The long non-coding RNA SMART regulates human megakaryopoiesis by modulating rRNA processing in the nucleolus

Abstract The process of megakaryopoiesis is complex and regulated by cytokines, transcription factors and non-coding RNAs. Long non-coding RNA (lncRNA) is a class of noncoding RNA >200 bp in length that has essential roles in regulating a variety of biological processes in mammals. Notably, lncRNA expression is species-specific and lineage-specific. Many lncRNAs have been reported in murine megakaryocyte-erythroid precursors cells and have been studied for their roles in erythroblast differentiation as well as human erythropoiesis. However, there is a lack of systematic understanding of the role and mechanism of lncRNA in human megakaryopoiesis. We induced human umbilical cord blood CD34+ cells to differentiate into MKs, performed Ribominus RNA-seq. Bioinformatics analysis identified a lncRNA named snoRD114-Capped Megakaryopoiesis-Associated RNA Transcript (SMART), which is derived from DLK1-DIO3 locus on human chromosome 14 and is 1042 nt in length. Northern blot confirmed the existence of SMART in megakaryocytic cell lines. RT-qPCR analysis revealed SMART is upregulated during megakaryocyte differentiation. RNA FISH and subcellular fractionation analyses showed that SMART is mainly localized in the nucleolus. Antisense oligonucleotides (ASOs), ribonuclease targeting chimeras (RIBOTACs), and CRISPR-Cas system were used to knockdown SMART in MKs derived from CD34+ cells. Knockdown of SMART impaired megakaryocytic differentiation and the expression of surface markers of megakaryocytes decreased. Biotin RNA pulldown, tRSA RNA pulldown and RIP confirmed that SMART interacts with nucleolar proteins. Three-dimensional structured illumination microscopy and Northern blot showed that SMART promotes pre-rRNA processing in the nucleolus and knockdown of SMART impaired rRNA processing during megakaryopoiesis. Our study not only reveals the crucial role of lncRNA and rRNA processing in megakaryopoiesis, but also provides novel insights into the molecular mechanisms underlying human megakaryocyte development and platelet formation.