Our current understanding of induced pluripotent stem cell (iPSC) generation has almost entirely been shaped by studies performed on reprogramming fibroblasts. However, whether the resulting model universally applies to the reprogramming process of other cell types is still largely unknown. By characterizing and profiling the reprogramming pathways of fibroblasts, neutrophils, and keratinocytes, we unveil that key events of the process, including loss of original cell identity, mesenchymal to epithelial transition, the extent of developmental reversion, and reactivation of the pluripotency network, are to a large degree cell-type specific. Thus, we reveal limitations for the use of fibroblasts as a universal model for the study of the reprogramming process and provide crucial insights about iPSC generation from alternative cell sources.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a ubiquitous and abundant protein that participates in cellular energy production. GAPDH normally exists in a soluble form; however, following necrosis, GAPDH and numerous other intracellular proteins convert into an insoluble disulfide-cross-linked state via the process of “nucleocytoplasmic coagulation.” Here, free radical-induced aggregation of GAPDH was studied as an in vitro model of nucleocytoplasmic coagulation. Despite the fact that disulfide cross-linking is a prominent feature of GAPDH aggregation, our data show that it is not a primary rate-determining step. To identify the true instigating event of GAPDH misfolding, we mapped the post-translational modifications that arise during its aggregation. Solvent accessibility and energy calculations of the mapped modifications within the context of the high resolution native GAPDH structure suggested that oxidation of methionine 46 may instigate aggregation. We confirmed this by mutating methionine 46 to leucine, which rendered GAPDH highly resistant to free radical-induced aggregation. Molecular dynamics simulations suggest that oxidation of methionine 46 triggers a local increase in the conformational plasticity of GAPDH that likely promotes further oxidation and eventual aggregation. Hence, methionine 46 represents a “linchpin” whereby its oxidation is a primary event permissive for the subsequent misfolding, aggregation, and disulfide cross-linking of GAPDH. A critical role for linchpin residues in nucleocytoplasmic coagulation and other forms of free radical-induced protein misfolding should now be investigated. Furthermore, because disulfide-cross-linked aggregates of GAPDH arise in many disorders and because methionine 46 is irrelevant to native GAPDH function, mutation of methionine 46 in models of disease should allow the unequivocal assessment of whether GAPDH aggregation influences disease progression.

The establishment and maintenance of pluripotency depend on precise coordination of gene expression. We establish serine-arginine-rich splicing factor 3 (SRSF3) as an essential regulator of RNAs encoding key components of the mouse pluripotency circuitry, SRSF3 ablation resulting in the loss of pluripotency and its overexpression enhancing reprogramming. Strikingly, SRSF3 binds to the core pluripotency transcription factor Nanog mRNA to facilitate its nucleo-cytoplasmic export independent of splicing. In the absence of SRSF3 binding, Nanog mRNA is sequestered in the nucleus and protein levels are severely downregulated. Moreover, SRSF3 controls the alternative splicing of the export factor Nxf1 and RNA regulators with established roles in pluripotency, and the steady-state levels of mRNAs encoding chromatin modifiers. Our investigation links molecular events to cellular functions by demonstrating how SRSF3 regulates the pluripotency genes and uncovers SRSF3-RNA interactions as a critical means to coordinate gene expression during reprogramming, stem cell self-renewal and early development.