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.

FIGURE 1.

Analysis of [³⁵S]-methionine/cysteine-labeled nuclear proteins by sucrose gradient fractionation and immunoprecipitation using anti-U1 70K antibody, mAb 2.73. Nucleoplasmic proteins from [³⁵S]-methionine/cysteine-labeled 293T cells were separated by centrifugation on 5%–30% sucrose density gradients. Alternate fractions were immunoprecipitated using mAb 2.73 and the immunoprecipitated proteins were separated by 12% SDS-PAGE and autoradiographed. The position of the U1 snRNP specific proteins U1 70K, U1A, and U1C and the Sm core proteins, B/B′, D1, D2, D3, E, and F are shown.

FIGURE 2.

Analysis of unlabeled nuclear proteins by sucrose gradient fractionation and immunoprecipitation using anti-U1 70k antibody, mAb 2.73. Unlabeled nucleoplasmic extract was fractionated on a 5%–30% sucrose gradient. Every other fraction was immunoprecipitated with mAb 2.73 as in Figure 1 ▶. The precipitates were then separated on a 10% SDS-PAGE gel and subjected to Western analysis probing with mAb 2.73. The position of U1 70K protein, the unknown 120–130-kD proteins (X), and the immunoglobulin heavy chain (hc) are indicated.

FIGURE 3.

The anti-U1 70K antibody, mAb 2.73, immunoprecipitates CF Im from the light fractions. (A) The CF Im-rich light fractions were immunoprecipitated with mAb 2.73, mAb 12E12, and mAb 1E1. The immunoprecipitated proteins were separated by 10% SDS-PAGE and subjected to Western analysis probing with antibodies to both the 68-kD and 25-kD subunits of CF Im. The positions of the 72-, 68-, 59-, and 25-kD Cf Im subunits are shown. (B, C) ³⁵S-met-labeled 68-kD (B) and 25-kD (C) subunits, prepared by in vitro transcription and translation, were immunoprecipitated with mAb 2.73 and mAb 1E1. The immunoprecipitated proteins were separated by 10% SDS-PAGE and autoradiographed. The input lane represents 1/10th of the amount of labeled proteins used for the immunoprecipitations.

FIGURE 4.

Association of CF Im subunits with snRNPs. Unlabeled nucleoplasmic extract was fractionated on a 5%–30% gradient. Every other fraction was immunoprecipitated with mAb 2.73 as in Figure 2 ▶. The precipitates were then separated by 10% SDS-PAGE and subjected to Western analysis sequentially probing with anti-CPSF 160 (top panel), mAb 2.73 (second panel), anti-CF Im 68 (third panel), and Anti-U1A (mAb 12E12; bottom panel). The Westerns were stripped between each probing; however, there was some carryover. The positions of significant proteins are indicated as well as the position of the immunoglobulin heavy chain (hc).

FIGURE 5.

In vitro synthesized CF Im 68- and 25-kD subunits associate with heavy complexes, including U1 snRNP. [^35-S]-methionine-labeled CF Im 68-kD and 25-kD subunits were synthesized by in vitro transcription and translation and subjected to 5%–30% sucrose density gradient centrifugation either alone (A) or after mixing with nuclear extract (B and C). In panels A and B, every other gradient fraction was immunoprecipitated using mAb 2.73. In panel C, every other fraction was precipitated with anti-U1A (mAb 1E1); note that panel C is offset from the others because it starts with fraction 2. The precipitated proteins were separated by 10% SDS-PAGE, the gels dried and autoradiographed.

FIGURE 6.

CF Im subunits coimmunoprecipitate with snRNPs. (A) The snRNP-containing heavy fractions were immunoprecipitated with mAb 2.73, mAb 1E1, Y12, and mAb 12E12. The first lane (light) is the light fraction immunoprecipitated with mAb 2.73 for visualization of the 72-, 68-, and 59-kD CF Im subunits (the amount of light fraction precipitated was one-fourth of that used for the heavy fraction precipitation). The precipitated proteins were separated by 10% SDS-PAGE and subjected to Western analysis probing with antibody against the 68-kD subunit of CF-Im (top panel) or antibody against U1A protein (bottom panel). (B) In vitro synthesized, [^35-S]-met-labeled 68- and 25-kD subunits were incubated with nuclear extract. The total snRNP fraction was isolated using affinity chromatography (see text and Materials and Methods), equivalent proportions of the depleted nuclear extract and purified snRNPs were separated by 10% SDS PAGE, and the gel dried and autoradiographed. Positions of significant proteins and the immunoglobulin heavy chain (hc) are indicated.

FIGURE 7.

The association of CF Im subunits with U1 snRNP is not due to RNA tethering. Nuclear extracts were either mock treated (No RNase) or ribonuclease A treated (RNase) and then serially immunoprecipitated, first using anti-U1A mAb 1E1. The supernatant was then precipitated with anti-snRNP Y12. Precipitates were separated by 10% SDS-PAGE and subjected to Western analysis probing with antibody against the 68-kD subunit of CF Im (top panel) mAb 2.73 (middle panel), and 1E1 (bottom panel). The positions of significant proteins are indicated.

FIGURE 8.

GST-25-kD CF Im subunit binds U1 70K protein. The first three lanes show the intensity of one-half of the input amounts of in vitro synthesized ³⁵S-met-labeled luciferase, 68-kD CF Im subunit, and U1 70K protein. The next three lanes show the bead controls where the labeled proteins were incubated with uncharged glutathione beads. The next three lanes show the binding resulting from incubating the labeled proteins with glutathione beads charged with GST-25-kD CF Im subunit (GST CF I 25). The last two lanes show the binding resulting from incubating the labeled proteins with glutathione beads charged with a nonspecific GST fusion protein, GST-IκB. Bound proteins were eluted from the beads and separated by SDS-PAGE (see Materials and Methods).