Ribosomal protein L35 is required for 27SB pre-rRNA processing in Saccharomyces cerevisiae

Abstract

Ribosome synthesis involves the concomitance of pre-rRNA processing and ribosomal protein assembly. In eukaryotes, this is a complex process that requires the participation of specific sequences and structures within the pre-rRNAs, at least 200 trans-acting factors and the ribosomal proteins. There is little information on the function of individual 60S ribosomal proteins in ribosome synthesis. Herein, we have analysed the contribution of ribosomal protein L35 in ribosome biogenesis. In vivo depletion of L35 results in a deficit in 60S ribosomal subunits and the appearance of half-mer polysomes. Pulse-chase, northern hybridization and primer extension analyses show that processing of the 27SB to 7S pre-rRNAs is strongly delayed upon L35 depletion. Most likely as a consequence of this, release of pre-60S ribosomal particles from the nucleolus to the nucleoplasm is also blocked. Deletion of RPL35A leads to similar although less pronounced phenotypes. Moreover, we show that L35 assembles in the nucleolus and binds to early pre-60S ribosomal particles. Finally, flow cytometry analysis indicated that L35-depleted cells mildly delay the G1 phase of the cell cycle. We conclude that L35 assembly is a prerequisite for the efficient cleavage of the internal transcribed spacer 2 at site C2.

Functional analysis of the uL11 protein impact on translational machinery

“…Additionally, we have noticed the half-mers in the polysome profile, which is often associated with a pre-60S maturation defect. [62][63][64] Although in such cases nuclear entrapment of proteins from large ribosomal subunits is usually observed, we did not see any mislocalization of the marker uL23 protein in microscopic observations. Additionally, we saw only small reduction of 60S, which departed from the prominent behavior observed in cells with a 60S biogenesis defect.…”

Ribosome Biogenesis in the YeastSaccharomyces cerevisiae

“…Consistent with this work, most r-proteins copurify with the earliest pre-rRNAs and assembly factors (Ferreira-Cerca et al 2007;Babiano and De La Cruz 2010;Babiano et al 2012;Jakovljevic et al 2012;Gamalinda et al 2013). However, the order of assembly of r-proteins relative to each other cannot be distinguished at any higher resolution by existing methods.…”

Ribosomal proteins L7 and L8 function in concert with six A3 assembly factors to propagate assembly of domains I and II of 25S rRNA in yeast 60S ribosomal subunits

“…Functional analysis of a few yeast and human r-proteins, and more recently, systematic examination, have begun to reveal roles of r-proteins in eukaryotic subunit assembly in vivo (Rotenberg et al 1988;Moritz et al 1990;Deshmukh et al 1993;van Beekvelt et al 2001;Jakovljevic et al 2004;Léger-Silvestre et al 2004;Ferreira-Cerca et al 2005;Martín-Marcos et al 2007;Rosado et al 2007;Robledo et al 2008;Pöll et al 2009;Babiano and de la Cruz 2010;O'Donohue et al 2010). Most investigations of r-protein function in vivo have focused on their participation in prerRNA processing.…”