Frameshift mutation in p53 regulator RPL26 is associated with multiple physical abnormalities and a specific pre-ribosomal RNA processing defect in diamond-blackfan anemia

Abstract

Diamond-Blackfan anemia (DBA) is an inherited form of pure red cell aplasia that usually presents in infancy or early childhood and is associated with congenital malformations in ∼30-50% of patients. DBA has been associated with mutations in nine ribosomal protein (RP) genes in about 53% of patients. We completed a large-scale screen of 79 RP genes by sequencing 16 RP genes (RPL3, RPL7, RPL8, RPL10, RPL14, RPL17, RPL19, RPL23A, RPL26, RPL27, RPL35, RPL36A, RPL39, RPS4X, RPS4Y1, and RPS21) in 96 DBA probands. We identified a de novo two-nucleotide deletion in RPL26 in one proband associated with multiple severe physical abnormalities. This mutation gives rise to a remarkable ribosome biogenesis defect that affects maturation of both the small and the large subunits. We also found a deletion in RPL19 and missense mutations in RPL3 and RPL23A, which may be variants of unknown significance. Together with RPL5, RPL11, and RPS7, RPL26 is the fourth RP regulating p53 activity that is linked to DBA.

Figure 1. Detection of RPL19 mutant form by Western blot

Immunoblot shows RPL19 protein in all family members (with and without deletion (c. 562_563delTT) in the RPL19 gene). RPL19 mutant protein was detected in the probands and his clinically unaffected mother and brother with RPL19 deletion. Mut, mutation; Pt, patient; M*, mother; B*, brother; F, father; Sa, sister a; Sb, sister b.

Figure 2. Pre-rRNA processing in human cells

The early 47S precursor is processed along different pathways depending on the order of the cleavages. Given the higher abundance of 30S pre-rRNA relative to the 41S species, cleavage of the ITS1 at site 2 appears to occur most frequently before processing of the 5′-ETS. Arrowheads indicate the pre-rRNA endonucleolytic cleavage sites, although some processing steps involve exonucleases rather than endonucleases. Dashed lines indicate the hypothetical pathway induced by RPL26 depletion.

Figure 3. Pre-rRNA processing in DBA patients

(A) Sucrose gradient analysis of cytoplasmic ribosomes extracted from HeLa cells treated with siRNAs against RPL3, RPL19 and RPL26. The decrease of the free 60S ribosomal subunit and the formation of half-mers (arrows) indicate a strong defect in 60S subunit production. (B) Northern blot analysis of pre-rRNAs in HeLa cells upon depletion of RPL3, RPL19 and RPL26. siRNAs targeting RPL11, another 60S subunit protein involved in DBA, were used as a comparison [Doherty et al., 2010]. Precursors to the 18S rRNA are detected with the 5′-ITS1 probe, while the ITS2 probe reveals precursors to the 60S subunit RNAs. Arrowheads indicate the 36S pre-rRNA and a shorter precursor detected in RPL26 depleted cells (see panel D). (C) Northern blot analysis of pre-rRNAs in DBA patients and their relatives. RNAs were extracted from lymphoblastoid cells. Controls correspond to non-DBA individuals. RNAs from a DBA harboring a mutation in RPL11 were included as a comparison. These results were reproduced on three independent northern blots. (D) A probe hybridizing to the core of the ITS1 reveals accumulation of 36S pre-rRNA together wih a shorter precursor (arrowheads). (E) A similar doublet is detected in cells from the RPL26 mutated patient. Similar results were obtained on two independent northern blots. (F) Quantification of the Northern blot data by phosphorimager. For each ratio, the values were divided by the means of the values obtained for the controls. Measurements were performed three times on independent Northern blots with similar results, except for the 36S/18S ratio which was only measured on one blot. This ratio takes into account both the 36S pre-rRNA and the band migrating right below (see panel E).