Transcriptional regulation by the 7SK snRNP during neuronal differentiation – SEVEN-UP

Promoter-proximal pausing is a key regulatory step in the transcription of protein-coding genes by RNA polymerase II (RNAPII). The transition to productive elongation relies on the positive transcription elongation factor b (P-TEFb), a kinase that promotes synthesis of full-length messenger RNAs through releasing paused RNAPII into gene bodies. The activity of P-TEFb is primarily controlled by the 7SK snRNA that, together with MePCE, LARP7 and HEXIM1 proteins, sequesters P-TEFb into a large kinase-inactive 7SK/P-TEFb snRNP. The 7SK snRNP controls the availability of P-TEFb through releasing or capturing active P-TEFb according to the transcriptional requirements of the cell, making it potentially one of the most effective ways to control RNAPII pausing in human cells. Until now, the 7SK/P-TEFb snRNP was mainly considered as a reservoir of P-TEFb which can be mobilized under stress conditions to increase P-TEFb activity and enable adaptive transcriptional reprogramming through modulation of RNAPII pausing. However, our unpublished observations point to an important function of the 7SK/P-TEFb snRNP during long developmental processes such as cell differentiation. We believe that, in addition to the stress response, the 7SK snRNP may also modulate the availability of P-TEFb and regulate the timing of key transcriptional transitions during embryogenesis. During differentiation, cells acquire specialized transcriptional programs to give rise to appropriate lineages. How P-TEFb activity and RNAPII pausing are modulated during this process remains poorly understood, but the expression of 7SK snRNA is higher in differentiated cells, particularly in the central nervous system. In addition, the loss of 7SK’s chaperone LARP7, which drastically reduces the steady-state level of 7SK snRNA, causes Alazami syndrome, a neurodevelopmental disorder characterized by intellectual disability, primordial dwarfism and distinctive facial dysmorphism. These findings prompted us to investigate the importance of 7SK snRNP in the neuronal lineage. Supported by the literature and by promising preliminary data, SEVEN-UP focuses on the ability of 7SK snRNP to control P-TEFb activity during neuronal differentiation, and aims to understand how it modulates RNAPII pausing and helps shape transcription during development. To address these important questions, we will generate a set of knock-out (KO) cells (7SK- and LARP7-deficient cells) and 7SK snRNP inducible degradation systems in two complementary models of human neuronal differentiation (embryonic carcinoma stem cells and induced pluripotent stem cells ; iPSCs). Using deep sequencing-based methods, we will evaluate the impact of 7SK snRNP loss on RNAPII pausing and transcriptional reprograming during neuronal differentiation. The requirement of the 7SK snRNP in the developing neocortex will also be assessed by microscopy and single-cell transcriptomic analyses in iPSC-derived 3D human neural organoids, which will improve our understanding of Alazami syndrome. A better understanding of the impact of P-TEFb availability on gene expression will provide valuable new information on the need to precisely regulate RNAPII pausing during development in human cells, and on the proposed function of RNAPII during differentiation. This ambitious research program will be carried out by the groups of S. EGLOFF (Centre de Biologie intégrative, Toulouse), A. DAVY (Centre de Biologie intégrative, Toulouse) and S. MURPHY (University of Oxford, UK), who have a long-standing interest in the study of regulatory non-coding RNAs, neurodevelopment and transcription, and who possess complementary expertise (i.e. molecular biology ; developmental biology and organoids ; bioinformatics). In summary, SEVEN-UP lies at the interface of two very active research areas: RNA-mediated gene regulation and cell fate determination, and we believe that the synergy of the consortium ensures a successful research programme.