Final Activity Report Summary - WTIC (Functional Significance of Cytoplasmic WT1)
The Wilms tumour suppressor, WT1, is a multifunctional protein essential for the development of genitor-urinary system and epicardium. Mutations in Wt1 gene are implicated in a number of developmental syndromes and some types of malignancies in humans. It is also reported to be involved during the development of retina and olfactory bulbs; however its localisation and function in other domains of the nervous system are not studied. WT1 can bind deoxyribonucleic acid (DNA) and is a well known transcription factor. It is mostly localised in the nucleus of the cell, but up to 10 % of the protein can be found in the cytoplasm. WT1 can also bind ribonucleic acid (RNA) and was recently implicated in RNA metabolism as it was found in RNA spicing complexes in the nucleus and on polysomes that actively translate proteins from mRNAs in the cytoplasm. Nevertheless, no RNA targets of WT1 are identified so far in vivo. Thus the main goal of the project was to identify RNA interactions of WT1 and their functional significance in neuronal cells where WT1 was predominantly cytoplasmic.
We studied WT1 localisation using a mouse model of neuron specific WT1 depletion. We showed WT1 nuclear localisation in the ventral motoneurons of the spinal cord and in the cells of the roof of the IV ventricle. We found cytoplasmic localisation of WT1 in the basal root ganglia as well as in otic epithelium. GFP WT1 knock-in mouse line, where fluorescent GFP protein was expressed alongside with WT1 from one of the WT1 alleles, helped us to confirm the mentioned findings and suggested that WT1 was expressed in the nervous system from very early stages.
We also used a model of ESC to investigate WT1 possible role in neuronal differentiation. About 5 % of wild type ESC differentiated with all trans-retinoic acid (RA) and became neurons. WT1 could be seen in the cell body and axons. WT1-null cells failed to develop neuronal morphology. Moreover, we used a model with a selection step for nestin-positive neuron precursors. Surprisingly WT1 expression was very low in this case and there was no difference in the number of neurons between wild type and WT1-null cultures. However, if we added RA in the protocol wild type ESC could develop up to 30 % of differentiated neurons whereas WT1-nulls only 5 to 8 %. Thus, WT1 was involved in RA driven neuronal differentiation.
In order to address WT1 role in the cytoplasm, we tried to identify its RNA and novel protein targets. We confirmed the interaction of WT1 with RNA-binding protein hnRNPU and located the interaction domains. We found that WT1 also interacted with actin via its Zn-finger domain in vitro and in vivo. Not surprisingly, WT1 was found in the cytoplasm predominantly in cytoskeleton-bound polysome fraction. Actin depolymerisation disturbed WT1 nucleo-cytoplasmic shuttling and displaced WT1 from the polysomes, stressing the functional importance of the interaction. Both actin and hnRNPU were implicated in the RNA processing and transport. Physical interaction of WT1 with them supported the hypothesis of its involvement into RNA metabolism. The next goal was to look for RNA targets of WT1 in the cytoplasm. In native immunoprecipitations (IP) with anti-WT1 antibody from differentiated ESC, we could enrich for RALDH2, Crabp2, Igfbp4, Dzip, Caldesmon and Tropomyosin mRNAs. We found a few RNA partners of WT1 which were coding proteins involved in RA signalling and neuron maturation. Specificity of this RNA binding dropped with actin de polymerisation. WT1 affected the distribution of RALDH2 and DZIP mRNAs in the polysome fractions, suggesting its role in the translation regulation. We finally showed, using formalin cross linking, that DZIP mRNA could be a direct WT1 target as it was able to bind it both in vitro and in vivo.
Correlations between the obtained data suggested that WT1 might be involved in RA driven neuron maturation taking part in the regulation of processing and actin-dependant transport of RNAs which involved RA signalling.
We studied WT1 localisation using a mouse model of neuron specific WT1 depletion. We showed WT1 nuclear localisation in the ventral motoneurons of the spinal cord and in the cells of the roof of the IV ventricle. We found cytoplasmic localisation of WT1 in the basal root ganglia as well as in otic epithelium. GFP WT1 knock-in mouse line, where fluorescent GFP protein was expressed alongside with WT1 from one of the WT1 alleles, helped us to confirm the mentioned findings and suggested that WT1 was expressed in the nervous system from very early stages.
We also used a model of ESC to investigate WT1 possible role in neuronal differentiation. About 5 % of wild type ESC differentiated with all trans-retinoic acid (RA) and became neurons. WT1 could be seen in the cell body and axons. WT1-null cells failed to develop neuronal morphology. Moreover, we used a model with a selection step for nestin-positive neuron precursors. Surprisingly WT1 expression was very low in this case and there was no difference in the number of neurons between wild type and WT1-null cultures. However, if we added RA in the protocol wild type ESC could develop up to 30 % of differentiated neurons whereas WT1-nulls only 5 to 8 %. Thus, WT1 was involved in RA driven neuronal differentiation.
In order to address WT1 role in the cytoplasm, we tried to identify its RNA and novel protein targets. We confirmed the interaction of WT1 with RNA-binding protein hnRNPU and located the interaction domains. We found that WT1 also interacted with actin via its Zn-finger domain in vitro and in vivo. Not surprisingly, WT1 was found in the cytoplasm predominantly in cytoskeleton-bound polysome fraction. Actin depolymerisation disturbed WT1 nucleo-cytoplasmic shuttling and displaced WT1 from the polysomes, stressing the functional importance of the interaction. Both actin and hnRNPU were implicated in the RNA processing and transport. Physical interaction of WT1 with them supported the hypothesis of its involvement into RNA metabolism. The next goal was to look for RNA targets of WT1 in the cytoplasm. In native immunoprecipitations (IP) with anti-WT1 antibody from differentiated ESC, we could enrich for RALDH2, Crabp2, Igfbp4, Dzip, Caldesmon and Tropomyosin mRNAs. We found a few RNA partners of WT1 which were coding proteins involved in RA signalling and neuron maturation. Specificity of this RNA binding dropped with actin de polymerisation. WT1 affected the distribution of RALDH2 and DZIP mRNAs in the polysome fractions, suggesting its role in the translation regulation. We finally showed, using formalin cross linking, that DZIP mRNA could be a direct WT1 target as it was able to bind it both in vitro and in vivo.
Correlations between the obtained data suggested that WT1 might be involved in RA driven neuron maturation taking part in the regulation of processing and actin-dependant transport of RNAs which involved RA signalling.