INTERACTION BETWEEN THE U1 SNRNP-A PROTEIN AND THE 160-KD SUBUNIT OF CLEAVAGE-POLYADENYLATION SPECIFICITY FACTOR INCREASES POLYADENYLATION EFFICIENCY IN-VITRO

We have previously shown that the U1 snRNP-A protein (U1A) interacts w ith elements in the SV40 late polyadenylation signal and that this ass ociation increases polyadenylation efficiency. It was postulated that this interaction occurs to facilitate protein-protein association betw een components of the U1 snRNP and proteins of the polyadenylation com plex. We have now used GST fusion protein experiments, coimmunoprecipi tations and Far Western blot analyses to demonstrate direct binding be tween U1A and the 160-kD subunit of cleavage-polyadenylation specifici ty factor (CPSF). In addition, Western blot analyses of fractions from various stages of CPSF purification indicated that U1A copurified wit h CPSF to a point but could be separated in the highly purified fracti ons. These data suggest that U1A protein is not an integral component of CPSF but may be able to interact and affect its activity. In this r egard, the addition of purified, recombinant U1A to polyadenylation re actions containing CPSF, poly(A) polymerase, and a precleaved RNA subs trate resulted in concentration-dependent increases in both the level of polyadenylation and poly(A) tail length. In agreement with the incr ease in polyadenylation efficiency caused by U1A, recombinant U1A stab ilized the interaction of CPSF with the AAUAAA-containing substrate RN A in electrophoretic mobility shift experiments. These findings sugges t that, in addition to its function in splicing, U1A plays a more glob al role in RNA processing through effects on polyadenylation.