Systemic studies of RNA binding proteins in stem cell differentiation and pluripotency

What mechanisms govern and maintain cell states during the process of differentiation is a pivotal question in science. What factors govern the commitment of developmental progenitors from pluripotent stem cells is a representative example of this question. Studies of transcriptional, signaling and chromatin regulation have been highly instrumental for elucidating mechanisms pluripotency maintenance. Nevertheless, current knowledge falls short in explaining the exit from pluripotency and its coupling to lineage commitment. It is unclear how pluripotency and differentiation become stabilized in a mutually exclusive manner. Here, I deepen our knowledge concerning post-transcriptional mechanisms in pluripotency-differentiation transition. For this purpose I first characterize by quantitative mass spectrometry the changes that occur in the mRNA bound proteome (RBPome) and identify extensive dynamic rearrangements of the RBPome during early embryonic development, from naive to primed stem cell state and to purified primitive streak progenitors (Chapter I). In parallel I identified developmental post-transcriptional processing landscape and show that the dynamic mRNA binding of the RNA-binding protein TDP-43 is critical in pluripotent stem cells (PSCs) for the choice between self-renewal and differentiation/ pluripotency breakdown (Chapter II). In detail, I discovered that TDP-43 directly regulates an evolutionary conserved switch in alternative polyadenylation (APA) of hundreds of transcripts during early differentiation of mouse and human PSCs. Functional analysis revealed that TDP-43 integrates into pluripotency circuitry by repressing the production of lengthened transcripts of the pluripotency factor SOX2, which is targeted for degradation by miR-21. Furthermore, in pluripotent stem cells TDP-43 also promotes self-renewal by repressing the formation of paraspeckles, membraneless nuclear compartments found only in differentiated cells, by enhancing production of short isoform of the lncRNA NEAT1. Conversely, reduction of TDP-43 during differentiation triggers a short-to-long isoform switch of NEAT1, which polymerizes paraspeckles that in turn recruit TDP-43 and relocalise it away from its other RNA targets. Consistent with this cross-regulation, TDP-43 inhibits differentiation and improves somatic cell reprogramming, while paraspeckles promote early differentiation. These findings reveal how the exit of pluripotency is regulated by a complex posttranscriptional network, which is functionally independent from lineage choices. Apart from its role in the exit of pluripotency, this cross-regulation between paraspeckles and TDP-43 has implications in cancer and neurodegeneration