Recursive splicing and mRNA polyadenylation regulatory circuits govern homeostasis and cell cycle potency of pluripotent cells.

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Mammalian transcriptomes are extremely complex, with large amounts of RNA copied from non-coding regions, like introns that occupy most of the length of protein-coding genes and are now understood to contribute to gene expression regulation. Our lab was of the first to uncover the existence of recursive splicing in human cells, an elegant mechanism for the stepwise processing of long introns. However, despite significant recent advances, including our work in the first funding round of this Priority Program, many aspects of recursive splicing remain unknown, and are the focal point of this proposal. We need to decipher how specific RS sites are actually selected in the production of mature mRNAs, and we need to dissect the contribution of recursive splicing in cell homeostasis and disease. For this proposal, we present preliminary data, generated within the SPP1935, uncovering a link between recursive splicing sites and disease manifestation across human cell types, as well as results highlighting recursive splicing regulatory circuits in human pluripotent stem cells that are governed by RNPs involving mRNAs and variant U1 transcripts. Thus, we now aim at dissecting the mRNP code behind these circuits by combining genomics, biochemical, and CRISPR/Cas9 genome editing approaches with in vitro differentiation of human pluripotent cells intro different lineages. We see this as a prerequisite for understanding the impact of recursive splicing in cell homeostasis and development, as well as in the revaluation of the functional partition of nuclear space as regards the transcription cycle of human genes. We are convinced that this novel layer of gene expression regulation presents a entryway into deciphering the complexity of mammalian mRNA processing and has implications for both development and disease.