Program: Oral and Poster Abstracts
Session: 101. Red Cells and Erythropoiesis, Structure and Function, Metabolism, and Survival, Excluding Iron: Poster I
In this study, freshly drawn RBC were density separated on discontinuous percoll gradients. The top two fractions, fraction 1 (F1) representative of the top layer containing the youngest, reticulocyte-like cells, and a second fraction with greater density and non-reticulocyte properties (F2), were isolated. Cell populations were analyzed to compare RBC protein levels using western blotting (WB). qRT-PCR was used to evaluate band 3 RNA. Argonaute (AGO2) RNA immunoprecipitation (RIP), was used to ascertain RISC mediated transcriptional repression of band 3 using qRT-PCR.
Band 3 protein expression was elevated in the denser F2 compared to F1, in freshly drawn normal RBCs. RNA levels of band 3 complemented protein levels, as increased band 3 RNA was observed in F2. RIP experiments revealed elevated post-transcriptional control of band 3 through increased association of its RNA with AGO2 in F1 compared to F2 thereby, correlating with greatly reduced, sometimes undetectable protein levels of band 3 in F1.
We confirm the presence of AGO2 in circulating RBCs. We report, for the first time, the post-transcriptional control of band 3, an integral RBC transmembrane protein, in circulating reticulocyte-like cells. These results suggest a possible subpopulation of reticulocytes, a pro-reticulocyte, with ongoing control of band 3 expression following enucleation, and a potential role of miRNA-RISC in band 3 pathology.
Disclosures: No relevant conflicts of interest to declare.
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