' substructure (containing the 59 and central domains of 18S rRNA) is formed first, followed by the ''head'' substructure (containing the 39 major domain of 18S rRNA).…”

“…To our knowledge, no global picture of the hierarchy of 60S subunit assembly from the beginning to the end has been described thus far. We reasoned that the correlation between the location and function of RPLs might reflect sequential construction of stable pre-60S assembly intermediates, analogous to the biogenesis of small ribosomal subunits (Held et al 1974;Ferreira-Cerca et al 2007;Mulder et al 2010;O'Donohue et al 2010).…”