Ribosomes are complex macromolecular machines that catalyze the fundamental process of translation. Assembly of ribosomes is a complicated multi-step process that involves transcription, folding, modification and processing of pre-rRNAs, as well as the concomitant assembly of ribosomal proteins (r-proteins) (1–4
). In eukaryotes, assembly of both 40S and 60S ribosomal subunits begins in the nucleolus with the transcription of pre-35S and pre-5S rRNA precursors by RNA polymerase I (Pol I) and RNA polymerase III (Pol III), respectively (A). Assembly proceeds through a dynamic flux of protein–protein, protein–rRNA and rRNA–rRNA interactions toward final maturation in the cytoplasm (1
). More than 200 trans-
acting assembly factors facilitate these remodeling events that trigger the successive nucleolytic removal of transcribed spacer sequences and the hierarchical addition of r-proteins into nascent subunits (6
) (A and B).
Figure 1. Ribosome assembly pathway in Saccharomyces cerevisiae. (A) Pre-rRNA processing pathway indicating exonucleolytic trimming and endonucleolytic reactions. Note that cotranscriptional processing at the A3 site also occurs (5) (not shown). (B) Maturation (more ...)
Pioneering in vitro
reconstitution studies of bacterial 30S subunits established the hierarchical succession of small subunit r-protein assembly with 16S rRNA (11–13
). rRNA structural probing showed that binding of an initial subset of r-proteins causes structural rearrangements in 16S rRNA. These rearrangements form binding sites that allow subsequent association of another set of r-proteins and stabilize rRNA configurations (14–19
). What emerged from these studies is the principle that ribosome synthesis is highly cooperative and occurs in parallel intermediate pathways (20–22
). However, ribosome assembly in vivo
is much more complex, involving (i) coordination of transcription with assembly, (ii) removal of spacer sequences, (iii) export of pre-ribosomes from the nucleus to the cytoplasm and (iv) controlled action of assembly factors.
The discovery of more than 200 ribosome biogenesis factors has led to extensive investigations on how they facilitate processing of pre-rRNAs and structural rearrangements to steer maturation of assembling pre-ribosomal complexes. Although the field has learned a great deal from studying them, trans
-acting factors are only one part of the grand design that is ribosome biogenesis. Ribosomes are multimolecular complexes wherein r-proteins embellish a core of rRNA; hence, r-proteins likely serve as molecular scaffolds necessary not only for ribosome function but also assembly. Comprehensive studies of how r-proteins facilitate ribosomal subunit biogenesis are greatly underrepresented. Recent work has identified for which steps in pre-rRNA processing different r-proteins are required, but it is not clear exactly how they participate in these steps (23–35
). It is presumed that they do not function directly in pre-rRNA processing; r-proteins are thought to have structural rather than enzymatic functions. The roles of r-proteins in establishing pre-ribosomal particles and recruitment of assembly factors and other r-proteins to enable subsequent maturation are even less well studied. Thus far, examination of how r-proteins affect the composition of pre-ribosomes to propel pre-rRNA maturation has only been reported for a handful of r-proteins from both r-subunits (36–39
). Hence, more studies of this kind are needed to fully comprehend the assembly process.
Initial systematic investigation of 60S subunit assembly categorized r-proteins based on their requirement for early, intermediate, and late pre-rRNA maturation steps [(30
), unpublished data]. Following up on Pöll et al.
), we recently characterized in more detail a subset of r-proteins that function early in ribosome assembly, during the exonucleolytic trimming of ITS1 sequences in 27SA3
). In this work, we have focused on r-proteins L17, L35 and L37, which had been implicated in removal of ITS2, and are adjacent to each other in the structure of mature 60S subunits (40–42
) (). L17 and L35 are eukaryotic homologs of bacterial L22 and L29, respectively, whereas L37 does not have a bacterial homolog. Together, these three r-proteins interact with all six secondary structure domains of 5.8S/25S rRNA, having multiple contact sites with each domain (40–42
). They may therefore be important in promoting and stabilizing long-range inter-domain interactions that ultimately influence the architecture of assembling ribosomes. Thus, these r-proteins may help create local or global structural prerequisites for pre-rRNA processing and association of proteins. They are also of interest because, together with the nonessential r-protein L26 (43
), L17, L35 and L37 are missing from pre-ribosomes in mutants unable to process early pre-rRNA intermediates (39
). This is attributed to misfolding of rRNA domains to which they bind, as well as reflecting the coupling of pre-rRNA processing with r-protein assembly. In addition, since these r-proteins lie around the rim of the nascent polypeptide exit tunnel, one might be able to relate the construction of this rRNP neighborhood to intermediate pre-rRNA maturation steps.
Figure 2. Localization of L17, L35, and L37 around the polypeptide exit tunnet (PET). (Top left) Location of L17, L35, and L37 in the structure of mature 60S ribosomal subunits (PDB: 3U5D and 3U5E) (41). CP denotes the central protuberance. (Top right) The 60S (more ...)
We wanted to take a comprehensive look at the function of these three r-proteins and how they affect ribosome assembly. We tested their role in recruiting assembly factors to begin to understand how their absence leads to a distinct pre-rRNA processing defect. We have compared the phenotypes upon depleting each of these r-proteins and found that they exhibit very similar defects in biogenesis of 60S subunits even though they are not interdependent for association with pre-ribosomal particles (pre-rRNPs). Our data indicate that these r-proteins affect recruitment of a specific set of assembly factors necessary for 27SB pre-rRNA processing, and in their absence pre-rRNPs are gradually turned over.