ALTERNATIVE-3' SPLICE-SITE SELECTION USING HELA-CELL NUCLEAR EXTRACTSPREPARED WITH HIGH-IONIC BUFFERS

To investigate the role of cellular trans-acting factors in alternativ e 3' splice-site selection, a series of HeLa cell nuclear extracts wer e generated with salt washes ranging from 0.4 to 0.8 M salt. These ext racts were tested with human beta-globin pre-mRNAs containing tandem 5 ' or 3' splice-site duplications as the substrates. High-salt (0.6 M a nd higher)-based buffers generated nuclear extracts that differentiall y processed pre-mRNAs containing competing 3' splice sites. High-salt extracts increased the usage of the distal 3' splice site, whereas no shift in 5' splice-site usage could be detected. Western analysis sugg ested that this shift in alternative 3' splice-site selection was not due to changes in the U2 snRNP auxiliary factor or polypyrimidine trac t-binding protein levels.