Phosphorylated RNA polymerase II stimulates pre-mRNA splicing

Abstract

RNA polymerase II (RNAP II) is responsible for transcription of mRNA precursors in eukaryotic cells. Recent studies, however, have suggested that RNAP II also participates in subsequent RNA processing reactions through interactions between the carboxy-terminal domain (CTD) of the RNAP II largest subunit and processing factors. Using reconstituted in vitro splicing assays, we show that RNAP II functions directly in pre-mRNA splicing by influencing very early steps in assembly of the spliceosome. We demonstrate that the phosphorylation status of the CTD dramatically affects activity: Hyperphosphorylated RNAP IIO strongly activates splicing, whereas hypophosphorylated RNAP IIA can inhibit the reaction.

Figure 1

Purification and characterization of the RNAP IIO and IIA. (a) Silver-stained 10% SDS–polyacrylamide gel containing 80 ng of purified hyperphosphorylated (IIO, lane 1) and hypophosphorylated (IIA, lane 2) RNAP II. The positions of size marker proteins are indicated at left; individual RNAP II subunits are indicated at right. The band of ∼40 kD in the IIA sample reflects a breakdown product of a larger subunit, as it was not observed reproducibly in separate analyses of the same preparation. (b) Western blot analysis of 40 ng of purified IIO (lane 1) and IIA (lane 2) probed with mAb 7G5. (c) Western blot analysis of 2 μg of HeLa NE (lane 1), 2 μg of HeLa cell S100 (lane 2), 100 ng of baculovirus recombinant ASF/SF2 (lane 3), 0.5 μg of purified SR proteins (lane 4), and 400 ng each of IIO (lane 5) or IIA (lane 6) probed with mAb 104. (d) In vitro splicing of β-globin pre-mRNA in S100 extract supplemented with buffer (lane 1), 250 (lane 2) and 500 ng (lane 3) of SR proteins, 40 (lane 4) and 80 ng (lane 5) RNAP IIA, and 40 (lane 6) and 80 ng (lane 7) of RNAP IIO. Reaction conditions were as described. Positions of the pre-mRNA, intermediates, and products of splicing are indicated schematically.

Figure 2

RNAP IIO activates and IIA inhibits splicing of β-globin pre-mRNA. (a) In vitro splicing of β-globin pre-mRNA in S100 supplemented with 250 ng of SR proteins (left) or 80 ng of ASF/SF2 (right) in the presence of buffer (lanes 2,13) or increasing amounts (40, 80, or 120 ng) of RNAP IIO (lanes 3–5,14–16), IIA (lanes 6–8,17–19) or GST (lanes 9–11,20). (Lanes 1,12) The precursor RNA and reaction products in S100 without SR proteins or ASF/SF2, respectively. (b) In vitro splicing of β-globin pre-mRNA in NE in the presence of buffer (lane 1), increasing amounts (40, 80, or 120 ng) of RNAP IIO (lanes 2–4), IIA (lanes 5–7), or GST (lanes 8–10).

Figure 3

Characterization of RNAP II’s effects on splicing. Splicing reactions with β-globin pre-mRNA were reconstituted with S100 extract plus 250 ng of SR proteins and the indicated additional components. (a) Extra SR proteins do not affect splicing. Reaction mixtures were supplemented with 120 ng of RNAP IIA (lane 2), RNAP IIO (lane 3), or 120 ng of additional HeLa SR proteins (lane 4). (b) The CTD is necessary but not sufficient. Reactions mixtures were supplemented with 100 ng of RNAP IIA (lanes 2,10), 100 ng of RNAP IIO (lanes 3,11), 60 ng of GST (lanes 4,15), 30 or 60 ng of GST–CTD (lanes 5,6), 30 or 60 ng of phosphorylated GST–CTD (lanes 7,8), or 30, 60, or 120 ng of RNAP IIB (lanes 12–14). (c) Time course of splicing. Reaction mixtures were supplemented with 100 ng of RNAP IIO (lanes 5–8) and incubated at 30°C for the indicated times.

Figure 4

RNAP II affects splicing of two other pre-mRNAs. IgM M1–M2 (a) and HIV tat (b) pre-mRNAs were incubated in S100 plus SR proteins (left) or in NE (right) as in Fig. 2. Reaction mixtures were supplemented with the indicated proteins as follows: (a) IgM M1–M2 splicing; 80 (lane 2) or 120 ng (lanes 3,7) of RNAP IIA, or 80 (lane 4) or 120 ng (lanes 5,8) of RNAP IIO. (b) HIV tat splicing; 40, 80, or 120 ng of RNAP IIA (lanes 3–5), RNAP IIO (lanes 6–8) or 120 ng of GST (lane 9), or 60 or 120 ng of RNAP IIA (lanes 11,12), RNAP IIO (lanes 13,14), or GST (lanes 15,16). (*) The band is a truncated pre-mRNA resulting from artifactual cleavage (Krainer et al. 1990).

Figure 5

RNAP IIO facilitates, but IIA inhibits, spliceosome formation. Standard splicing reaction mixtures with β-globin pre-mRNA and S100 plus 250 ng of SR proteins in the presence of buffer (lanes 1–5), 80 ng of RNAP IIO (lanes 7–12) or 80 ng of RNAP IIA (lanes 13–18) were incubated for the time indicated above each lane, and splicing complexes were processed and fractionated by native gel electrophoresis as described. The positions of the free precursor RNA, nonspecific complex H, prespliceosomal complex A, early spliceosomal complex B, and catalytically active late spliceosomal complex C are indicated at right.