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, 12 (20), 3217-25

WT1 Interacts With the Splicing Factor U2AF65 in an Isoform-Dependent Manner and Can Be Incorporated Into Spliceosomes

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WT1 Interacts With the Splicing Factor U2AF65 in an Isoform-Dependent Manner and Can Be Incorporated Into Spliceosomes

R C Davies et al. Genes Dev.

Abstract

WT1 is essential for normal kidney development, and genetic alterations are associated with Wilms' tumor, Denys Drash (DDS), and Frasier syndromes. Although generally considered a transcription factor this study has revealed that WT1 interacts with an essential splicing factor, U2AF65, and associates with the splicing machinery. WT1 is alternatively spliced and isoforms that include three amino acids, KTS, show stronger interaction with U2AF65 in vitro and better colocalization with splicing factors in vivo. Interestingly a mutation associated with DDS enhanced both -KTS WT1 binding to U2AF65 and splicing-factor colocalization. These data illustrate the functional importance of WT1 isoforms and suggest that WT1 plays a role in pre-mRNA splicing.

Figures

Figure 1
Figure 1
WT1 interacts with U2AF65. (A) Yeast two-hybrid analysis. Yeast was transformed with plasmids encoding the indicated proteins, and individual colonies grown on LeuTrp (left) prior to the assessment of β-galactosidase activity (right). (B) In vitro binding assay. The indicated in vitro-translated proteins (left) were mixed with GST or GST–U2AF65 purified from bacteria, bound protein resolved by SDS-PAGE, and detected by fluorography (right). (C) WT1 coimmunoprecipitates with U2AF65. WT1-expressing M15 cells were immunoprecipitated with antibodies against the indicated proteins or normal mouse IgG, the complexes resolved by SDS-PAGE, and the presence of WT1 determined by Western blotting.
Figure 1
Figure 1
WT1 interacts with U2AF65. (A) Yeast two-hybrid analysis. Yeast was transformed with plasmids encoding the indicated proteins, and individual colonies grown on LeuTrp (left) prior to the assessment of β-galactosidase activity (right). (B) In vitro binding assay. The indicated in vitro-translated proteins (left) were mixed with GST or GST–U2AF65 purified from bacteria, bound protein resolved by SDS-PAGE, and detected by fluorography (right). (C) WT1 coimmunoprecipitates with U2AF65. WT1-expressing M15 cells were immunoprecipitated with antibodies against the indicated proteins or normal mouse IgG, the complexes resolved by SDS-PAGE, and the presence of WT1 determined by Western blotting.
Figure 1
Figure 1
WT1 interacts with U2AF65. (A) Yeast two-hybrid analysis. Yeast was transformed with plasmids encoding the indicated proteins, and individual colonies grown on LeuTrp (left) prior to the assessment of β-galactosidase activity (right). (B) In vitro binding assay. The indicated in vitro-translated proteins (left) were mixed with GST or GST–U2AF65 purified from bacteria, bound protein resolved by SDS-PAGE, and detected by fluorography (right). (C) WT1 coimmunoprecipitates with U2AF65. WT1-expressing M15 cells were immunoprecipitated with antibodies against the indicated proteins or normal mouse IgG, the complexes resolved by SDS-PAGE, and the presence of WT1 determined by Western blotting.
Figure 2
Figure 2
U2AF65 binding is influenced by isoform. (A) Quantitation of β-galactosidase activity following transformation of yeast with the indicated constructs. (Solid bars) U2AF65; (open bars) SNF1; (hatched bars) WT1+/+ (WT1+/+). (B) WT1 binding to GST–U2AF65 compared to GST alone under different salt concentrations. (Solid bars) 100 mm; (hatched bars) 250 mm; (open bars) 500 mm.
Figure 3
Figure 3
U2AF65 binding is independent of dimerization, and affected by point mutation. (A) Schematic representation of WT1 and deletion constructs. Four major isoforms are generated by inclusion or exclusion of 17 amino acids and KTS, respectively. (*) Arg-to-Trp substitution associated with DDS. (B,C). Quantitation of β-galactosidase activity following transformation of yeast with U2AF65 (solid bars), SNF1 (open bars), and WT1+/+ (hatched bars, WT1+/+). Constructs carrying the DDS mutation are marked D. The ability of CΔF1, CTF0, and F+KTS to dimerize was not determined. (D) The percentage of WT1-transfected Cos7 cells showing good colocalization with splice factors.
Figure 4
Figure 4
Both the amino-terminal RS domain and linker region of U2AF65 are required for WT1 binding. (A) Schematic representation of U2AF65 and deletion constructs. (B) Yeast was transformed with plasmids encoding the indicated proteins, and individual colonies grown on LeuTrp media (left) prior to the assessment of β-galactosidase activity (right).
Figure 4
Figure 4
Both the amino-terminal RS domain and linker region of U2AF65 are required for WT1 binding. (A) Schematic representation of U2AF65 and deletion constructs. (B) Yeast was transformed with plasmids encoding the indicated proteins, and individual colonies grown on LeuTrp media (left) prior to the assessment of β-galactosidase activity (right).
Figure 5
Figure 5
WT1 is incorporated into spliceosomes. (A) Nuclear extracts were prepared from WT1-expressing M15 cells, and their ability to splice a model pre-mRNA compared to that of HeLa extract. Control reaction C was set up with inactive M15 extract. Splicing intermediates were resolved by PAGE and detected by autoradiography. (B) Radioactive tracing was used to follow the fractionation of large-scale splicing reactions set up with sense (S) and anti-sense (AS) pre-mRNAs. The indicated fractions were pooled and streptavidin beads used to capture proteins associated with the 32P-labeled biotinylated RNA. Immunoblotting with the indicated antibodies against WT1 or U2-B were used to determine protein distribution. Nuclear extract (NE) was loaded as a positive control. (C) Native gel analysis of a fractionated splicing reaction. A, B+C, and H complexes are indicated. (*) Nonspecific aggregates.
Figure 5
Figure 5
WT1 is incorporated into spliceosomes. (A) Nuclear extracts were prepared from WT1-expressing M15 cells, and their ability to splice a model pre-mRNA compared to that of HeLa extract. Control reaction C was set up with inactive M15 extract. Splicing intermediates were resolved by PAGE and detected by autoradiography. (B) Radioactive tracing was used to follow the fractionation of large-scale splicing reactions set up with sense (S) and anti-sense (AS) pre-mRNAs. The indicated fractions were pooled and streptavidin beads used to capture proteins associated with the 32P-labeled biotinylated RNA. Immunoblotting with the indicated antibodies against WT1 or U2-B were used to determine protein distribution. Nuclear extract (NE) was loaded as a positive control. (C) Native gel analysis of a fractionated splicing reaction. A, B+C, and H complexes are indicated. (*) Nonspecific aggregates.
Figure 5
Figure 5
WT1 is incorporated into spliceosomes. (A) Nuclear extracts were prepared from WT1-expressing M15 cells, and their ability to splice a model pre-mRNA compared to that of HeLa extract. Control reaction C was set up with inactive M15 extract. Splicing intermediates were resolved by PAGE and detected by autoradiography. (B) Radioactive tracing was used to follow the fractionation of large-scale splicing reactions set up with sense (S) and anti-sense (AS) pre-mRNAs. The indicated fractions were pooled and streptavidin beads used to capture proteins associated with the 32P-labeled biotinylated RNA. Immunoblotting with the indicated antibodies against WT1 or U2-B were used to determine protein distribution. Nuclear extract (NE) was loaded as a positive control. (C) Native gel analysis of a fractionated splicing reaction. A, B+C, and H complexes are indicated. (*) Nonspecific aggregates.

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