Association of polyadenylation cleavage factor I with U1 snRNP

Abstract

Splicing and polyadenylation factors interact for the control of polyadenylation and the coupling of splicing and polyadenylation. We document an interaction between the U1 snRNP and mammalian polyadenylation cleavage factor I (CF Im), one of several polyadenylation factors needed for the cleavage of the pre-mRNA at the polyadenylation site. Sucrose density gradient centrifugation demonstrated that CF Im separated into two fractions, a light fraction which contained the known CF Im subunits (72, 68, 59, and 25 kD), and a heavy fraction, rich in snRNPs, which contained predominately the 68- and 25-kD CF Im subunits. Using specific antibodies we found that the heavy fraction contains U1 snRNP/CF Im coprecipitable complexes. These complexes were insensitive to RNase treatment, suggesting that the coprecipitation is not due to RNA tethering. In vitro binding experiments show that both the 68- and 25-kD subunits bind to and comigrate with U1 snRNP. In addition, the 25-kD CF Im subunit binds specifically to the 70K protein of U1 snRNP (U1 70K). This binding may account for the CF Im/U1 snRNP interaction. During these studies we found that mAb 2.73 (mAb 2.73), an established U1 70K antibody, efficiently precipitates the bulk of the CF Im from cellular extracts. Because mAb 2.73 has been used in a number of previous studies related to the U1 snRNP and the U1 70K protein, the precipitation of CF Im must be considered in evaluating past and future data based on the use of mAb 2.73.