Embryonic stem cell (ESC) fate decisions are regulated by a complex molecular circuitry that requires tight and coordinated gene expression regulations at multiple levels from chromatin organization to mRNA processing. Recently, ribosome biogenesis and translation have emerged as key regulatory pathways that efficiently control stem cell homeostasis. However, the molecular mechanisms underlying the regulation of these pathways remain largely unknown to date. Here, we analyzed the expression of over 300 genes involved in ribosome biogenesis in mouse ESCs and identified RSL24D1 as the most differentially expressed between self-renewing and differentiated ESCs. RSL24D1 is highly expressed in multiple mouse pluripotent stem cell models and its expression profile is conserved in human ESCs. RSL24D1 is associated with nuclear pre-ribosomes and is required for the maturation and the synthesis of 60S subunits in mouse ESCs. Interestingly, RSL24D1 depletion significantly impairs global translation, particularly of key pluripotency factors, including POU5F1 and NANOG, as well as components of the polycomb repressive complex 2 (PRC2). Consistently, RSL24D1 is required for mouse ESC self-renewal and proliferation. Taken together, we show that RSL24D1-dependant ribosome biogenesis is required to both sustain the expression of pluripotent transcriptional programs and silence developmental programs, which concertedly dictate ESC homeostasis.

Embryonic stem cell (ESC) fate decisions are regulated by a complex circuitry that coordinates gene expression at multiple levels from chromatin to mRNA processing. Recently, ribosome biogenesis and translation have emerged as key pathways that efficiently control stem cell homeostasis, yet the underlying molecular mechanisms remain largely unknown. Here, we identified RSL24D1 as highly expressed in both mouse and human pluripotent stem cells. RSL24D1 is associated with nuclear pre-ribosomes and is required for the biogenesis of 60S subunits in mouse ESCs. Interestingly, RSL24D1 depletion significantly impairs global translation, particularly of key pluripotency factors and of components from the Polycomb Repressive Complex 2 (PRC2). While having a moderate impact on differentiation, RSL24D1 depletion significantly alters ESC self-renewal and lineage commitment choices. Altogether, these results demonstrate that RSL24D1-dependant ribosome biogenesis is both required to sustain the expression of pluripotent transcriptional programs and to silence PRC2-regulated developmental programs, which concertedly dictate ESC homeostasis.

Bi-specific T-cell engager antibodies (BITEs) are synthetic soluble molecules derived from antibodies that induce active contact between T-cells and other target cells in the body. BITE therapeutics have shown great promise for the treatment of various forms of cancer; however, the current development process for BITEs is time consuming and costly. BITE development requires empirical testing and characterization of the individual antigen binding domains, followed by extensive engineering and optimization in bi-specific molecular format to generate a molecule with strong biological activity and appropriate characteristics for clinical development. Here, we sought to create a cost efficient high-throughput method for creating and evaluating BITEs using a simplified function first approach to identify bioactive molecules without purification. Using a plasmid with a modular structure to allow high efficiency exchange of either binder arm, we established a simple method to combine many novel tumour-targeting single chain variable (scFv) domains with the well-characterized OKT3 scFv CD3-targeting domain. After generating these novel plasmids, we demonstrate two systems for high throughput functional screening of BITE molecules based on Jurkat T cells (referred to as BITE-J). Using BITE-J we evaluate four EGFRvIII BITEs, identifying two constructs with superior activity. We then confirmed this activity in primary T cells, where novel EGFRvIII-BITEs induced T cell activation and antigen selective tumor killing. We also demonstrate that we can similarly exchange the CD3-interacting element of our bi-modular plasmid. By testing several novel CD3-targeting scFv elements for activity in EGFRvIII-targeted BITEs, we were able to identify highly active BITE molecules with desirable properties for downstream development. In summary, BITE-J presents a low cost, high-throughput method for the rapid assessment of novel BITE molecules without the need for purification and quantification.