rRNA Maturation in Yeast Cells Depleted of Large Ribosomal Subunit Proteins

Abstract

The structural constituents of the large eukaryotic ribosomal subunit are 3 ribosomal RNAs, namely the 25S, 5.8S and 5S rRNA and about 46 ribosomal proteins (r-proteins). They assemble and mature in a highly dynamic process that involves more than 150 proteins and 70 small RNAs. Ribosome biogenesis starts in the nucleolus, continues in the nucleoplasm and is completed after nucleo-cytoplasmic translocation of the subunits in the cytoplasm. In this work we created 26 yeast strains, each of which conditionally expresses one of the large ribosomal subunit (LSU) proteins. In vivo depletion of the analysed LSU r-proteins was lethal and led to destabilisation and degradation of the LSU and/or its precursors. Detailed steady state and metabolic pulse labelling analyses of rRNA precursors in these mutant strains showed that LSU r-proteins can be grouped according to their requirement for efficient progression of different steps of large ribosomal subunit maturation. Comparative analyses of the observed phenotypes and the nature of r-protein – rRNA interactions as predicted by current atomic LSU structure models led us to discuss working hypotheses on i) how individual r-proteins control the productive processing of the major 5′ end of 5.8S rRNA precursors by exonucleases Rat1p and Xrn1p, and ii) the nature of structural characteristics of nascent LSUs that are required for cytoplasmic accumulation of nascent subunits but are nonessential for most of the nuclear LSU pre-rRNA processing events.

A ribosome-specialized translation initiation pathway is required for cap-dependent translation of vesicular stomatitis virus mRNAs

“…As in prior studies, rpL40 depletion modestly decreased the total levels of 28S and 18S RNAs (4% and 17%, respectively) but did not abolish processing of mature ribosomal RNAs (Fig. S2A) (34). This result confirms that rpL40 depletion does not inhibit VSV mRNA translation by altering ribosomal RNA processing.…”

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

“…In cells depleted of middle-acting RPLs L9 and L23, aberrant 27SB pre-rRNAs are also eventually targeted for degradation but more slowly than when early-acting RPLs are depleted. In contrast, after depletion of late-acting RPLs L10 and L28, 27S pre-rRNAs do not undergo significant turnover and are processed into downstream rRNA species, although more slowly than in wild-type cells (Poll et al 2009). This suggests that final rearrangements in preribosomes occur in a less efficient fashion when L10 or L28 is absent.…”

“…L3 is the largest yeast RPL (;44 kDa) and has multiple contacts with rRNA (Ben-Shem et al 2011). However, such is not the case; depletion of L4, the second largest RPL (;40 kDa), does not lead to such an extreme early defect (Poll et al 2009), although L4 exhibits equally extensive interactions with rRNA (Ben-Shem et al 2011). Rather, we posit that the location of L3 in proximity to both the 59 and 39 ends of 25S/5.8S rRNA (Supplemental Fig.…”

“…RP depletions in yeast and siRNA-mediated knockdowns in cultured mammalian cells showed that most RPs are essential for ribosome biogenesis (Ferreira-Cerca et al 2005;Robledo et al 2008;Poll et al 2009;Babiano and de la Cruz 2010;O'Donohue et al 2010;Jakovljevic et al 2012;Gamalinda et al 2013). However, phenotypic analyses of these mutants were primarily limited to assaying effects on pre-rRNA processing and nucleocytoplasmic export of preribosomes.…”

“…The segregation of large subunits into distinct structural domains is not as apparent as for small subunits (Ban et al 2000); the six rRNA domains are more intertwined in large subunits (Holbrook 2008). Hence, it was initially striking to find that depletion of individual yeast large subunit RPs (RPLs) led to specific rather than global pre-rRNA processing defects (Ferreira-Cerca et al 2005;Hofer et al 2007;Robledo et al 2008;Poll et al 2009; Fernandez-Pevida et al 2012;Jakovljevic et al 2012;Gamalinda et al 2013), which are grouped into early, middle, and late classes (Table 1). From these results, we observed a pattern previously not described: RPLs belonging to these phenotypic classes are remarkably clustered into distinct structural neighborhoods of the yeast 60S subunit ( Fig.…”