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Efficient globin production during terminal erythropoiesis depends on the cooperative action of TENT5C poly(A) polymerase and LARP4B in mice

ABSTRACT: To identify erythroid-specific TENT5C interactors, we employed a modified TurboID proximity biotinylation assay followed by MS/MS analysis of fetal liver erythroblasts differentiated ex vivo. We took advantage of Tent5c-EGFP knock-in mouse line and employed lentiviral vector expressing nanobody anti-EGFP fused with TurboID biotinylase. By introducing this construct to either WT or Tent5c-EGFP erythroblasts, we were able to quantitatively determine the specificity of enriched proteins found in proximity to TENT5C-EGFP compared to the background found in the WT. This approach does not require the introduction of exogenous biotinylase-tagged TENT5C, which expression would be out of place in the earliest stages of erythropoiesis. Importantly, it was designed for the knock-in model in which the expression timing for TENT5C is in accord with the developmental timeline of erythroblasts. Hence, we expected the enriched proximal proteins to be physiologically relevant interactors of TENT5C.

INSTRUMENT(S):

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Erythrocyte, Liver

DISEASE(S): Disease Free

SUBMITTER: Andrzej Dziembowski

LAB HEAD: Andrzej Dziembowski

PROVIDER: PXD072661 | Pride | 2026-06-29

REPOSITORIES: Pride

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Project description:The supply of red blood cells (RBCs) is not sufficient in many developing countries or in developed countries for patients who need chronic transfusion from best-matched donors. Ex vivo expansion and maturation of human erythroid precursor cells (erythroblasts) could represent a potential solution. Proliferating erythroblasts can be expanded from human umbilical cord blood mononuclear cells (CB MNCs) ex vivo for 10^6-10^7 fold (in ~50 days) before undergoing senescence. Here, we report that ectopic expression of three to four genetic factors that have been used for iPS cell derivation enables CB-derived erythroblasts to undergo extended ex vivo expansion (M-bM-^IM-%10^51 fold in ~9 months) in a defined suspension culture condition without change of cell identity or function. These vastly expanding erythroblasts maintain homogeneously immature erythroblast phenotypes, a normal diploid karyotype and dependence on specific combination of cytokines and hormone for survival and proliferation throughout the continuous expansion period. When switched to a culture condition for terminal maturation, these immortalized erythroblasts gradually exit cell cycle, decrease cell size, accumulate hemoglobin, condense nuclei and eventually give rise to enucleated hemoglobin-containing erythrocytes. Our result may ultimately lead to the development of unlimited sources of cultured RBCs for optimally-matched or personalized transfusion medicine. We compared the global gene expression profiles of different human cell types: iE: immortalized erythroblasts generated by genetic reprogramming from pCBE; pCBE: primary cord blood-derived erythroblasts; CD34+: CD34+ purified hematopoietic stem/progenitor cells from adult blood or fetal liver; TF-1: a human erythroleukemia cell line; ESC: human embryonic stem cells; iPSCs: human induced pluripotent stem cells. We want to see the relationship among these cell types. We included multiple samples (biological replicates) for most cell types.

			Action	DRS
	TENT5C.sdrf.tsv	Tabular
	TurboID_MFL_TENT5C_GFP1.raw	Raw
	TurboID_MFL_TENT5C_GFP2.raw	Raw
	TurboID_MFL_TENT5C_GFP3.raw	Raw
	TurboID_MFL_WT1.raw	Raw

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Efficient globin production during terminal erythropoiesis depends on the cooperative action of TENT5C poly(A) polymerase and LARP4B in mice

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