Impact of Nutraceuticals on the TERT gene encoded protein




Xu Liu

Introduction

During DNA replication, Okazaki fragments are formed at the growing replication fork to make sure both DNA strands grow in the 5’-to-3’ direction. However, when the replication fork reaches to the end of the linear chromosome, there is no enough space to produce the RNA primer starting the last Okazaki fragment at the very tip of a linear DNA molecule. Thus, eukaryotes use a RNA-protein complex, telomerase, to solve the problem.
Telomerase Reverse Transcriptase (TERT), the protein subunit of Telomerase, is an enzyme and syntheses DNA using Telomerase RNA subunit as template. Then the synthesized DNA will be added to the parental DNA strand to complete the last Okazaki fragment.
For normal somatic cells, the activity of hTERT has been turned town to avoid uncontrolled cell proliferation, since the enzyme couldn’t quite keep up with the chromosome replication. While it has been shown that increased TERT expression will contribute to the cell transformation and TERT knockdown also induces cancer cells apoptosis.[1] Therefore, TERT is a potential gene target for the cancer treatment.
Given that approximately all the eukaryotic genes are affected by alternative splicing, expanding the coding capability of the genome, and it has been shown that nutraceuticals could induce alternative splicing of RNAs. Therefore, in this study, we tend to detect the impact of nutraceuticals on the TERT encoded protein.

Materials and Methods

Caco-2 cells were treated with various nutraceuticals compounds before Protein and RNA were extracted.
Western-Blot was done for the extracted proteins, using rabbit polyclonal TERT antibody. 5 ng/μL extracted RNA stocks were prepared for each sample. Reverse Transcriptase PCR was done with overlapping primers spanning all exons of TERT, using the extracted RNAs as templates.
Products of RT-PCR were then visualized using gel electrophoresis with 1% agarose gel.
Finally, products were purified and sent out for sequencing to make sure the products amplified are the RNAs of TERT. BLAST was done then to verify the existence of spliced TERT transcripts.

Results
After treating Caco-2 cells with 8 nutraceutical compounds, Western Blot was carried for the extracted proteins using rabbit polyclonal TERT antibody(figure.1). For the cell samples treated with nutraceutical combo 1, 6 and 8, we could clearly find a robust band indicating increased Full-length TERT protein expression at the site around 120Kd when compared with untreated (control) sample and other nutraceutical combo treatments.

RT-PCR was carried out using the primer pair whose PCR product is located from Exon 6 to 9 and the result is visualized by Gel Electrophoresis(figure.2). After purifying and sequencing those bands, we find two alternative TERT transcripts, around 240bp and 420bp, respectively, despite the existence of a non-specific band pointed by the yellow arrow.

After blasting and comparing, we find the upper band, which is around 420bp and not expressed in untreated cells, is the expected PCR products. However, the lower band that is around 240bp and expressed in all cell samples lacks the total Exon 7 and Exon 8(figure.3).

Discussion
We have demonstrated alternative splicing of the transcripts generated by TERT gene. The deletion of Exon 7 and 8 will induce the reading frame shift and then there will be a premature stop codon in Exon 10. When only the Exon 7 and 8 are deleted, the translated product will be 807 Amino Acid length, around 90Kd. Since the TERT antibody we applied will recognize the C-terminal domain of the full-length TERT protein, so the translated product is not visible on the Western Blot results.
In spite of the function of the transcript variant without Exon 7 and 8 is unknown, it is highly possible that such kind of deletion will affect its reverse transcriptase activity. It has been found that the motif A and B of the full-length TERT, encoded by Exon 5/6/7/8/9, are responsible for its enzyme activity[2]. Therefore, the reading frame shift induced by deletion of Exon 7 and 8 will affect the TERT motif B, probably altering the reverse-transcriptase activity(figure.4). In this study, although we didn’t test the changes of the reverse-transcriptase activity between different TERT transcript variants, what we could still confirm is that those Caco-2 cells treated with some nutraceutical combos express increased TERT protein, with more Exon 7 and 8 non-deletion transcripts.

Figures


Figure 1-Western blot results of proteins extracted from cell samples treated with 8 Nutraceutical combos and untreated one (Control).


Figure 2-Gel Electrophoresis Results of RT-PCR.


Figure 3-Alternatively spliced TERT transcripts observed in this study.


Figure 4-Diagram of TERT protein motifs being responsible for its reverse transcriptase activity.


Summary
Telomerase is a Ribonucleo-protein polymerase that plays essential role during DNA replication and maintains the chromosome length. Telomerase Reverse Transcriptase (TERT) is the protein component of telomerase, whose activity is usually turned down to avoid the uncontrolled cell proliferation. While the increased TERT expression is always related with the immortality of cancer cells, thus the TERT gene is a potential target for tumor treatment. Nearly all the eukaryotic cells apply alternative splicing to expand the coding capability of the genome and nutraceuticals are supposed to induce alternatively spliced RNAs. Thus, in this study, we treat Caco-2 cells with different nutraceutical combos and find some Caco-2 cells treated with nutraceuticals express more non-deletion transcript variants, hence more full-length TERT protein.

Reference
[1] Konnikova L, Simeone MC, Kruger MM. et al. Signal transducer and activator of transcription 3 (STAT3) regulates human telomerase reverse transcriptase (hTERT) expression in human cancer and primary cells. Cancer Res. 2005;65:6516–20.
[2] Akincilar, Semih Can, Bilal Unal, and Vinay Tergaonkar. “Reactivation of telomerase in cancer.” Cellular and Molecular Life Sciences (2016): 1-12.

Full Paper

Acknowledgments

I would like to thank my Teaching Assistants, Anthony Evans and Catharina Grubaugh for all of their patient guidance, advices and suggestions, as well as all the valuable work they put in to assure the experiment went as smoothly as possible. Finally, I would also like to thank Dr. Berish Rubin for his help and support to make this project possible.


This document was last modified 05/16/2016.
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