Neonatal expression of RNA-binding protein IGF2BP3 regulates the human fetal-adult megakaryocyte transition
Kamaleldin E. Elagib, … , Camelia Iancu-Rubin, Adam N. Goldfarb
Kamaleldin E. Elagib, … , Camelia Iancu-Rubin, Adam N. Goldfarb
Published May 8, 2017
Citation Information: J Clin Invest. 2017;127(6):2365-2377. https://doi.org/10.1172/JCI88936.
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Research Article Hematology

Neonatal expression of RNA-binding protein IGF2BP3 regulates the human fetal-adult megakaryocyte transition

Abstract

Hematopoietic transitions that accompany fetal development, such as erythroid globin chain switching, play important roles in normal physiology and disease development. In the megakaryocyte lineage, human fetal progenitors do not execute the adult morphogenesis program of enlargement, polyploidization, and proplatelet formation. Although these defects decline with gestational stage, they remain sufficiently severe at birth to predispose newborns to thrombocytopenia. These defects may also contribute to inferior platelet recovery after cord blood stem cell transplantation and may underlie inefficient platelet production by megakaryocytes derived from pluripotent stem cells. In this study, comparison of neonatal versus adult human progenitors has identified a blockade in the specialized positive transcription elongation factor b (P-TEFb) activation mechanism that is known to drive adult megakaryocyte morphogenesis. This blockade resulted from neonatal-specific expression of an oncofetal RNA-binding protein, IGF2BP3, which prevented the destabilization of the nuclear RNA 7SK, a process normally associated with adult megakaryocytic P-TEFb activation. Knockdown of IGF2BP3 sufficed to confer both phenotypic and molecular features of adult-type cells on neonatal megakaryocytes. Pharmacologic inhibition of IGF2BP3 expression via bromodomain and extraterminal domain (BET) inhibition also elicited adult features in neonatal megakaryocytes. These results identify IGF2BP3 as a human ontogenic master switch that restricts megakaryocyte development by modulating a lineage-specific P-TEFb activation mechanism, revealing potential strategies toward enhancing platelet production.

Authors

Kamaleldin E. Elagib, Chih-Huan Lu, Goar Mosoyan, Shadi Khalil, Ewelina Zasadzińska, Daniel R. Foltz, Peter Balogh, Alejandro A. Gru, Deborah A. Fuchs, Lisa M. Rimsza, Els Verhoeyen, Miriam Sansó, Robert P. Fisher, Camelia Iancu-Rubin, Adam N. Goldfarb

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Figure 5

IGF2BP3 interacts with and regulates 7SK snRNP.

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IGF2BP3 interacts with and regulates 7SK snRNP. (A) Interaction of endog...
(A) Interaction of endogenous IGF2BP3 and HEXIM1. K562 cellular extracts underwent IP with anti-HEXIM1 or control antibody followed by IB analysis as indicated. (B) Binding of 7SK snRNA by IGF2BP3. Extracts from 293 transfectants expressing Myc epitope–tagged IGF2BP3 underwent IP with anti-Myc or control antibody followed by reverse-transcriptase PCR (RT-PCR) with gel and real-time qPCR assessment. Graph depicts mean qPCR signal relative to input from 3 independent experiments. **P < 0.01, t test. Line in gel indicates position of cropping of empty lanes. (C) Interaction of endogenous IGF2BP3 and 7SK. IGF2BP3 iCLIP-seq hits of Palanichamy et al. (36) from RS4;11 cells were arranged in order of read frequency. Rank and read counts are shown for 7SK, MYC, and CD44. (D) IGF2BP3 binds 7SK within HP4, a stability target for LARP7. Human 7SK folding was modeled using RNAfold server (ViennaRNA Web Services, Universitat Wien, Vienna, Austria). IGF2BP3 contact regions from iCLIP peak and known LARP7-binding region are depicted by ovals. (E) Alteration of LARP7 complexes by IGF2BP3. Extracts from 293 cells transfected with control or IGF2BP3 expression vector underwent fractionation by glycerol gradient sedimentation, with IB analysis of representative fractions. (F) IGF2BP3 contribution to 7SK stability in neonatal megakaryocytes. Neonatal progenitors transduced with lentiviral shRNA (sh) vectors underwent megakaryocyte culture and qRT-PCR as in Figure 2F. Graphs represent mean ± SEM of 7SK normalized to GAPDH in 3 independent experiments. ***P < 0.005, t test. See also Supplemental Figure 7.