INTERACTION BETWEEN THE U1 SNRNP-A PROTEIN AND THE 160-KD SUBUNIT OF CLEAVAGE-POLYADENYLATION SPECIFICITY FACTOR INCREASES POLYADENYLATION EFFICIENCY IN-VITRO
Cs. Lutz et al., INTERACTION BETWEEN THE U1 SNRNP-A PROTEIN AND THE 160-KD SUBUNIT OF CLEAVAGE-POLYADENYLATION SPECIFICITY FACTOR INCREASES POLYADENYLATION EFFICIENCY IN-VITRO, Genes & development, 10(3), 1996, pp. 325-337
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.