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

Karen Gu; Carina Walpole; Shayarana Gooneratne; Xin Liu; Oscar L Haigh; Kristen J Radford; Mark Mw Chong

doi:10.1002/cti2.1361

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

Clin Transl Immunology. 2022 Jan 23;11(1):e1361. doi: 10.1002/cti2.1361. eCollection 2022.

Authors

Karen Gu^{1

2}, Carina Walpole³, Shayarana Gooneratne¹, Xin Liu¹, Oscar L Haigh³, Kristen J Radford³, Mark Mw Chong^{1

2}

Affiliations

¹ St Vincent's Institute of Medical Research Fitzroy VIC 3065 Australia.
² Department of Medicine (St Vincent's) University of Melbourne Fitzroy VIC 3065 Australia.
³ Mater Research Institute Translational Research Institute The University of Queensland Woolloongabba QLD 4102 Australia.

Abstract

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.

Keywords: DICER; DROSHA; haematopoietic stem cells; mRNA; microRNA.