Mmu-Mir-181a Regulates Terminal Erythroid Nuclear Maturation By Regulating Levels of Xpo7

Mammalian terminal erythroid differentiation involves cell cycle arrest, termination of transcription, and finally progressive chromatin condensation culminating in enucleation. The mechanisms underlying these final events are not completely understood. We have previously published that an erythroid-specific variant of the protein Exportin 7 (Xpo7) plays an important role in terminal erythroid nuclear maturation. Xpo7 is highly induced and highly abundant in late erythroblasts. Xpo7 knockdown in mouse erythroblasts inhibits the final stages of normal erythroid development such as chromatin condensation and enucleation without affecting such important processes as cell surface Ter119 acquisition or hemoglobin accumulation. Migration of nuclear proteins such as core histones into the cytoplasm during normal erythropoiesis is also inhibited by Xpo7 knockdown (1).

In order to interrogate the function of Xpo7 in erythropoiesis, we next investigated Xpo7’s genomic structure and how it is regulated during mouse red blood cell development. Xpo7 exerts its effect on the erythroid nucleus after erythroblasts have left the cell cycle and transcription has ended so the timing of its induction must be tightly regulated.

We have previously published that Xpo7 has an erythroid–specific start site and first exon (1). Using 5’ RACE, we determined that the erythroid variant of Xpo7 has a different 5’ UTR than the non-erythroid transcript. In contrast, the 3’ UTR is identical in both erythroid and non-erythroid transcripts, and contains a miR-181a binding site. MiR-181a has been previously shown to be abundant in hematopoietic stem cells and to regulate normal B-cell (2) and megakaryocyte development (3). However, in erythropoiesis, miR-181a decreases in expression just as Xpo7 increases, with lowest levels of miR-181a when Xpo7 is most abundant and presumably exerting its effect on erythroid nuclear maturation. Through luciferase reporter assay experiments, we have found that miR-181a directly binds the 3-UTR binding site of Xpo7. Moreover, miR-181a overexpression results in decreased Xpo7 transcript levels via qPCR and decreased enucleation via flow cytometry. Based on these results, we conclude that miR-181a regulates enucleation by repressing Xpo7 expression while the nucleus is active during terminal erythroid development. Our next step is to examine how miR-181a itself is downregulated during erythroid development, such as perhaps through the expression of a complementary long-noncoding RNA.

1. Hattangadi, SM, et. al. Histones to the cytosol: Exportin 7 plays an important role in terminal erythroid nuclear maturation. Blood 2014, in press.

2. Chen,CZ; Li, L; Lodish, HF; Bartel,DP. MicroRNAs modulate hematopoietic lineage differentiation. Science 2004; 303: 83–86 3. Li, X; Zhang J; Gao, L; McClellan, S; Finan, MA; Butler TW; Owen, BL; Piazza, GA; Xi, Y. MiR-181 mediates cell differentiation by interrupting the Lin28 and let-7 feedback circuit. Cell Death and Differentiation (2012) 19, 378–386.