Diamond-Blackfan anemia (DBA, MIM 105650) is a congenital red cell aplasia characterized by severe normochromic-macrocytic anemia often associated with physical
abnormalities. Although it is rare (frequency 6/million live births in Italy), it holds an important place in hematology as a paradigm of an intrinsic genetic disorder of the
committed erythroid progenitor.
DBA patients display high EPO levels, irrespective of the degree of their anemia. The failure of their hemopoietic progenitors to respond to EPO suggests EPO insensitivity,
and a defect in the EPO-R pathway has thus been widely accepted as an explanation of defective erythropoiesis. This has also been suggested by the inducement of phenotype
reversal in vitro by the addition of SCF (which uses a different transduction pathway from EPO) to IL-3 and EPO to CD34+ bone marrow cells from DBA patients.
IL-9 and thrombopoietin potentiate the effect of SCF. Clinical trials using EPO or IL-3 have been ineffective in most patients, while SCF has not been used in vivo
because of its severe side-effects. However, EPO-R and other genes encoding for erythropoietic growth factors have been ruled out as potential candidates. More than 50%
of patients respond to steroid therapy, though the mechanisms involved are unknown. Options in steroid-resistant patients are chronic red cell transfusions or allogenic
stem cells transplantation. In these cases, life expectancy is drastically reduced. The recent report that metoclopramide or valproate may modulate erythropoiesis in DBA
has yet to be validated by larger studies. A number of patients also experience spontaneous remission.
Most patients are sporadic, but the disease is familial in 20% of cases and displays a dominant transmission. An international collaborative group to which we belong has
identified a DBA locus on 19q13.2. The DBA gene on 19q encodes for a structural ribosomal protein, RPS19, the first RP known to cause an inherited human disease.
Causal mutations have been characterised in 25% of patients; all are present on a single allele and most are clearly loss-of-function mutations pointing to
haploinsufficiency. They are either gene deletions, chromosomal rearrangements involving the RPS19 gene, or mutations causing NMD (Nonsense-Mediated Decay. So far,
11 missense mutations have been identified and preliminary data show that these mutations hamper both nucleolar localization and inclusion on the ribosome.
Rare patients carry mutations in RPS24 gene and a further DBA locus has been identified on chromosome 8p. It is interesting to note that immunoelectron microscope
studies locate RPS19 to the external surface of the 40S subunit, where it is in close vicinity to RPS24, a region that interacts with eIF-2 during ribosomal scanning and
translation initiation. Indeed, we have shown that RPS19 interacts with RPS24 in a large proteomic study. Thus, it is possible that RPS19 and RPS24 participate in the same
function.
Other inherited bone marrow aplastic syndromes are due to defects in genes expected to have a role in ribosomal function. This suggests a similar pathogenetic mechanism
for DBA.
The link between erythropoiesis and RPs may be a defect in general protein synthesis in tissues with high cell-turnover. On the other hand, the finding of extraribosomal
functions for ribosomal proteins may represent the missing link.
A defect in protein synthesis has been postulated as the cause of malformations and low stature in DBA patients. Animal models known so far for mutations in ribosomal
components include: i) "minute" mutants (min) of Drosophila, which carry alterations in RP genes; ii) the "anucleolate" and the
"partial nucleolus" mutants in Xenopus laevis, showing complete or partial deletion of rRNA genes cluster. They display several developmental
abnormalities and growth retardation. These phenotypes support the hypothesis that some of the features observed in DBA reflect a dysfunction of the translation apparatus.
Unfortunately, an animal model for DBA is not available, because KO mice show a lethal phenotype in homozygosity, but do not show any abnormality in heterozygosity.
This may be due to the fact that RPS19 expression is not decreased in heterozygotes, as compared to wildtype.
It has been shown that RPS19 expression from the yeast genes RPS19 A and B is necessary for ribosomal 40S subunit assemblage: the knock-out of one of them is
sufficient to impair ribosomal 40S subunit assembly and is associated to 21S rRNA accumulation. RPS19B yeast mutants carrying mutations at the same residues as
found in DBA patients show the same phenotype as ko mutants. The same defect has been observed in DBA patients. These results point to a role in ribosome biogenesis
and function.
A general or specific defect in translation has been found both in patients that carry mutations in RPS19 and mutation-negative patients. A decreased expression of
ribosomal proteins and proteins involved in translation has been reported both in RPS19 mutated and in non mutated DBA patients. It has also been suggested that a
deficiency of 40S subunits might lead to altered polysome-recruitment of specific mRNAs important for erythroid development. This may affect the ratio of different
transcription factor isoforms that are translated from transcripts with alternative in-frame translation initiation sites. Alternatively, RPS19 may regulate the fate of
translation of specific transcripts, similarly to RPL13a and RPL26.
In our laboratory we have chosen to study indepth the hypothesis of impaired extraribosomal functions of RPS19. In order to clarify the role of RPS19 and the pathways in
which it is involved, we have performed a high-throughput analysis of its interactome.
First of all, we did a two-hybrid screening analysis and identified the serine threonine kinase PIM1 as a RPS19 interactor. PIM1 is highly expressed in hematopoietic
tissues and is induced by EPO; up to now, it represents the only link between RPS19 and erythropoiesis. We also showed that PIM1 is able to phosphorylate RPS19 in a
kinase assay in vitro and that PIM1 binds the 40S subunit. Experiments performed on missense RPS19 mutants showed that all can still bind PIM1, but with
different binding affinities. Consequences of these differences are still object of study.
Recently, we have analysed RPS19 interactome through µLC-MS/MS, in collaboration with the laboratory of prof. Margherita Ruoppolo (University "Federico
II", Napoli). Spectrometry has been performed on eluates obtained from pulldown experiments with GST-RPS19 recombinant protein on erythroleukemic K562 cells
lysate. This strategy has lead to complementary results in regard to the two-hybrid analysis, due to the different biological systems.
In order to elucidate RPS19 role in the translational regulation of specific transcripts, we next analysed its mRNAs interactors: RNase-free whole cell extracts from K562
have been loaded onto RPS19- or control-conjugated resins. RNAs eluted from the resins were quantified and used as a probe on a whole-genome mouse cDNA microarray
(RIKEN). Three experiments have been performed in duplicate and 600 transcripts have been found to be enriched by RPS19 binding. Data were normalized to the K562
transcriptome. We have used the following categories to focus the validation experiments on the more pertinent interactors: 1) we have considered the most represented
transcripts in the eluate as compared to the cell lysate; 2) we have considered only transcripts that show a low expression in K562 cells, but were significantly enriched in
the eluate: 120 transcripts showed this pattern. Microarray analysis has been performed in collaboration with the laboratory of prof. Stefano Gustincich (International
School for Advanced Studies I.S.A.S.-S.I.S.S.A., Trieste).
Our work is performed in collaboration with different research groups: