Post-transcriptional regulation of neural stem cell fate by non-canonical Drosha functions

The ability of stem cells to differentiate in to a variety of cell types is the fundamental basis for life in all organisms. But also later in life, the contribution and ability of stem cells to differentiate in tissue specific cells is crucial and needs to be preserved in adulthood. Yet, stem cells can only be found in a suitable environment called stem cell niches. The niche maintains the stem cell properties of the cells or provides factors required for their differentiation. In the adult brain, there are two distinct niches of adult neural stem cells (NSCs) that can be found in the subventricular zone (SVZ) on the outside walls of the lateral ventricle and the subgranular zone of the hippocampal dentate gyrus (DG). In the latter, NSCs differentiate into granular neurons and astrocytes that may contribute to learning and memory formation. This process is not only strictly regulated on a genetic level, but also post-transcriptional regulation of RNA transcripts strongly contributes to the extremely fine-tuned regulation of adult neurogenesis. A key factor in this process is the microprocessor, a complex best-known for its involvement in the microRNA (miRNA) pathway. The microprocessor consists of Drosha and DGCR8, and processes the pri-miRNA into pre-miRNA by targeting and cleaving distinct hairpin (HP) structures in these transcripts. The main effector of this process is the RNAse III Drosha. However, Drosha has been shown to participate in many additional processes that are not related to miRNA biogenesis and thus are referred to as non-canonical functions. Of special interest for this study is the finding that Drosha is able to target evolutionary conserved HPs that are located in the untranslated regions (UTRs) of mRNAs coding for transcription factors that drive NSCs into specific cell fates. In the adult hippocampal DG, Drosha targets and cleaves the 3’ UTR HP of the mRNA of NFIB, which leads to de destabilization and degradation of the whole mRNA transcript. Drosha thus prevents NFIB protein expression and hinders the cell to undergo oligodendrogenesis. This downregulation of NFIB is required to maintain the DG NSC pool and guarantee proper neuronal development and differentiation. Interestingly, NFIB contains two RNA HPs, located in the 5’ and 3’ UTRs. Both HPs are targeted by Drosha but only the 3’ UTR HP is cleaved. As Drosha is ubiquitously expressed in the entire brain, it remained to be shown how transcripts can escape Drosha cleavage and express NFIB in a cell-specific manner when needed, for example during oligodendrogenesis in other brain regions. In order to answer this question, we developed strategies to identify regulatory proteins that control Drosha-mediated NFIB cleavage in DG NSCs. By using protein immunoprecipitation (IP) followed by dual mass spectrometry (MS2), we identified Drosha interacting proteins in DG NSCs. Moreover, we also determined NFIB HP interacting proteins by RNA pulldown assays and MS2. This data did not only allow us to investigate closer some putative regulators of Drosha cleavage, but also allowed us to characterize the Drosha interactome in NSCs. Additionally, we analyzed the differences between NFIB 3’ UTR and 5’ UTR interaction partners and could show that especially the 5’ UTR HPs interacts closely with ribosomal protein and thus seems to be deeper involved in translation than the 3’ UTR HP. In order to investigate the functional relevance of the Drosha and NFIB mRNA bound proteins, we developed an in vitro GFP reporter assay in DG NSCs to directly monitor Drosha activity upon overexpression of putative cleavage regulating partners. We found that overexpression of certain candidate proteins modulates Drosha cleavage, especially gain-of-function of Scaffold Attachment Factor B1 (Safb1) significantly reduced the read-out GFP signal on mRNA as well as protein levels. This reduction was found to be Drosha-dependent as Drosha cKO in DG NSCs could rescue the reduced GFP levels caused by Safb1 overexpression. Moreover, Safb1 overexpression also reduced the mRNA level of endogenous NFIB. We could confirm direct binding of Safb1 to Drosha by protein IP and to NFIB by crosslinking and immunoprecipitation (CLIP) experiments. To investigate whether Safb1 gain-of-function and the consequent increase in Drosha cleavage has an influence of cell fate determination we overexpressed Safb1 in SVZ NSCs, an oligodendrogenic population, and found reduced oligodendrogenesis in the transfected cells. Thus we identified Safb1 as a key regulator of Drosha-mediated cleavage of NFIB mRNAs, and could show that Safb1 levels influence NSC differentiation and oligodendrogenesis