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

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.

List of scientific papers

I. Anna Vilborg, Jacob A. Glahder, Margareta T. Wilhelm, Cinzia Bersani, Martin Corcoran, Salah Mahmoudi, Maiken Rosenstierne, Dan Grandér, Marianne Farnebo, Bodil Norrild, and Klas G. Wiman. The p53 target Wig-1 regulates p53 mRNA stability through an AU-rich element. Proc Natl Acad Sci USA. 2009 Sep 15;106(37):15756-61
https://doi.org/10.1073/pnas.0900862106

II. Anna Vilborg, Cinzia Bersani, Malin Wickström, Lova Segerström, Per Kogner, Klas G. Wiman. Wig-1, a novel regulator of N-Myc mRNA and N-Myc-driven tumor growth. Cell Death Dis. 2012 Apr 19;3:e298
https://doi.org/10.1038/cddis.2012.33

III. Cinzia Bersani, Lidi Xu, Anna Vilborg, Weng-Onn Lui, Klas G. Wiman. Wig-1 regulates cell cycle arrest and cell death through the p53 targets FAS and 14-3-3!. Oncogene. 2014 Aug 28;33(35):4407-17.
https://doi.org/10.1038/onc.2013.594

IV. Cinzia Bersani, Mikael Huss, Stefania Giacomello, Lidi Xu, Anna Vilborg, Andrey Alexeyenko, Weng-Onn Lui and Klas G. Wiman Genome-wide identification of Wig-1 mRNA targets by RIP-Seq analysis. [Manuscript]