RNA Sequestration Controls Hematopoietic Stem Cell Quiescence and Function

Hematopoietic stem cells (HSCs) lie at the pinnacle of the hematopoietic hierarchy, balancing self-renewal and differentiation to sustain lifelong production of all blood cell lineages. Under steady-state conditions, HSCs are highly quiescent, protecting their longevity by restricting metabolism and protein synthesis. In contrast, regenerative stress triggers HSCs to exit quiescence and increase metabolism and protein synthesis to fuel growth, expansion, and differentiation. Despite remarkable advances in our knowledge, it remains unclear how HSCs orchestrate the rapid shifts in protein levels necessary for this phenotypic plasticity. The overall goal of this study is to elucidate how fine-tuning of mRNA translation safeguards HSC function during normal and regenerative hematopoiesis.

Post-transcriptional mechanisms govern the protein output of the cellular transcriptome and play crucial roles in tissue homeostasis and disease. Our lab recently discovered that the post-transcriptional regulator DDX6 sustains acute myeloid leukemia by repressing the translation of mRNAs encoding tumor suppressors (Kodali et al., Nature Cell Biology, in press). However, the role of DDX6 in homeostatic and regenerative hematopoiesis is unknown.

To address this knowledge gap, we generated novel a Ddx6^fl/fl mouse model crossed with an Mx1-Cre driver to specifically delete DDX6 in the hematopoietic compartment. To investigate the role of mRNA sequestration in steady state hematopoiesis, Mx1-Cre/Ddx6^fl/fl and control mice were injected with poly(I:C) for seven days, every other day (15 mg/kg, n=6/group). Trending peripheral blood lineages every two weeks revealed that DDX6 is dispensable in maintaining mature hematopoietic populations under steady state conditions (p>0.2). Similarly, upon sacrifice four months after Cre induction, there were no significant changes in cellularity in the spleen, bone marrow or LSK frequency (p>0.2). Strikingly, however, DDX6-depleted HSCs robustly expanded nearly three-times relative to wildtype counterparts (p<0.001), as confirmed by Ki67 staining. Intriguingly, we also observed increased mitochondrial mass and metabolism in DDX6-/- HSCs by MitoTracker and TMRM staining, respectively (p<0.01). Transcriptomic analysis echoed these findings through upregulated cell cycle progression and metabolomic programs specifically among DDX6-depleted HSCs compared to MPPs. Similar results were achieved using an alternative Rosa26-Cre-ERT2 driver. Together, these findings suggest that loss of DDX6-mediated RNA sequestration triggers loss of HSC quiescence.

It is well established that quiescence is inextricably linked to HSC function. We therefore asked if DDX6^-/- HSCs are functionally impaired when challenged with regenerative stress. To answer this question, we isolated HSCs from the steady state depletion mice upon sacrifice and competitively transplanted them against wildtype HSCs into lethally irradiated hosts at a 1:1 ratio. Loss of DDX6 severely impaired transplanted cells’ ability to contribute to the recipient hematopoietic population at the progenitor and mature levels (p<0.001). A parallel transplant experiment controlled for homing defects yielded the same result. Together, these data suggest that mRNA sequestration is essential for preserving HSC quiescence and function.

Mechanistically, our early insights indicate that in HSCs, DDX6 orchestrate the translational suppression and sequestration of transcripts encoding key regulators of quiescence exit in P-bodies. Overall, our study uncovered a novel post-transcriptional node that controls the balance between quiescence and proliferation in HSCs, opening new avenues for targeting RNA processing in therapeutic settings.