The role of the RNA-binding protein Wig-1 in post-transcriptional regulation of gene expression

Sammanfattning: The p53 transcription factor is activated by cellular stress. This triggers transcriptional activation of a number of p53 target genes, leading to responses such as cell cycle arrest and/or induction of apoptosis. Wig-1 is a p53 target gene and its RNA and protein levels increase after p53 protein activation. Wig-1 is a RNA-binding zinc finger protein with affinity to double-stranded RNA and it is involved in regulation of mRNA stability. In this thesis, I focused on the characterization of the Wig-1 protein function, on the identification of its bound RNA targets and on the elucidation of the biological implication of their regulation. We found that Wig-1 belongs to the group of proteins known as AU-rich element binding proteins (ARE-BPs) and plays a role in regulation of post-transcriptional gene expression targeting mRNAs containing AU-rich elements (ARE) in their 3’UTRs. In paper I, we showed that Wig-1 stabilizes p53 mRNA by preventing its deadenylation and that this regulation is mediated through direct binding of Wig-1 to a U-rich element (a subgroup of AREs) in the 3’UTR of p53 mRNA. In paper II, we found that Wig-1 binds to N-Myc mRNA and positively regulates it through an ARE in the 3’UTR. We also showed that Wig-1 knockdown in neuroblastoma cells carrying amplified N-Myc leads to cell differentiation and repressed cell growth as a consequence of Wig-1 regulation of N-Myc RNA stability. In paper III we performed microarray gene expression analysis after Wig-1 knockdown in the colon cancer cell line HCT116 and found a large group of mRNAs that are directly or indirectly affected by Wig-1. We also discovered that Wig-1 knockdown is affecting cell cycle and the apoptotic response to stress through regulation of the p53 target genes FAS and 14-3-3!. We could demonstrate that FAS mRNA regulation is dependent on Wig-1 binding to an ARE on FAS 3’UTR. At last, in paper IV, we performed RNA-immunoprecipitation followed by deep sequencing in order to identify genome-wide Wig-1 associated mRNAs. The analysis revealed that Wig-1 binds a large number of mRNAs most of which are functionally connected to the cell cycle pathway. Moreover, sequence analysis revealed that AREs are highly enriched in the 3’UTRs of these Wig-1-bound mRNAs. In conclusion, this thesis provides a comprehensive view of the RNA-binding properties of Wig-1 and helps to better define the Wig-1-RNA interaction network. Our data establish Wig-1 as an AU-rich element binding protein involved in regulation of post-transcriptional gene expression of many mRNAs such as the p53 tumor suppressor and its transcriptional target FAS, the N-Myc oncogene and several other targets, ultimately affecting cell cycle progression and cell proliferation. Moreover, we provide additional insights into preferred Wig-1 RNA binding motifs. Additionally, as Wig-1 is a target of the p53 transcription factor, we gained further understanding of the p53-mediated tumor suppression through its target Wig-1, extending the frontiers of gene expression control from transcriptional to posttranscriptional level.

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