Human ribosomes from cells with reduced dyskerin levels are intrinsically altered in translation

Abstract

Dyskerin is a pseudouridine (c) synthase involved in fundamental cellular processes including uridine modification in rRNA and small nuclear RNA and telomere stabilization. Dyskerin functions are altered in X-linked dyskeratosis congenita (X-DC) and cancer. Dyskerin's role in rRNA pseudouridylation has been suggested to underlie the alterations in mRNA translation described in cells lacking dyskerin function, although relevant direct evidences are currently lacking. Our purpose was to establish definitely whether defective dyskerin function might determine an intrinsic ribosomal defect leading to an altered synthetic activity. Therefore, ribosomes from dyskerin-depleted human cells were purified and 1) added to a controlled reticulocyte cell-free system devoid of ribosomes to study mRNA translation; 2) analyzed for protein contamination and composition by mass spectrometry, 3) analyzed for global pseudouridylation levels. Ribosomes purified from dyskerindepleted cells showed altered translational fidelity and internal ribosome entry site (IRES)-mediated translation. These ribosomes displayed reduced uridine modification, whereas they were not different in terms of protein contamination or ribosomal protein composition with respect to ribosomes from matched control cells with full dyskerin activity. In conclusion, lack of dyskerin function in human cells induces a defect in rRNA uridine modification, which is sufficient to alter ribosome activity.-Penzo, M., Rocchi, L., Brugiere, S., Carnicelli, D., Onofrillo, C., Couté, Y., Brigotti, M., Montanaro, L. Human ribosomes from cells with reduced dyskerin levels are intrinsically altered in translation. FASEB J. 29, 3472-3482 (2015). www.fasebj.org

mRNA pseudouridylation affects RNA metabolism in the parasite Toxoplasma gondii

“…In this way, Ψ's can stabilize tRNA folding and increase the fidelity of codon-anticodon interactions, stabilize certain stem-loop conformations of snRNAs, and enable ribosome assembly (Yarian et al 1999;Greenbaum 2001, 2002b;Nobles et al 2002;Cabello-Villegas and Nikonowicz 2005;Denmon et al 2011;Jack et al 2011;Penzo et al 2015). The majority of indications that pseudouridylation plays an important biological role stems indirectly from the observation that Ψ's are modulated in response to different cellular conditions.…”

“…First, mutations in the human rRNA PUS (called DKC1) can lead to impaired binding affinity to both IRESs and tRNAs, leading to reduced translation of specific anti-tumor factors and reduced translational fidelity (Jack et al 2011;Penzo et al 2015). These mutations manifest as the disease X-linked dyskeratosis congenital (X-DC), which ultimately manifests as cancer (Marrone and Mason 2003).…”

A reconstituted cell-free assay for the evaluation of the intrinsic activity of purified human ribosomes

“…We have recently developed a reconstituted RRL-derived cell-free assay for evaluating the intrinsic activity of ribosomes purified from mammalian cells (including those of human origin; see 'Ribosome sources' in Experimental design for a list of cell types that we have successfully used in the assay) 4 . Unfractionated RRL is the most popular and efficient cell-free eukaryotic system 5 used to measure the translation of mRNA into proteins, as, under appropriate conditions, it recapitulates all the functions of the complex protein synthesis machinery.…”

“…However, a fractionated system is more versatile and allows for better control of different experimental parameters, such as addition of exogenous mRNAs and use of ribosomes from different sources. Therefore, we chose to use a fractionated approach to study the intrinsic activity of human ribosomes by adding highly purified ribosomes from human cells to a fractionated RRL system deprived of endogenous ribosomes 4 .…”

Pseudouridine: the fifth RNA nucleotide with renewed interests

“…Using an unbiased proteomics approach, the Ruggero lab discovered a specific defect in IRES (internal ribosome entry site)-dependent translation of mRNAs in the X-DC patients. Such a defect was found to cause impaired translation of IRES-containing mRNAs, some of which encode important anti-apoptotic factors and tumor suppressors [15,33,34]. Following this elegant study, Ruggero, Dinman and coworkers further revealed that DKC1 mutations decreased the rRNA binding affinity to IRES, thus impairing the IRES-dependent translation initiation.…”