Expression in Xenopus oocytes shows that WT1 binds transcripts in vivo, with a central role for zinc finger one.
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 C(2)-H(2) 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 CTDeltaF1 (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.
Ladomery
Michael
M
MRC Human Genetics Unit, Western General Hospital, Crewe Rd, Edinburgh EH4 2XU, UK.
Sommerville
John
J
Woolner
Sarah
S
Slight
Joan
J
Hastie
Nick
N
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D045744:Cell Line, Tumor; D002466:Cell Nucleolus / Q000378:metabolism; D002522:Chlorocebus aethiops; D002852:Chromatography, Ion Exchange; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D051766:Early Growth Response Protein 1; D005260:Female; D006367:HeLa Cells; D006801:Humans; D017874:Immediate-Early Proteins; D051379:Mice; D008969:Molecular Sequence Data; D009865:Oocytes / Q000378:metabolism*; D011485:Protein Binding; D012313:RNA / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012261:Ribonucleoproteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D025721:WT1 Proteins / Q000235:genetics / Q000378:metabolism*; D029867:Xenopus Proteins; D014982:Xenopus laevis; D016335:Zinc Fingers / Q000235:genetics*
D004268:DNA-Binding Proteins; C486111:EGR1 protein, Xenopus; C497325:EGR1 protein, human; D051766:Early Growth Response Protein 1; C486109:Egr1 protein, mouse; D017874:Immediate-Early Proteins; D012261:Ribonucleoproteins; D014157:Transcription Factors; D025721:WT1 Proteins; D029867:Xenopus Proteins; D012313:RNA