Early Hematopoietic Progenitors of Dkc1 Hypomorphic Mutant Mice Display Decreased Proliferation Rate and an Impaired Control of Serine/Arginine-Rich Splicing Factor 4 (Srsf4) Translation

The X-linked form of dyskeratosis congenita (X-DC) is caused by mutations in DKC1, which encodes for dyskerin: a putative pseudouridine synthase that mediate the posttranscriptional modification of ribosomal RNA (rRNA) through the conversion of uridine (U) to pseudouridine (Ψ). Patients with X-DC display defects in the pseudouridylation of ribosomal RNA that leads to translational upregulation of IRES-containing mRNAs and affects the affinity of the ribosome for these mRNAs. Studies in vivo and in vitro suggest that the pseudouridylation of ribosomal RNA is the underlying mechanism responsible for the enhanced susceptibility to cancer in these patients. Ruggero et al. have previously reported (Ruggero et al. Science. 2003 Jan 10;299(5604):259-62) that hypomorphic Dkc1m (Dkc1m) mice present pancytopenia associated with hypocellularity of the bone marrow (BM) and increased susceptibility to cancer, therefore constituting a reliable model to study the effect of impaired ribogenesis on hematopoiesis and oncogenesis. However, the cellular and molecular mechanisms leading to BM failure in X-DC remain unknown. Here, we describe the in vivo analysis of the proliferation rate of hematopoietic progenitors in Dkc1m mice and compare the proteomic profile of hematopoietic progenitors between Dkc1m mice and wild-type (WT) controls. For in vivo proliferation assays, 1mg of bromodeoxyuridine (BrdU) was injected intraperitoneally, every 6 hours during 24 hours, in 16 mice (eight WT and eight Dkc1m), and BM cells were harvested by flushing bone cavity, followed by immunofluorescence staining of incorporated BrdU and flow cytometric analysis. No differences were detected in the number of lineage-negative (Lin−), Sca1-positive, c-kit-negative (LSK−) cells, multipotent precursors (MPP), common myeloid progenitors (CMP), common lymphoid progenitors (CLP) and immature B (B lin) and erythroid (Eryt) cells between Dkc1m and WT mice. Nevertheless, the BrdU incorporation was lower in LSK cells and CMPs from Dkc1m mice (P<0.05), indicating a lower proliferation rate. Using in vitro stable isotope labelling of amino acids (SILAC) hematopoietic progenitor cells were cultured in complete medium containing 10% fetal bovine serum and cytokines (6 ng/ml mIL-3, 10 ng/ml mIL-6 and 100 ng/ml mSCF). Of note, SILAC is one of the most applied approaches for quantitative proteomics, which uses labeled amino acids contain atoms of different isotopes in cell culture. Briefly, one cell population is cultured in unlabeled medium (control), while a second population is grown in medium substituted with a heavy amino acid (usually arginine 13C and/or lysine 15N). After 2-3 weeks culture, murine hematopoietic progenitors were collected and equal amounts of cells from Dkc1m and WT mice (labeled or not with heavy amino acid) were mixed for protein extraction and analyses. Using this approach, we identified about 3,500 differentially expressed proteins; including proteins related to mRNA assembling and splicing, chromatin remodeling, apoptosis and cell cycle arrest. Interestingly, one of the most differentially expressed proteins between WT and Dkc1m mice (WT light /Dkc1m heavy ratio: 18-fold) was the Serine/arginine-rich splicing factor 4(Srsf4); a member of the splicing factor family (SRSF1, SRSF3 and SRSF4) frequently associated with alternative splicing of genes related to hematopoietic progenitor cell differentiation. Altogether, our preliminary results reveal defects in the transcription/translation of specific mRNAs in Dkc1m cells. Additionally, it is conceivable that the down-regulation of Srsf4 protein could be associated with the low proliferative rate in DKC1m mice and explain the impairment of hematopoiesis in X-DC patients.