OC 48.1 - Vps34 localizes to the nucleolus and mediates megakaryocyte maturation via ribosome biogenesis

Background: A considerable increase in size is one of the most characteristic features of the maturation of megakaryocytes (MKs), together with the polyploidization of the nucleus. Ribosome biogenesis, which takes place in the nucleolus, is essential for protein synthesis and cell growth. Recently, we have shown that Vps34 kinase contributes significantly to the final steps of MK maturation by mediating late endosomal/lysosomal fusion with the plasma membrane mediating proplatelet formation(Bertovic et al 2020 JTH). Unexpectedly, in the majority of young MKs Vps34 was found in the nucleolus.

Aims: In the present study, we aimed to investigate if ribosomal biogenesis is regulated by the Vps34 kinase of primary mouse MKs.

Methods: As a model, we used bone marrow and fetal liver-derived MKs and the Balb3T3 cell line to study nucleolus by confocal microscopy.

Results: In both bone marrow and fetal liver, Vps34 colocalized with upstream binding factor (UBF), a transcription factor required for ribosomal RNA (rRNA) expression that controls a rate-limiting step in ribosome biogenesis. Cell fractionation confirmed Vps34 confinement to the nucleolus. Furthermore, nucleolar localization of Vps34 was observed in in situ MK of murine bone marrow sections and in Balb3T3. By pull-down of GFP-UBF, Vps34 was co-immunoprecipitated from Balb3T3 cells. In MKs, inhibition of RNA polymerase I (Pol I) with actinomycin D abolished the nucleolar localization of Vps34 and the binding of Vps34 to UBF. Furthermore, pharmacologic inhibition of Vps34 decreased nascent RNA production and inhibited transcription of 45S rRNA, indicating impaired ribosomal biogenesis. Furthermore, inhibition of RNA Pol I function with the Vps34 inhibitor correlated dose-dependently with reduced cell size and expression of the MK maturation marker GPIb, indicating reduced MK development.

Conclusion(s): Taken together, these data suggest that Vps34 may play an important, as yet undescribed role in nucleolar function that could control the early stages of MK development via ribosome biogenesis.