COMBINED APPLICATION OF IN-VIVO UV-CROSS-LINKING AND IN-VITRO LABEL TRANSFER IN THE EXAMINATION OF AU-RICH SEQUENCE BINDING-PROTEIN RNA INTERACTIONS

A combination of in vivo UV light-induced crosslinking of nucleic acid s to proteins and in vitro label transfer assay war applied to investi gate specific interactions between AU-rich sequences (ARE) in the 3' U TR of lymphokine mRNAs and cytoplasmic AU-rich sequence element bindin g proteins (AUBP) in normal human lymphoblasts and MLA 144 gibbon lymp hoid tumor cells. We demonstrate that a pool of cytoplasmic AUBP can b e effectively crosslinked to RNA in vivo, suggesting a close associati on of these proteins with ARE sequences in the cytoplasm. We also show that the UV-crosslinked AUBP pool is markedly reduced in malignantly transformed MLA 144 cells compared with normal lymphoblasts, indicatin g weaker interaction between lymphokine ARE and AUBP in these tumor ce lls. Similar differences in AUBP-RNA associations were found between t he membrane-bound polysomal subfractions of the two cell types where m ost of the AUBP activity was localized. We suggest that the decreased AUBP-mRNA association in MLA 144 cells might reflect a process concern ed with disturbances of mRNA metabolism in the neoplastic phenotype.