Human ribosomal protein L13a is dispensable for canonical ribosome function but indispensable for efficient rRNA methylation

Abstract

Previously, we demonstrated that treatment of monocytic cells with IFN-g causes release of ribosomal protein L13a from the 60S ribosome and subsequent translational silencing of Ceruloplasmin (Cp) mRNA. Here, evidence using cultured cells demonstrates that Cp mRNA silencing is dependent on L13a and that L13a-deficient ribosomes are competent for global translational activity. Human monocytic U937 cells were stably transfected with two different shRNA sequences for L13a and clonally selected for more than 98% abrogation of total L13a expression. Metabolic labeling of these cells showed rescue of Cp translation from the IFN-g mediated translational silencing activity. Depletion of L13a caused significant reduction of methylation of ribosomal RNA and of cap-independent translation mediated by Internal Ribosome Entry Site (IRES) elements derived from p27, p53, and SNAT2 mRNAs. However, no significant differences in the ribosomal RNA processing, polysome formation, global translational activity, translational fidelity, and cell proliferation were observed between L13a-deficient and wild-type control cells. These results support the notion that ribosome can serve as a depot for releasable translation-regulatory factors unrelated to its basal polypeptide synthetic function. Unlike mammalian cells, the L13a homolog in yeast is indispensable for growth. Thus, L13a may have evolved from an essential ribosomal protein in lower eukaryotes to having a role as a dispensable extra-ribosomal function in higher eukaryotes.

Polypyrimidine tract binding protein regulates IRES-mediated translation of p53 isoforms

“…A recent study has also suggested the role of another ribosomal protein, L13a in regulating the cap independent translation of p53, p27 and SNAT2 mRNAs. 34 These observations, together with the current study demonstrating the interaction of PTB with the p53 IRESs, suggest that p53 translation is subject to a multiplicity of controls by differential protein binding that regulates p53 expression. The blots were probed with antibodies against PTB and also antibody corresponding for actin (loading control).…”

Interleukin-1 Receptor-associated Kinase 2 Is Critical for Lipopolysaccharide-mediated Post-transcriptional Control

“…Polysome Fraction Analysis-Cytoplasmic extracts were prepared from BM-derived macrophages stimulated with LPS (1 g/ml) as described (31). Cytoplasmic exacts were carefully layered over 5-50% linear sucrose gradients in polysome buffer (10 mM HEPES, pH 7.5, 100 mM KCl, 2.5 mM MgCl 2 , 1 mM dithiothreitol, 50 units of recombinant RNasin (Promega), and 0.1% Igepal-CA630 (Sigma)) and centrifuged at 17,000 rpm in a Beckman SW32.1 Ti rotor for 18 h at 4°C.…”

Uncoupling ribosome biogenesis regulation from RNA polymerase I activity during herpes simplex virus type 1 infection

“…Simultaneously, pre-rRNA are subjected to extensive chemical modifications such as methylations and pseudouridylations (Fromont-Racine et al 2003) at defined sites, and particularly on nucleotides involves in catalysis. The exact function of these modifications is not yet elucidated, but it appears more and more clear that they play a major role in controlling the translational activity of the ribosomes (Yoon et al 2006;Baxter-Roshek et al 2007;Chaudhuri et al 2007). …”