Identification of a novel Wilms' tumour 1 binding protein

Abstract

The paediatric, nephroblastoma Wilms' Tumour, affecting 1 in 10,000 children, is a disease where the disruption in the normal events of kidney development leads to tumour formation. The WT1 gene has been shown by knockout experiments to be crucial in kidney and gonadal development. The structure of the protein, which contains four zinc fingers of the Kriippeltype, together with in vitro work, suggests WT1 is a transcription factor. Other work however also suggests that the protein may be involved in post-transcriptional regulation as WT1 localises and co-immunoprecipitates with splicing factors. The structure of the protein also backs up this possible role as WT1 has a putative RNA recognition motif and the zinc fingers can bind RNA in vitro.The genuine functions of WT1 are thus not clear so this project set out to address what WT1 is doing at the molecular level. By looking for WT1-binding proteins, 1 aimed to investigate which components of the cellular machinery WT1 is interacting with, as it is only once we comprehend the molecular mechanism behind development that we will begin to understand the link with tumourigenesis.Using a yeast two hybrid screen I identified a protein that interacts with the C-terminus of WT1. As this protein is novel, with no homologues found in the yeast or invertebrate databases, it was called NAL (Novel Associating Ligand). NAL was mapped, using FISH, to human chromosome 6q26-27, a location which possibly harbours a tumour suppressor gene. The mouse homologue was cloned and, at the amino acid level, is 94% identical to the human clone. This sequence conservation suggests that whatever the role of this novel protein may be, its structure must be important for function. Using FISH and the EUCIB resource, the mouse homologue was mapped close to the centromere on chromosome 17, a region which has conserved synteny with human 6q26-27 and which maps close to the murine T-locus.In vitro assays showed that the interaction between NAL and WT1 could occur outside of the yeast two hybrid system and in vivo co-immunoprecipitation experiments revealed that the interaction is physiologically relevant as it occurs in WT1-expressing cells. Expression studies using RT-PCR and whole mount in situ hybridisation showed that NAL is ubiquitously expressed. The expression pattern of NAL during development of the kidney and testis was also investigated to see if, like WT1, NAL is confined to the developing nephron and Sertoli cells respectively. Immunohistochemistry revealed that NAL is expressed in all cells of these organs and thus is not exclusively localised to cells expressing WT1, suggesting that NAL may play a housekeeping role.Immunofluorescence indicated that N AL is a nuclear protein and its distribution within the nucleus was interesting as it resembled that of WT 1: a speckled pattern within an overall diffuse staining. Immunocytochemistry confirmed that this protein, like WT1, does indeed co-localise with splicing factors. Thus it will be of interest to see if this protein really is involved in splicing. Unfortunately no further functional assays could be carried out and so future work needs to be carried out to determine its true role and the potential significance of its interaction with WT1.