Poster Presentation 41st Lorne Genome Conference 2020

A proteomic characterisation of the microprocessor complex in mouse embryonic stem cells (#216)

Lawrence Mok 1 , Karen Gu 1 , Ashfaqul Hoque 1 , Jon S Oakhill 1 , Nicholas A Williamson 2 , Mark M.W Chong 1
  1. St Vincent's Institute of Medical Research, Fitzroy, Victoria, Australia
  2. Bio21 Molecular Science and Biotechnology Institute, Parkville, Victoria, Australia

The microprocessor complex is a core component of the microRNA (miRNA) biogenesis machinery, and comprises of two core proteins, Drosha, and Dgcr8. This protein complex binds to and cleaves stem loop structures in long primary miRNA precursor transcripts within the nucleus. This stem-loop is exported into the cytoplasm and processed further to become a mature miRNA which bind to protein coding genes, repressing translation. While the requirement for this complex in miRNA biogenesis is well established, it has also been shown to be involved in mRNA cleavage. The protein complex binds to and cleaves mRNAs preventing translation. Interestingly, despite the ubiquitous presence of both of these proteins within cells, cleavage activity is only active within stem cells. This has been shown to occur in, and critical for the maintenance and differentiation of neural, and embryonic stem (ES) cells.

We propose that post translational phosphorylation or methylation of the microprocessor mediates this stem cell restriction cleavage. Here, we undertake a proteomic characterisation of Drosha and Dgcr8. Both proteins were immunoprecipitated from LIF maintained and retinoic acid differentiated mouse ES cells. Following resolution with SDS-PAGE, gel bands were excised, in-gel trypsin digested and subjected to MS/MS. Phosphorylation and methylation sites were determined using MASCOT, with each potential modification manually checked to ensure sufficient b- and y- ions are detected.

Phosphorylation analysis identified 14 and 15 sites in Drosha and Dgcr8 respectively, with two sites unique to the differentiated state in both. For methylation, there were 18 for Drosha and 24 for Dgcr8, with some multiply methylated. Similarly, there were methylated residues unique to the differentiated state in both proteins. The identification of these modifications will provide an insight on how this stem-cell restriction is established and will be critical for understanding how this cleavage mechanism contributes to stem cell maintenance and differentiation.

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  2. Knuckles P, et al. Drosha regulates neurogenesis by controlling neurogenin 2 expression independent of microRNAs. Nat Neurosci. 2012;15:962-9.
  3. Rolando, C. et al. Multipotency of Adult Hippocampal NSCs In Vivo Is Restricted by Drosha/NFIB. Cell Stem Cell 19, 2016;19:653–62.