Combined application of in vivo UV-crosslinking 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 acids to proteins and in vitro label transfer assay was applied to investigate specific interactions between AU-rich sequences (ARE) in the 3' UTR of lymphokine mRNAs and cytoplasmic AU-rich sequence element binding proteins (AUBP) in normal human lymphoblasts and MLA 144 gibbon lymphoid tumor cells. We demonstrate that a pool of cytoplasmic AUBP can be effectively crosslinked to RNA in vivo, suggesting a close association 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, indicating weaker interaction between lymphokine ARE and AUBP in these tumor cells. Similar differences in AUBP-RNA associations were found between the membrane-bound polysomal subfractions of the two cell types where most of the AUBP activity was localized. We suggest that the decreased AUBP-mRNA association in MLA 144 cells might reflect a process concerned with disturbances of mRNA metabolism in the neoplastic phenotype.
Henics
T
T
VA Medical Center, White River Junction, VT 05009, USA.
Nagy
E
E
Rigby
W F
WF
D000818:Animals; D001483:Base Sequence; D016207:Cytokines / Q000378:metabolism; D000882:Haplorhini; D006801:Humans; D008969:Molecular Sequence Data; D012313:RNA / Q000737:chemistry / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D017421:Sequence Analysis; D014407:Tumor Cells, Cultured; D014466:Ultraviolet Rays
D016207:Cytokines; D016601:RNA-Binding Proteins; D012313:RNA