ldentification of pTINCR: a new ubiquitin-like protein that promotes epithelial differentiation and suppresses tumor growth

Abstract

[eng] Recent advances in computational analyses, peptidomics and ribosome profiling have revealed that many regions previously annotated as non-coding are in fact translated into a myriad of bioactive proteins, largely overlooked until now. These small proteins, shorter than 100 amino acids, are called microproteins, micropeptides or SEPs (from sORF-encoded peptides). To date, only a subset of them have been functionally characterized, and they have been shown to play essential functions regulating a plethora of fundamental processes such as DNA repair, RNA splicing, or cell metabolism. Importantly, mounting evidence suggest that microproteins also have a role in response to stress, such as oxidative stress, viral and bacterial infection, tissue injury and even tumor initiation. The acquisition of cell identity by cell differentiation is essential for proper tissue function. In epithelial tissues, cell polarity plays a pivotal role during tissue morphogenesis and in the maintenance of the specialized functions and differentiated status of epithelial cells. On the other hand, loss of epithelial cell identity is nowadays considered an essential initial step in tumorigenesis, and epithelial cell polarity has been positioned as a key tumor suppressive mechanism in epithelial cancers. In this doctoral thesis, we aimed to find and characterize novel microproteins with an important role in cell identity and cancer. We have identified pTINCR, an 87-amino acid microprotein encoded by TINCR, a gene misannotated as a long non-coding RNA. pTINCR is an evolutionary conserved microprotein expressed in skin and several epithelial tissues. By gain- and loss-of-function studies, we have demonstrated that pTINCR is a key regulator of epithelial cell differentiation in vitro and promotes epidermal differentiation in vivo. Additionally, pTINCR is upregulated upon cellular damage by the tumor suppressor p53 and it is required for the DNA damage-induced differentiation response. Consistent with its tumor suppressive role, pTINCR expression is lost in human cutaneous squamous cell carcinomas (cSCCs), and its overexpression reduces malignancy in cSCC patient-derived xenografts (PDXs). Moreover, the expression of TINCR correlates with better prognosis in several epithelial cancers. At the molecular level, pTINCR is a novel ubiquitin-like protein (UBL) which can bind to SUMO through its SUMO interacting motif (SIM) and regulate SUMO conjugation. Importantly, we have identified two pTINCR binding partners, NONO and CDC42, and showed that pTINCR enhances their SUMOylation. NONO is a nuclear protein involved in transcriptional regulation, pre-mRNA splicing and nuclear retention of defective RNAs. We have shown that pTINCR triggers a specific splicing program related to epithelial cells, probably through its interaction with NONO. On the other hand, CDC42 is a Rho-GTPase critical for actin cytoskeleton remodeling and the establishment of cell polarity. We demonstrate that pTINCR binds to CDC42 and promotes its SUMOylation and activation, triggering an epithelial pro-differentiation cascade. In summary, we have identified pTINCR as a novel UBL that regulates epithelial differentiation and has tumor suppressor activity. Our results further expand our knowledge on the yet unexplored field of microproteins and suggest that the proteome hidden in previously assumed non-coding RNAs can indeed be a source of new regulators of cell identity relevant for cancer.