Volume 8 Issue 1 - March 20, 2009
EGF increases the interaction between nucleolin and heterogeneous nuclear ribonucleoprotein K/poly(C) binding protein 1 complex to regulate the gastrin mRNA turnover
Pin-Tse Lee1, Pao-Chi Liao2, Wen-Chang Chang1,3,4, Joseph T. Tseng3,4,5*

1 Institute of Basic Medical Sciences, 2 Department of Occupational Health, 3Department of Pharmacology, College of Medicine, and 4Center for Gene Regulation and Signal Transduction Research, and 5Institute of Biosignal Transduction, College of Bioscience and Biotechnology, National Cheng Kung University, Tainan, Taiwan
tctseng@mail.ncku.edu.tw

Molecular Biology of the Cell, 18, 5004-5013 (2007)

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Gene expression was regulated by sequential control mechanisms in eukaryotic cells (Figure 1). The first step is producing the mRNA via transcriptional regulation and mRNA processing control mechanism in nucleus. And them the mRNA is exported to the cytoplasm or specific location to synthesize protein. Finally, the protein translation regulation and mRNA degradation mechanisms determine the fate of mRNA to synthesize protein or metabolize.
Figure 1. The regulation steps in gene expression.

Transcriptional regulation is well documented in controlling gene expression; however, it is becoming increasingly clear that the post-transcriptional regulation mechanism is also an important control point in determining the abundance of cellular transcripts (1). mRNA turnover and translational regulation were two major control mechanisms in the field of post-transcriptional regulation. The aberrant control of mRNA turnover has been implicated in disease states, including cancer, chronic inflammatory responses and coronary disease (2,3).

Gastrin is a classical gut peptide hormone, which was identified originally as a stimulant of gastric acid secretion. And now it has been confirmed as a growth factor for gastrointestinal tract malignancies. High expression of gastrin mRNA was observed in pancreatic and colorectal cancer, however, the mechanism is unclear (4). EGF was reported to activate the gastrin mRNA expression through up-regulation of transcriptional activity. In our study, we also demonstrated that EGF increases gastrin mRNA stability, indicating mRNA turnover regulation mechanism is involved in the control of gastrin mRNA expression. In order to address this hypothesis, biotin-labeled RNA probe pull-down assay combined with mass spectrometry analysis were performed. We identified the heterogeneous nuclear ribonucleoprotein K (hnRNP K) and poly(C) binding protein 1 (PCBP1) bound with the C-rich region in gastrin mRNA 3’UTR. Using SiRNA technology to reduce the protein expression of hnRNP K and PCBP1, the gastrin mRNA expression was also decreased which indicated hnRNP K and PCBP1 stabilize the gastrin mRNA. However, the EGF-induce gastrin mRNA expression was not abolished by reducing the hnRNP K and PCBP1 protein expression. To identify other regulation factors, we examined the possible RNA-binding motifs existing in the gastrin mRNA 3’UTR. Interestingly, we found a motif sequence AGCCCU located between the C-rich region similar to the reverse orientation of nucleolin binding sequence (U/G)CCCG(A/G). Then the biotin-labeled probe pull-down combined with Western-blot analysis was conducted to confirm the interaction between nucleolin and gastrin mRNA. We found nucleolin bound with the AGCCCU motif located at gastrin mRNA 3’UTR and also interacted with hnRNP K.

Under EGF treatment, we observed the amount of nucleolin interacting with hnRNP K and gastrin mRNA increased.
Figure 2. The binding model of nucleolin interacted with gastrin mRNA.
Using SiRNA technology to define the functional role of nucleolin, we found nucleolin plays a crucial role in mediating the increased gastrin mRNA stability induced by EGF signaling. Besides, we also observed hnRNP K/PCBP1 complex bound with the C-rich region in the gastrin mRNA increased nucleolin binding with gastrin mRNA. Finally, a novel binding model was proposed (Figure 2). To sum up, this novel mechanism in regulation gastrin mRNA may provide us another direction to study the mechanism which maintains high expression levels of gastrin mRNA in gastrointestinal cancer. And it may provide a new method to treat with the gastrointestinal cancer.

References
  • Wilusz, C. J., and Wilusz, J. (2004). Bringing the role of mRNA decay in the control of gene expression into focus. Trends Genet. 20, 491-497.
  • Audic, Y., and Hartley, R. S. (2004). Post-transcriptional regulation in cancer. Biol. Cell 96, 479-498.
  • Hollams, E. M., Giles, K. M., Thomson, A. M., and Leedman, P. J. (2002). mRNA stability and the control of gene expression: implications for human disease. Neurochem. Res. 27, 957-980.
  • Ferrand, A., and Wang, T. C. (2006). Gastrin and cancer: a review. Cancer Lett. 238, 15-29.
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