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Rationale: RBPs (RNA binding proteins) play critical roles in the cell by regulating mRNA transport, splicing, editing, and stability. The RBP SRSF3 (serine/arginine-rich splicing factor 3) is essential for blastocyst formation and for proper liver development and function. However, its role in the heart has not been explored. Objective: To investigate the role of SRSF3 in cardiac function. Methods and Results: Cardiac SRSF3 expression was high at mid gestation and decreased during late embryonic development. Mice lacking SRSF3 in the embryonic heart showed impaired cardiomyocyte proliferation and died in utero. In the adult heart, SRSF3 expression was reduced after myocardial infarction, suggesting a possible role in cardiac homeostasis. To determine the role of this RBP in the adult heart, we used an inducible, cardiomyocyte-specific SRSF3 knockout mouse model. After SRSF3 depletion in cardiomyocytes, mice developed severe systolic dysfunction that resulted in death within 8 days. RNA-Seq analysis revealed downregulation of mRNAs encoding sarcomeric and calcium handling proteins. Cardiomyocyte-specific SRSF3 knockout mice also showed evidence of alternative splicing of mTOR (mammalian target of rapamycin) mRNA, generating a shorter protein isoform lacking catalytic activity. This was associated with decreased phosphorylation of 4E-BP1 (eIF4E-binding protein 1), a protein that binds to eIF4E (eukaryotic translation initiation factor 4E) and prevents mRNA decapping. Consequently, we found increased decapping of mRNAs encoding proteins involved in cardiac contraction. Decapping was partially reversed by mTOR activation. Conclusions: We show that cardiomyocyte-specific loss of SRSF3 expression results in decapping of critical mRNAs involved in cardiac contraction. The molecular mechanism underlying this effect likely involves the generation of a short mTOR isoform by alternative splicing, resulting in reduced 4E-BP1 phosphorylation. The identification of mRNA decapping as a mechanism of systolic heart failure may open the way to the development of urgently needed therapeutic tools.

Abstract:Embryonic stem cells (ESC) have the potential to generate all the cell lineages that will form the body. However, the molecular mechanisms underlying ESC differentiation and especially the role of alternative splicing in this process remain poorly understood. Here, we show that the alternative splicing regulator Mbnl1 promotes the generation of the atypical calcineurin Aβ variant CnAβ1 in mouse ESCs (mESC). CnAβ1 has a unique C-terminal domain that drives its localization mainly to Golgi apparatus by interacting with Cog8. CnAβ1 regulates the intracellular localization and activation of the mTORC2 complex. CnAβ1 knockdown results in delocalization of mTORC2 from the membrane to the cytoplasm, inactivation of the AKT/GSK3β/β-catenin signaling pathway and defective mesoderm specification. In summary, we unveil here the structural basis for the mechanism of action of CnAβ1 and its role in the differentiation of mESCs to the mesodermal lineage. Graphical Abstract Regulation of mesoderm specification by CnAβ1Gómez-Salinero et al. 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 Regulation of mesoderm specification by CnAβ1Gómez-Salinero et al. knockdown results in delocalization of mTORC2 from the membrane to the cytoplasm, inactivation of the AKT/GSK3β/-catenin signaling pathway and defective mesoderm specification. In summary, we unveil here the structural basis for the mechanism of action of CnAβ1 and its role in the differentiation of mESCs to the mesodermal lineage. Page 2 of 36

Cellular senescence is an antiproliferative response with essential functions in tumor suppression and tissue homeostasis. Here we show that SIX1, a member of the SIX family of homeobox transcriptional factors, is a novel repressor of senescence. Our data show that SIX1 is specifically downregulated in fibroblasts upon oncogenic stress and other pro-senescence stimuli, as well as in senescent skin premalignant lesions. Silencing of SIX1 in human fibroblasts suffices to trigger senescence, which is mediated by p16INK4A and lacks a canonical senescence-associated secretory phenotype. Interestingly, SIX1-associated senescence is further characterized by the expression of a set of development and differentiation-related genes that significantly overlap with genes associated with SIX1 in organogenesis or human tumors, and show coincident regulation in oncogene-induced senescence. Mechanistically, we show that gene regulation by SIX1 during senescence is mediated, at least in part, by cooperation with Polycomb repressive complexes. In summary, our results identify SIX1, a key development regulator altered in human tumors, as a critical repressor of cellular senescence, providing a novel connection between senescence, differentiation and tumorigenesis.