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, 18 (4), 1765-73

Interaction Between Subunits of Heterodimeric Splicing Factor U2AF Is Essential in Vivo

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Interaction Between Subunits of Heterodimeric Splicing Factor U2AF Is Essential in Vivo

D Z Rudner et al. Mol Cell Biol.

Abstract

The heterodimeric pre-mRNA splicing factor, U2AF (U2 snRNP auxiliary factor), plays a critical role in 3' splice site selection. Although the U2AF subunits associate in a tight complex, biochemical experiments designed to address the requirement for both subunits in splicing have yielded conflicting results. We have taken a genetic approach to assess the requirement for the Drosophila U2AF heterodimer in vivo. We developed a novel Escherichia coli copurification assay to map the domain on the Drosophila U2AF large subunit (dU2AF50) that interacts with the Drosophila small subunit (dU2AF38). A 28-amino-acid fragment on dU2AF50 that is both necessary and sufficient for interaction with dU2AF38 was identified. Using the copurification assay, we scanned this 28-amino-acid interaction domain for mutations that abrogate heterodimer formation. A collection of these dU2AF50 point mutants was then tested in vivo for genetic complementation of a recessive lethal dU2AF50 allele. A mutation that completely abolished interaction with dU2AF38 was incapable of complementation, whereas dU2AF50 mutations that did not effect heterodimer formation rescued the recessive lethal dU2AF50 allele. Analysis of heterodimer formation in embryo extracts derived from these interaction mutant lines revealed a perfect correlation between the efficiency of subunit association and the ability to complement the dU2AF50 recessive lethal allele. These data indicate that Drosophila U2AF heterodimer formation is essential for viability in vivo, consistent with a requirement for both subunits in splicing in vitro.

Figures

FIG. 1
FIG. 1
Coexpression plasmids used to purify recombinant U2AF heterodimers. (A) Two independent phage T7 promoters (indicated by arrows) were fused to the large and small U2AF subunits. Only one of the two subunits is (his)6-tagged (his). This expression plasmid is a dimeric plasmid containing two selectable markers, ampicillin resistance (amp) and kanamycin resistance (kan), and two origins of replication (ori). (B) A single phage T7 promoter was fused to a bicistron containing both U2AF subunits. Only one of the two subunits is (his)6 tagged.
FIG. 2
FIG. 2
Mapping the domain on dU2AF50 that is necessary and sufficient for interaction with dU2AF38. (A) An SDS–12% polyacrylamide gel of dU2AF50 wild-type and deletion mutant proteins stained with Coomassie blue. U2AF subunits were coexpressed in E. coli with the dimeric coexpression plasmid. dU2AF50 was (his)6 tagged (His-dU2AF50) in the dU2AF heterodimer, and hU2AF35 was (his)6 tagged in the hU2AF heterodimer (His-hU2AF35). dU2AF heterodimer formation was assessed by copurification of dU2AF38 on Ni2+-NTA-agarose. E. coli lysates from uninduced (−) and induced (+) cells as well as the eluate (EL) from Ni2+-NTA-agarose purification are shown. The position of dU2AF38 is indicated by an arrow. dU2AF38 runs heterogeneously due to carboxyl-terminal proteolysis. The identity of these polypeptides as dU2AF38 was confirmed by immunoblot analysis (data not shown; Fig. 4A and B). A similar heterogeneity was observed when an amino-terminal (his)6-tagged dU2AF38 was purified separately (data not shown). The sizes of the protein molecular size markers are indicated in kilodaltons. WT, wild type. (B) Schematic representation of the results of the copurification interaction assay. The (his)6 tag (His), RS domain (RS), and three RNA recognition motifs (RRM1–3) of dU2AF50 are indicated. The different dU2AF50 domains are not drawn to scale.
FIG. 3
FIG. 3
Amino acid sequence comparison of the U2AF large-subunit interaction domains and locations of the alanine-substitution mutations. The 28-amino-acid linker region from four U2AF large-subunit homologs is shown. Amino acid identities and similarities are shown in dark-gray and light-gray boxes, respectively. Dashes denote gaps. Amino acid positions are shown on the right. Triple-alanine substitution mutations (mut.) used to identify residues in dU2AF50 required for heterodimer formation are indicated above the sequence comparison. Amino acid sequence data are from Kanaar et al. (11) (Drosophila), Zamore et al. (36) (human), Zorio et al. (38) (C. elegans), and Potashkin et al. (18) (S. pombe).
FIG. 4
FIG. 4
Identification of point mutations that disrupt heterodimer formation in vitro. (his)6-tagged dU2AF50 or dU2AF50 interaction mutant derivatives were coexpressed with dU2AF38 with the bicistronic coexpression plasmid. Heterodimer formation was assessed by copurification of dU2AF38 on Ni2+-NTA-agarose. E. coli lysates from uninduced (−) and induced (+) cells and the eluate (EL) from Ni2+-NTA-agarose purification were electrophoresed on an SDS–10% polyacrylamide gel and stained with Coomassie blue (A) or transferred to nitrocellulose and probed with affinity-purified anti-dU2AF38 antibodies (B and C). The position of dU2AF38 is indicated with an arrow. Molecular size markers are indicated in kilodaltons.
FIG. 5
FIG. 5
dU2AF50 mutants bind pyrimidine tract RNA with similar affinity to wild-type dU2AF50 (WT). (A) Electrophoretic mobility shift analysis of dU2AF50 and dU2AF50 interaction mutants with the MINX pyrimidine tract. Wild-type dU2AF50 (WT) protein concentrations were 1.25, 0.25, and 0.05 μM (lanes 2 to 4). dU2AF50 Δinteraction domain (ΔI) protein concentrations were 2.5, 0.5, and 0.1 μM (lanes 5 to 7). dU2AF50 mutant 1 (mut.1) protein concentrations were 5, 1, and 0.2 μM (lanes 8 to 10). dU2AF50 mutant 2 (mut.2) protein concentrations were 2.5, 0.5, and 0.1 μM (lanes 12 to 14). dU2AF50 mutant 3 (mut.3) protein concentrations were 5, 1, and 0.2 μM (lanes 15 to 17). dU2AF50 mutant 4 (mut.4) protein concentrations were 10, 2, and 0.4 μM (lanes 18 to 20). 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 KDs were determined to be 7.1 × 10−6 M (WT), 9.0 × 10−6 M (ΔI), 8.1 × 10−6 M (mut.1), 8.5 × 10−6 M (mut.2), 8.9 × 10−6 M (mut.3), and 7.0 × 10−6 M (mut.4). (B) Sequence of the MINX pyrimidine tract. (C) An SDS–10% polyacrylamide gel of the recombinant (his)6-tagged dU2AF50 proteins stained with Coomassie blue. Molecular size markers are indicated in kilodaltons.
FIG. 6
FIG. 6
In vivo analysis of dU2AF50 interaction domain mutants. HA-tagged dU2AF50 and mutant-derivative transgenes were tested for complementation of a recessive lethal dU2AF50 allele. The amino acid sequence of the interaction domain (linker) is shown. The alanine substitution mutations are depicted in white. The average rescue of the rescuing transgene lines is shown. Mutant 4 could rescue the dU2AF50 mutant allele only when the transgene was present in two copies. The average rescue when two mutant 4 transgenes were present is shown in parentheses.
FIG. 7
FIG. 7
Protein expression levels of epitope-tagged dU2AF50 mutants. Immunoblot analysis of whole-fly extracts probed with anti-dU2AF50 (α-d50) and anti-HA (α-HA) antibodies. Whole-fly extracts are from w1118, dU2AF50+ (+), flies (lanes 1 and 10), w1118 flies carrying HA-dU2AF50 wild-type (lane 2) or interaction mutant derivatives (lanes 4, 5, 6, and 8) or dU2AF50 mutant flies (d50) rescued by HA-dU2AF50 wild-type (lane 3) or mutant transgene derivatives (lanes 7 and 9). The presence of the HA epitope on dU2AF50 results in the slower mobility observed. Molecular size markers are indicated in kilodaltons.
FIG. 8
FIG. 8
Analysis of heterodimer formation by coimmunoprecipitation from embryo extracts. (A) Immunoblot analysis of dU2AF50 and dU2AF38. The embryo extracts used in panel A are from wild-type dU2AF50+ (+) flies that either lack a transgene or contain a wild-type, HA-tagged dU2AF50 transgene (P[HA-d50WT]) or a mutant derivative (P[HA-d50mut]). The transgenes are indicated above the lanes. Lanes 1, 2, 3, and 13 are representative embryo extracts. Lanes 4, 6, and 8 are representative immunoprecipitates from embryo extracts with a control antibody (α-KP). Lanes 5, 7, 9, 10, 11, and 12, are immunoprecipitates from embryo extracts with the anti-HA antibody (α-HA). The immunoprecipitates and extracts were subjected to electrophoresis through an SDS–10% polyacrylamide gel in duplicate and probed with anti-dU2AF50 (α-d50) or anti-dU2AF38 (α-d38) antibodies. (B) Immunoblot analysis of coimmunoprecipitates from embryo extracts derived from rescued dU2AF50 mutant flies. The extracts used in panel B (except lanes 1 and 5) are from dU2AF50 mutant (d50) fly lines that are rescued by a wild-type HA-dU2AF50 transgene or HA-dU2AF50 mutant derivatives. The transgenes are indicated above the lanes. Lanes 1 to 4 are the embryo extracts, and lanes 5 to 8 are the immunoprecipitates with the anti-HA antibody (α-HA). The extracts and immunoprecipitates were subjected to electrophoresis through an SDS–10% polyacrylamide gel and transferred to nitrocellulose. The membrane was stained with Ponceau S and cut in half at the 45-kDa marker. The top half of the membrane was probed with anti-dU2AF50 antibodies, and the bottom half was probed with anti-dU2AF38 antibodies. The two pieces of nitrocellulose were aligned prior to enhanced chemiluminescence detection. The sizes of the protein molecular size markers are indicated in kilodaltons.

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