Drosha controls dendritic cell development by cleaving messenger RNAs encoding inhibitors of myelopoiesis

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Abstract

To investigate if the microRNA (miRNA) pathway is required for dendritic cell (DC) development, we assessed the effect of ablating Drosha and Dicer, the two enzymes central to miRNA biogenesis. We found that while Dicer deficiency had some effect, Drosha deficiency completely halted DC development and halted myelopoiesis more generally. This indicated that while the miRNA pathway did have a role, it was a non-miRNA function of Drosha that was particularly critical. Drosha repressed the expression of two mRNAs encoding inhibitors of myelopoiesis in early hematopoietic progenitors. We found that Drosha directly cleaved stem-loop structure within these mRNAs and that this mRNA degradation was necessary for myelopoiesis. We have therefore identified a mechanism that regulates the development of DCs and other myeloid cells.

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Figure 1: Drosha deficiency perturbs myeloid populations more severely than does Dicer deficiency.
Figure 2: Drosha deficiency impairs the DC development of cells cultured with Flt3L, but Dicer deficiency does not.
Figure 3: Drosha deficiency impairs DC development at an early stage of myelopoiesis.
Figure 4: The requirement for Drosha in myelopoiesis is not circumvented by inflammatory or cytokine challenge.
Figure 5: Gene-expression profiling of Drosha- or Dicer-deficient LSK cells.
Figure 6: Derepression of Myl9 or Todr1 in HSCs is responsible for the DC-developmental block in Drosha deficient mice.
Figure 7: The inhibitors of myelopoiesis Myl9 and Todr1 are direct endonucleolytic targets of Drosha in hematopoietic progenitors.

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Acknowledgements

We thank C. Yates for animal husbandry. Supported by the National Health and Medical Research Council, Australia (637338, 1004541, 1042211 and 1079586 to M.M.W.C.; 1037321, 1043414 and 1080321 to A.M.L.), the Juvenile Diabetes Research Foundation (5-2011-100 to M.M.W.C.), the Diabetes Australia Research Trust (Y13G-CHOM to M.M.W.C.), the Australian Research Council (M.M.W.C.) and the Victorian State Government Operational Infrastructure Support and Australian National Health and Medical Research Council Research Institute Infrastructure Support Scheme.

Author information

T.M.J. performed most of the experiments with help from A.A.K., J.H.C.Y. and Y.Z.; M.C. and A.K. performed the bioinformatics analyses; and T.M.J., A.M.L., Y.Z. and M.M.W.C. designed the study, analyzed the data and wrote the manuscript.

Correspondence to Mark M W Chong.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Residual splenic DCs in Drosha-deficient chimeras have escaped deletion.

The residual splenic DCs in Tamoxifen-treated Droshafl/fl CreER chimeras were sorted by FACS, then Drosha mRNA levels were measured by quantitative RT-PCR. Source data

Supplementary Figure 2 The accumulation of LSKs and CMPs caused by Drosha deficiency is cell intrinsic.

Comparison of LSK and CMP populations between the CD45.2 experimental and CD45.1 wild type supporting component in the bone marrow of chimeric mice following Tamoxifen treatment. The data shown is the mean +/- S.D. of twelve (control, Droshafl/fl CreER) or nine (Dicerfl/fl CreER) animals for each genotype from three independent experiments. Source data

Supplementary Figure 3 Drosha ablation and miRNA expression are equivalent in LSK-, CMP-, CLP- and CDP-derived cultures.

Quantitative RT-PCR or Taqman-based quantitative RT-PCR determined levels of Drosha mRNA or a panel of mature miRNAs, respectively. The data shown is the mean +/- S.D. of one or two (LSK culture Drosha levels) independent experiments. Source data

Supplementary Figure 4 Identification of cleavage sites within Myl9 mRNA and Todr1 mRNA.

(a) Schematic of 5’P capture experiment. PolyA transcripts were purified from total RNA isolated from lineage-depleted bone marrow cells. An adaptor was ligated specifically to transcripts exhibiting a 5’ monophosphate (a mark of endonucleolytic cleavage) and reverse transcribed. Tiled PCRs were then performed using a forward primer in the adaptor and reverse primers within Myl9 and Todr1 mRNA. Thus, PCR products could only be obtained if Myl9 and Todr1 contained a 5’ phosphate. PCR products were Sanger sequenced. The junction between the adaptor and mRNA denoted the original cleavage site. (b) Location of cleavage sites detected in Myl9 and Todr1 mRNA. Blue and red text denote stem or loop structure, respectively.

Supplementary Figure 5 Mutagenesis of the Todr1 stem-loop to disrupt base pairing.

Site-directed mutagenesis was used to introduce mismatch mutations into the sequence of the 5’ arm of the putative stem-loop structure within Todr1 mRNA.

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Supplementary Figures 1–5 and Supplementary Tables 1 and 2. (PDF 1482 kb)

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Johanson, T., Keown, A., Cmero, M. et al. Drosha controls dendritic cell development by cleaving messenger RNAs encoding inhibitors of myelopoiesis. Nat Immunol 16, 1134–1141 (2015) doi:10.1038/ni.3293

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