RNA binding activity of heterodimeric splicing factor U2AF: at least one RS domain is required for high-affinity binding

Abstract

The pre-mRNA splicing factor U2AF (U2 small nuclear ribonucleoprotein particle [snRNP] auxiliary factor) plays a critical role in 3' splice site selection. U2AF binds site specifically to the intron pyrimidine tract between the branchpoint and the 3' splice site and targets U2 snRNP to the branch site at an early step in spliceosome assembly. Human U2AF is a heterodimer composed of large (hU2AF65) and small (hU2AF35) subunits. hU2AF65 contains an arginine-serine-rich (RS) domain and three RNA recognition motifs (RRMs). hU2AF35 has a degenerate RRM and a carboxyl-terminal RS domain. Genetic studies have recently shown that the RS domains on the Drosophila U2AF subunit homologs are each inessential and might have redundant functions in vivo. The site-specific pyrimidine tract binding activity of the U2AF heterodimer has previously been assigned to hU2AF65. While the requirement for the three RRMs on hU2AF65 is firmly established, a role for the large-subunit RS domain in RNA binding remains unresolved. We have analyzed the RNA binding activity of the U2AF heterodimer in vitro. When the Drosophila small-subunit homolog (dU2AF38) was complexed with the large-subunit (dU2AF50) pyrimidine tract, RNA binding activity increased 20-fold over that of free dU2AF50. We detected a similar increase in RNA binding activity when we compared the human U2AF heterodimer and hU2AF65. Surprisingly, the RS domain on dU2AF38 was necessary for the increased binding activity of the dU2AF heterodimer. In addition, removal of the RS domain from the Drosophila large-subunit monomer (dU2AF50DeltaRS) severely impaired its binding activity. However, if the dU2AF38 RS domain was supplied in a complex with dU2AF50DeltaRS, high-affinity binding was restored. These results suggest that the presence of one RS domain of U2AF, on either the large or small subunit, promotes high-affinity pyrimidine tract RNA binding activity, consistent with redundant roles for the U2AF RS domains in vivo.

FIG. 1

SDS-polyacrylamide gel electrophoresis of purified recombinant U2AF proteins. Samples from the final preparations of U2AF monomers and heterodimers were run on an SDS–12% polyacrylamide gel and stained with Coomassie blue. All individually purified monomers are His₆ tagged. dU2AF⁵⁰ is His₆ tagged in the dU2AF heterodimers, and hU2AF³⁵ is His₆ tagged in the hU2AF heterodimer. Marker sizes are indicated in kilodaltons.

FIG. 2

dU2AF³⁸ interacts weakly with pyrimidine tract RNA. For electrophoretic mobility shift analysis of dU2AF³⁸ with MINX (WT and MUT) pyrimidine tract RNA, 200 pM ³²P-labeled RNA oligonucleotide was incubated in the presence (+) or absence (−) of 5 μM dU2AF³⁸ complexed with the His₆-dU2AF⁵⁰ interaction domain (L/d38). Protein-RNA complexes (C) and unbound RNA (F) were separated by electrophoresis through a native polyacrylamide gel and visualized by autoradiography.

FIG. 3

The dU2AF heterodimer binds pyrimidine tract RNA with higher affinity than the dU2AF⁵⁰ monomer. (A) Electrophoretic mobility shift analysis of dU2AF heterodimer and dU2AF⁵⁰ monomer interaction with MINX-WT pyrimidine tract RNA. dU2AF⁵⁰ monomer protein concentrations were 5,000, 1,000, 200, 40, and 8 nM (lanes 2 to 6). dU2AF heterodimer concentrations were 5,000, 1,000, 200, 40, and 8 nM (lanes 7 to 11). Proteins were incubated with 100 pM ³²P-labeled RNA oligonucleotide. Protein-RNA complexes (C) and unbound RNA (F) were separated by electrophoresis through a native polyacrylamide gel and visualized by autoradiography. Occasionally, the dU2AF⁵⁰/dU2AF³⁸ heterodimer-RNA complex appeared to be retained in the sample well. However, this effect was variable. The sequence of the MINX-WT pyrimidine tract oligonucleotide is displayed below the autoradiogram. The apparent K_d for the dU2AF⁵⁰ monomer was ∼2.2 × 10⁻⁶ M. The apparent K_d for the dU2AF heterodimer was ∼1.0 × 10⁻⁷ M. (B) Electrophoretic mobility shift analysis of dU2AF heterodimer and dU2AF⁵⁰ monomer interaction with MINX-MUT pyrimidine tract RNA. Protein concentrations were identical to those in panel A. The sequence of the MINX-MUT pyrimidine tract oligonucleotide is displayed below the autoradiogram. The apparent K_d for the dU2AF⁵⁰ monomer could not be determined. The apparent K_d for the U2AF heterodimer was ∼1.1 × 10⁻⁶ M. Although it may appear that U2AF binding is cooperative, this is due to the broad titration of protein used in this experiment. When a finer titration is used, we detect no evidence of cooperative binding (Fig. 5B and data not shown) (9).

FIG. 4

The hU2AF heterodimer binds pyrimidine tract RNA with higher affinity than the hU2AF⁶⁵ monomer. (A) Electrophoretic mobility shift analysis of hU2AF heterodimer and hU2AF⁶⁵ monomer interaction with MINX-WT pyrimidine tract RNA. hU2AF⁶⁵ monomer protein concentrations were 2,000, 400, 80, 16, 3.2, 0.64, and 0.128 nM (lanes 2 to 8). hU2AF heterodimer concentrations were 400, 80, 16, 3.2, 0.64, and 0.128 nM (lanes 9 to 14). Proteins were incubated with 100 pM ³²P-labeled RNA oligonucleotide. Protein-RNA complexes (C) and unbound RNA (F) were separated by electrophoresis through a native polyacrylamide gel and visualized by autoradiography. The apparent K_d for hU2AF⁶⁵ was ∼3.8 × 10⁻⁸ M. The apparent K_d for hU2AF heterodimer was ∼2.5 × 10⁻⁹ M. (B) Electrophoretic mobility shift analysis of hU2AF heterodimer and hU2AF⁶⁵ monomer interaction with MINX-MUT pyrimidine tract RNA. Protein concentrations were identical to those in panel A. The apparent K_d for hU2AF⁶⁵ was ∼2.6 × 10⁻⁷ M. The apparent K_d for the hU2AF heterodimer was ∼6.1 × 10⁻⁹ M.

FIG. 5

At least one RS domain on U2AF is required for high-affinity RNA binding. (A) Electrophoretic mobility shift analysis of dU2AF⁵⁰ΔRS monomer and dU2AF⁵⁰ΔRS/dU2AF³⁸ heterodimer interaction with MINX-WT pyrimidine tract RNA. dU2AF⁵⁰ΔRS monomer protein concentrations were 5,000, 1,000, 200, and 40 nM (lanes 1 to 4). dU2AF⁵⁰ΔRS/dU2AF³⁸ heterodimer concentrations were 5,000, 1,000, 200, and 40 nM (lanes 5 to 8). Proteins were incubated with 100 pM ³²P-labeled RNA oligonucleotide. Protein-RNA complexes (C) and unbound RNA (F) were separated by electrophoresis through a native polyacrylamide gel and visualized by autoradiography. The apparent K_d for dU2AF⁵⁰ΔRS could not be determined. The apparent K_d for dU2AF⁵⁰ΔRS/dU2AF³⁸ was ∼1.7 × 10⁻⁷ M. (B) Electrophoretic mobility shift analysis of dU2AF⁵⁰ and dU2AF⁵⁰/dU2AF³⁸ΔRS heterodimer interaction with MINX-WT pyrimidine tract RNA. dU2AF⁵⁰ monomer protein concentrations were 4,000, 1,600, 640, 256, and 102 nM (lanes 2 to 6). dU2AF⁵⁰/dU2AF³⁸ΔRS heterodimer protein concentrations were 4,000, 1,600, 640, 256, and 102 nM (lanes 7 to 11). The apparent K_d for dU2AF⁵⁰ was ∼8.0 × 10⁻⁷ M. The apparent K_d for the dU2AF⁵⁰/dU2AF³⁸ΔRS heterodimer was ∼3.0 × 10⁻⁷ M.