LOCALIZATION AND CHARACTERIZATION OF THE RNA-BINDING PROTEIN TLS IN SKIN AND STRATIFIED MUCOSA

Translocated in liposarcoma (TLS), a member of the Ewing's sarcoma fam ily of RNA binding proteins, is targeted to the product of RNA POL II and functions in nuclear events as well as in nuclear-cytoplasmic tran sport of mRNA. It has been most extensively studied in cell lines, but was identified in several rat tissues by northern blot analysis, with adipose tissue showing the highest expression followed by whole skin. This paper describes a protein with amino acid sequence homology to T LS that was isolated from bovine tongue epithelium using an affinity c olumn made with an antibody to the cornified envelope precursor sciell in. Using reverse transcriptase polymerase chain reaction technology a nd total RNA isolated from bovine tongue epithelium, a cDNA was obtain ed whose nucleotide sequence coded for a protein homologous to human T LS. Nuclear staining in all layers of human epidermis and bovine strat ified epithelium was observed with an antibody to TLS, whereas periphe ral staining of the upper layers of these tissues was observed with th e antibody to sciellin. Cultured cells gave similar results; however, adult tissue required boiling in citrate buffer to unmask antigenic si tes before reacting with the TLS antibody. Western blots of extracts o f human and bovine keratinocytes using TLS and sciellin antibodies sho wed that the two proteins shared at least one epitope, but that they w ere different. TLS was lost from the nucleus following inhibition of R NA POL II activity and the protein was identified in CNBr extracts of purified keratinocytes cornified envelopes by western blot. These resu lts clearly indicate that TLS functions as an RNA binding protein in k eratinocytes in vivo and in vitro. Furthermore the sequestration of TL S to the cell envelope may play a role in regulating its nuclear-cytop lasmic transport and effect its role in transcription.