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First published online 4 March 2003
doi: 10.1242/jcs.00324

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Expression in Xenopus oocytes shows that WT1 binds transcripts in vivo, with a central role for zinc finger one

Michael Ladomery^1,*, John Sommerville², Sarah Woolner^1,, Joan Slight¹ and Nick Hastie^1,

¹ MRC Human Genetics Unit, Western General Hospital, Crewe Rd, Edinburgh EH4 2XU, UK
² School of Biology, Bute Medical Buildings, University of St Andrews, St Andrews, Fife KY16 9TS, UK
^* Present address: Centre for Research in Biomedicine, Faculty of Applied Sciences, University of the West of England, Coldharbour Lane, Bristol BS16 1QY, UK
Department of Anatomy and Developmental Biology, University College London, Gower Street, London WC1E 6BT, UK

Author for correspondence (e-mail: nick.hastie{at}hgu.mrc.ac.uk)

Accepted 16 December 2002

The Wilms' tumour suppressor gene WT1 encodes a protein involved in urogenital development and disease. The salient feature of WT1 is the presence of four `Krüppel'-type C₂-H₂ zinc fingers in the C-terminus. Uniquely to WT1, an evolutionarily conserved alternative splicing event inserts three amino acids (KTS) between the third and fourth zinc fingers, which disrupts DNA binding. The ratio of +KTS:–KTS isoforms is crucial for normal development. Previous work has shown that WT1 (+KTS) interacts with splice factors and that WT1 zinc fingers, particularly zinc finger one, bind to RNA in vitro. In this study we investigate the role of zinc finger one and the +KTS splice in vivo by expressing tagged proteins in mammalian cells and Xenopus oocytes. We find that both full-length +/–KTS isoforms and deletion constructs that include zinc finger one co-sediment with ribonucleoprotein particles (RNP) on density gradients. In Xenopus oocytes both isoforms located to the lateral loops of lampbrush chromosomes. Strikingly, only the +KTS isoform was detected in B-snurposomes, but not when co-expressed with –KTS. However, co-expression of the C-terminus (amino acids 233-449, +KTS) resulted in snurposome staining, which is consistent with an in vivo interaction between isoforms via the N-terminus. Expressed WT1 was also detected in the RNA-rich granular component of nucleoli and co-immunoprecipitated with oocyte transcripts. Full-length WT1 was most stably bound to transcripts, followed by the C-terminus; the least stably bound was CTF1 (C-terminus minus zinc finger one). Expression of the transcription factor early growth response 1 (EGR1), whose three zinc fingers correspond to WT1 zinc fingers 2-4, caused general chromosomal loop retraction and transcriptional shut-down. However, a construct in which WT1 zinc finger one was added to EGR1 mimicked the properties of WT1 (–KTS). We suggest that in evolution, WT1 has acquired the ability to interact with transcripts and splice factors because of the modification of zinc finger one and the +KTS alternative splice.

Key words: Wilms' tumour suppressor, C₂-H₂ zinc fingers, ribonucleoprotein particles (RNP), Density gradients, Xenopus oocytes, Lampbrush chromosomes, B-snurposomes, Nucleoli

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