Norad long noncoding RNA, a decoy for pumilio RNA-binding proteins, elevates fetal hemoglobin levels

Abstract NORAD long noncoding RNA (Noncoding RNA activated by DNA Damage) is a highly conserved, abundant, and widely expressed long non-coding RNA (lncRNA) in mammals. NORAD contains 20 Pumilio recognition elements (PREs), which are binding sites for PUM1/2 (Pumilio homolog 1/2), two members of the Pumilio family of RNA binding proteins. NORAD contains 12 sequence-similar NORAD Repeat Units (NRUs). NORAD lncRNA is known to sequester PUM1 away from its mRNA targets. However, the functions of NORAD in erythroid cells are thus far unknown. We previously showed that PUM1 knockdown leads to increased levels of fetal hemoglobin in both HUDEP2 cells and human primary hematopoietic stem and progenitor cells (Blood Advances, 2022). This increase was not accompanied by impairment of erythroid differentiation. Further, we identified a novel mutation in PUM1 RNA binding domain (p.(His1090Profs*16) in a patient who displayed increased levels of fetal hemoglobin, without indications of anemia. Since PUM1 is an RNA binding protein, it could serve as a target for the development of non-gene altering strategies to inhibit its function and thereby elevate fetal hemoglobin levels to alleviate the symptoms in individuals with sickle cell anemia and beta thalassemia. In the current study, we tested the hypothesis that overexpression of NORAD lncRNA can sequester PUM1, and thereby inhibit its function and induce fetal hemoglobin in erythroid cells. For this, we first overexpressed NORAD lncRNA in HUDEP2 cells, and this led to a significant increase in gamma globin mRNA and protein levels, and hemoglobin HPLC documented up to 40% fetal hemoglobin (HbF) levels compared to control cells that have less than 1% HbF. We further confirmed a similar induction in fetal gamma globin levels in human primary hematopoietic stem and progenitor cells (HSPCs) upon NORAD lncRNA overexpression. Interestingly, overexpression of NORAD lncRNA does not reduce the levels of BCL11A, a well-characterized gamma globin repressor. There are currently no published studies on the functions of NORAD in erythroid cells, and the functions of PUM1 in erythroid cells are unknown beyond our discovery on the impact of PUM1 knockdown on fetal hemoglobin induction (Blood Advances, 2023). Therefore, to understand the mechanism by which NORAD lncRNA overexpression leads to high fetal hemoglobin levels, and to evaluate if NORAD lncRNA can act as a decoy for Pumilio RNA binding proteins in erythroid cells, we first determined if PUM1 binds to NORAD in erythroid cells. Using RNA Editing Molecular Recording in Adenosines (REMORA) assay and expressing PUM1 fused to Adenosine base editor (rABE-PUM1) to identify PUM1's RNA binding sites through introduced adenosine deamination, we confirmed that PUM1 binds to endogenous NORAD lncRNA in erythroid cells. To further investigate the mechanism of fetal hemoglobin induction upon NORAD lncRNA overexpression, and to identify the minimal NORAD lncRNA region required for fetal gamma globin induction, we overexpressed a truncated version of NORAD (Mini-NORAD) containing NORAD repeat units (NRU) 7/8, in HUDEP2 cells. These repeat units which contain three Pumilio response elements (PREs/Pumilio binding sites) were shown in previous studies to be sufficient for sequestering Pumilio proteins away from their mRNA targets. We compared the impact of the overexpression of wildtype Mini-NORAD RNA and mutated Mini-NORAD RNA (harboring TGTA to ACAA mutation in their PREs), on fetal hemoglobin levels. Mini-NORAD RNA significantly elevated gamma globin expression in HUDEP2 cells, unlike PRE-mutated Mini-NORAD RNA. Furthermore, these observations were also consistent in our preliminary studies in human primary hematopoietic stem and progenitor cells (HSPCs). Lastly, we determined if NORAD lncRNA overexpression affects the progression of erythroid terminal differentiation. In both HUDEP2 cells and human HSPCs, the progress of erythroid terminal differentiation was not impeded upon NORAD lncRNA overexpression, similar to what we observed upon PUM1 knockdown. Taken together, our studies show that the overexpression of NORAD lncRNA, a decoy for Pumilio RNA binding proteins, robustly increases fetal hemoglobin (HbF) levels without impeding erythroid differentiation, and therefore could serve as a novel approach to treat beta hemoglobinopathies such as sickle cell anemia and beta thalassemia.