Project Summary/Abstract Background: Transcriptional mechanisms that regulate epidermal homeostasis and neoplasia have been well established but recently we have discovered that post- transcriptional mechanisms play prominent roles in maintaining epidermal self-renewal. We have shown that the 3'-5' mRNA degradation pathway mediated by the exosome complex is necessary to maintain epidermal self-renewal. Specifically, the exosome subunits, EXOSC7, EXOSC9, and EXSCO10 are necessary to prevent premature differentiation of epidermal stem cells by targeting and degrading transcripts that code for potent pro-differentiation transcription factors. Objective/hypothesis: This proposal seeks to understand the molecular mechanisms governing the progression from normal to neoplastic skin using a RAS driven human epidermal tumor model. Our preliminary data suggests that a 5 subunit exosome subcomplex is upregulated during tumor initiation and targets/degrades transcripts coding for factors that would inhibit tumor growth and survival. Our objective is to characterize the role of each tumor induced exosome subunit in the progression of normal to neoplastic skin. Furthermore we seek to determine the specific transcripts that each exosome subunit binds during tumor initiation to promote tumorigenesis. Specific Aims: (1) To determine the role of exosome subunits on the progression from normal to neoplastic skin and (2) to identify and characterize the transcripts associated with exosome subunits. Study Design: To study epidermal homeostasis in a more clinically relevant setting, we generate 3-dimensionally intact human skin, containing human epidermal cells (that have been permanently knocked down for exosome subunits) in the context of human dermal stroma and basement membrane, regenerated on immune compromised mice. By using this model, we can perform loss of function experiments on exosome subunits in regenerated human skin to characterize their role in epidermal growth, differentiation, and progression to neoplasia. We will use CLIP-Seq to determine the RNAs associated with the exosome subunits during the progression from normal to neoplastic epidermis.