DROSHA but not DICER is required for human haematopoietic stem cell function

• Published in: Clinical & translational immunology 2022, Vol.11 (1), p.e1361-n/a
• Main Authors: Gu, Karen, Walpole, Carina, Gooneratne, Shayarana, Liu, Xin, Haigh, Oscar L, Radford, Kristen J, Chong, Mark MW
• Format: Article
• Language: English
• Subjects: Biosynthesis; Blood; CD34 antigen; Cell cycle; Cord blood; Dendritic cells; DICER; DROSHA; Ectopic expression; Enzymes; Flow cytometry; Gene expression; Genes; haematopoietic stem cells; Hematopoietic stem cells; microRNA; MicroRNAs; miRNA; mRNA; Original; Pluripotency; Stem cell transplantation; Stem cells
• ISSN: 2050-0068; 2050-0068
• DOI: 10.1002/cti2.1361

Objectives DROSHA and DICER have central roles in the biogenesis of microRNAs (miRNAs). However, we previously showed that in the murine system, DROSHA has an alternate function where it directly recognises and cleaves protein‐coding messenger (m)RNAs and this is critical for safeguarding the pluripotency of haematopoietic stem cells (HSCs). Maintenance of murine HSC function is dependent on DROSHA‐mediated cleavage of two mRNAs, Myl9 and Todr1. The goal of this study is to determine whether this pathway is conserved in human HSCs. Methods DROSHA and DICER were knocked down in human cord blood CD34+ HSCs with short hairpin RNAs. The function of HSCs was analysed in vitro and in humanised mice. Analysis of mRNA cleavage was performed by capture of 5′ phosphorylated RNAs. Results Consistent with murine HSCs, DROSHA knockdown impaired the differentiation of human HSCs in vitro and engraftment into humanised mice, whereas DICER knockdown had no impact. DROSHA cleaves the MYL9 mRNA in human HSCs and DROSHA deficiency resulted in the accumulation of the mRNA. However, ectopic expression of MYL9 did not impair human HSC function. We were unable to identify a human homolog of Todr1. Conclusion A miRNA‐independent function of DROSHA is critical for the function of human HSCs. DROSHA directly recognises and degrades mRNAs in humans HSCs. However, unlike in murine HSCs, the degradation of the MYL9 mRNA alone is not critical for human HSC function. Therefore, DROSHA must be inhibiting other targets and/or has another miRNA‐independent function that is essential for safeguarding the pluripotency of human HSCs. In this study, we show that DROSHA but not DICER is required for the function of human haematopoietic stem cells. MicroRNA biogenesis requires both these RNase III enzymes. This therefore indicates that a non‐microRNA function of DROSHA is responsible for maintaining the pluripotency of these cells.