Abstract
Background:
In sickle cell disease (SCD), erythrocyte reactive oxygen species (ROS) production and oxidative stress play a critical role in vaso-occlusion, a hallmark of SCD. Small noncoding nucleolar RNAs (snoRNAs) of the Rpl13a locus have been described as regulators of ROS levels. However, whether Rpl13a snoRNAs are present in sickle red blood cells (RBCs) and regulate ROS levels and whether they contribute to SCD pathophysiology remain unknown.
Methods:
To determine whether sickle RBC ROS levels are associated with Rpl13a snoRNA levels and identify the mechanism by which they regulate ROS and snoRNAs' effects on SCD hemodynamics, we used human RBCs, Rpl13a snoRNA knockout sickle mice, K562 U32a, U33, U34, U35a, and the control U25 knockout mutants generated by CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 protein)-targeted genome editing, and genetic targeting with antisense oligonucleotides.
Results:
Excessive ROS production in sickle RBCs of patients with SCD is associated with high Rpl13a snoRNAs U32a, U33, U34, and U35a levels. U32a, U34, and U35a regulate ROS and hydrogen peroxide levels in sickle erythroid populations by modulating peroxidase activity. This was due to U32a- and U34-guided 2'-O-methylation on Prdx2 (peroxiredoxin 2) messenger RNA, a modification conveyed by fibrillarin during erythropoiesis, subsequently reducing Prdx2 expression and activity. The snoRNA U35a impaired Prdx2 expression/activity but independently of Prdx2 messenger RNA 2'-O-methylation. Excess sickle RBC ROS increased in turn Rpl13a snoRNAs levels. In vivo targeting combinations of U34+U35a and U32a+U34+U35a in sickle mice with antisense oligonucleotide blunted RBC ROS generation, improved erythropoiesis and anemia, alleviated leukocytosis and endothelial damage, diminished cell adhesion in inflamed vessels and vaso-occlusion, restored blood flow, and reduced animal mortality.
Conclusions:
Rpl13a snoRNAs U34 and U35a specifically increase ROS levels, which, in turn, regulate snoRNA expression, in sickle erythroid cells, modulating Prdx2 expression/activity, subsequently impairing hemodynamics. Targeted U34+U35a with antisense oligonucleotide may represent a novel and safe therapy to ameliorate erythropoiesis and downstream events in SCD.