S163: polysome interactome in rps14-deficient cells reveals the involvement of the mrna decay machinery in translation selectivity.

Background: During normal erythropoiesis, protein expression poorly correlates with transcript expression highlighting the importance of post-transcriptional regulatory mechanisms. During pathologic erythropoiesis, mRNA translation efficiency, which is governed by cis-regulatory mechanisms including the folding and length of UTRs and the optimal codon composition of coding sequences, is shaping the proteome in conditions of limited ribosome availability in cell models mimicking the 5q- syndrome or Blackfan-Diamond anemia by RPS14 or RPS19 gene haploinsufficiency. Aims: To further identify regulatory mechanisms of translation in trans, we investigated the implication of RNA binding proteins (RBP) in the control of mRNA stability/decay in normal and pathological conditions. Methods: We performed a mass spectrometry analysis of polysomes to identify deregulated RBPs upon shRPS14 induction in UT-7/EPO cell line. We applied an integrated analysis of more than 250 public Clip-Seq experiments to identify enrichments in targets of some RBPs. We performed the knock-downs (KD) of RBPs EDC3, DCP1a and DCP2, and of RNA helicase DDX6 implied in RNA decay and analyzed their impact in human cord blood CD34+ cells cultured in erythroid differentiation conditions. Results: Upon shRPS14 induction, the polysome interactome was characterized by the down-regulation of RBPs involved in mRNA decay, P-bodies (PB) and stress granules (SG) biogenesis. Knocking down RNA decay factor EDC3, DCP1a or DCP2 in normal CD34+ cells resulted in impaired erythroid proliferation and differentiation. Most of these deregulated proteins of the polysome interactome were shown to interact directly or indirectly with RNA helicase DDX6 involved in PB or SG biogenesis. Furthermore, the transcripts which translation was selectively increased upon shRPS14 induction were those reported to be normally sequestered in PB and SG or upregulated after DDX6 KD in HEK293T cells. Thus, we explored the role of DDX6 in normal erythropoiesis. DDX6 immunofluorescence analysis revealed the presence of PB at a frequency of 4-5 PB/erythroblast. These PB remain stable along erythroid differentiation till orthochromatic stage, where they disappear. DDX6 KD in colony forming cell (CFC) assay demonstrated a huge reduction in BFU-E formation without impacting CFU-GM. Erythroblasts deriving from DDX6 KD CD34+ cells displayed a 10-fold reduction of growth rate. Infusion of DDX6 KD CD34+ cells to NSG mice confirmed the default in erythroid differentiation with the absence of BFU-E after 3 months. Summary/Conclusion: Our analyses suggest that translation selectivity upon induction of RPS14 gene haploinsufficiency could be driven by the loss of control of RNA decay and/ or the release from storage of transcripts normally sequestered in PB and/or SG. DDX6 through its direct role in controlling mRNA decay or PB formation could be a key player in post-transcriptional regulation of mRNA and protein expression in erythropoiesis. Keywords: Erythropoieisis, Myelodysplastic syndrome, Anemia, Gene expression