<i>Rpl13a</i> snoRNAs <i>U34</i> and <i>U35a</i>: New Targets for Sickle Cell Disease Complications.

Abstract

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 <i>Rpl13a</i> locus have been described as regulators of ROS levels. However, whether <i>Rpl13a</i> snoRNAs are present in sickle red blood cells (RBCs) and regulate ROS levels and whether they contribute to SCD pathophysiology remain unknown. To determine whether sickle RBC ROS levels are associated with <i>Rpl13a</i> snoRNA levels and identify the mechanism by which they regulate ROS and snoRNAs' effects on SCD hemodynamics, we used human RBCs, <i>Rpl13a</i> snoRNA knockout sickle mice, K562 <i>U32a</i>, <i>U33</i>, <i>U34</i>, <i>U35a</i>, and the control <i>U25</i> knockout mutants generated by CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 protein)-targeted genome editing, and genetic targeting with antisense oligonucleotides. Excessive ROS production in sickle RBCs of patients with SCD is associated with high <i>Rpl13a</i> snoRNAs <i>U32a</i>, <i>U33</i>, <i>U34</i>, and <i>U35a</i> levels. <i>U32a</i>, <i>U34</i>, and <i>U35a</i> regulate ROS and hydrogen peroxide levels in sickle erythroid populations by modulating peroxidase activity. This was due to <i>U32a</i>- and <i>U34</i>-guided 2'-O-methylation on <i>Prdx2</i> (peroxiredoxin 2) messenger RNA, a modification conveyed by fibrillarin during erythropoiesis, subsequently reducing Prdx2 expression and activity. The snoRNA <i>U35a</i> impaired Prdx2 expression/activity but independently of <i>Prdx2</i> messenger RNA 2'-O-methylation. Excess sickle RBC ROS increased in turn <i>Rpl13a</i> snoRNAs levels. In vivo targeting combinations of <i>U34</i>+<i>U35a</i> and <i>U32a</i>+<i>U34</i>+<i>U35a</i> 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. <i>Rpl13a</i> snoRNAs <i>U34</i> and <i>U35a</i> specifically increase ROS levels, which, in turn, regulate snoRNA expression, in sickle erythroid cells, modulating Prdx2 expression/activity, subsequently impairing hemodynamics. Targeted U34+<i>U35a</i> with antisense oligonucleotide may represent a novel and safe therapy to ameliorate erythropoiesis and downstream events in SCD.