Presence of WT1, the Wilm's Tumor Suppressor Gene Product, in Nuclear Poly(A)+ Ribonucleoprotein

Michael R Ladomery,Joan Slight,Sharon Mc Ghee,Nicholas D Hastie

doi:10.1074/jbc.274.51.36520

Michael R Ladomery, Joan Slight + Show 2 more

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https://doi.org/10.1074/jbc.274.51.36520

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Abstract

The tumor suppressor gene WT1 encodes a zinc finger protein, which consists of four C-terminal C(2)-H(2) zinc fingers of the Krüppel type, and at the N terminus a Q/P-rich trans-regulatory domain, both characteristic of transcription factors. However, recent findings suggest that WT1 may also be involved in a post-transcriptional process. Specifically, WT1 isoforms containing the alternatively spliced exon 9 (+lysine-threonine-serine (KTS)) preferentially associate with nuclear speckles and co-immunoprecipitate splicing antigens (Larsson, S. H., Charlieu, J.-P., Miyagawa, K., Engelkamp, D., Rassoulzadegan, M., Ross, A., Cuzin, F., van Heyningen, V., and Hastie, N. D. (1995) Cell 81, 391-401); furthermore, WT1 has been shown to interact with the ubiquitous splicing factor U2AF65 (Davies, R. C., Calvo, C., Larsson, S. H., Lamond, A. I., and Hastie, N. D. (1998) Genes Dev. 12, 3217-3225) and binds to RNA in vitro (Caricasole, A., Duarte, A., Larsson, S. H., Hastie, N. D., Little, M., Holmes, G., Todorov, I., and Ward, A. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 7562-7566; Bardeesy, N., and Pelletier, J. (1998) Nucleic Acids Res. 26, 1784-1792). To extend these findings, we have fractionated nuclear extracts to see if particles containing WT1 have the properties of ribonucleoprotein (RNP). In summary, WT1 is enriched by oligo(dT) chromatography, as are U2AF65, the U5 small nuclear RNP-associated protein p116 and hnRNP A1. Gel filtration and sedimentation profiles suggest that WT1 is present in RNase-sensitive particles, >2 MDa in size, peaking at approximately 60 S, and approximately 1.27 g/cm(3) on Nycodenz. Similar results were obtained from two cell lines expressing WT1, fetal kidneys (day E17), and transiently transfected cells, suggesting that the presence of WT1 protein in nuclear poly(A)(+) RNP is a general aspect of WT1 function.

Highlights

The WT1 gene encodes a protein which includes, at its C terminus, four C2-H2 zinc fingers of the Kruppel-type, with close structural homology to zinc fingers in the early growth response family of transcription factors
The WT1 zinc fingers, the first out of the four, bound an RNA sequence encoded by exon 2 of the IGF2 gene [3], which overlaps with a putative DNA target sequence
We found WT1 to be highly enriched in total extract poly(A)ϩ fractions, as was U2AF65, a U2 snRNP-associated splicing factor that recognizes the polypyrimidine tract at the 3Ј splice site [35]; p116, a U5 snRNP-associated GTPase structurally related to the ribosomal translocase EF2 [36]; and hnRNP A1, a core hnRNP particle component (Fig. 1B)

Summary

Introduction

The WT1 gene encodes a protein which includes, at its C terminus, four C2-H2 zinc fingers of the Kruppel-type, with close structural homology to zinc fingers in the early growth response family of transcription factors. The possibility arose that WT1, and in particular the ϩKTS isoforms, may be involved in post-transcriptional events, splicing To extend these findings, Davies et al [2] subsequently described an interaction between WT1 and the ubiquitous splicing factor U2AF65. Davies et al [2] subsequently described an interaction between WT1 and the ubiquitous splicing factor U2AF65 This interaction was defined and analyzed using the yeast two-hybrid approach, coupled to in vitro binding and in vivo co-immunoprecipitation. A more recent paper describes a SELEX (in vitro ligand selection) experiment that used the zinc fingers of WT1 to define three candidate RNA target sequences [4] The significance of these findings remains to be investigated

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