Expression of functional alternative telomerase RNA component gene in mouse brain and in motor neurons cells protects from oxidative stress

Abstract

Telomerase, a ribonucleoprotein, is highly expressed and active in many tumor cells and types, therefore it is considered to be a target for anti-cancer agents. On the other hand, recent studies demonstrated that activation of telomerase is a potential therapeutic target for age related diseases. Telomerase mainly consists of a catalytic protein subunit with a reverse transcription activity (TERT) and an RNA component (TERC), a long non-coding RNA, which serves as a template for the re-elongation of telomeres by TERT. We previously showed that TERT is highly expressed in distinct neuronal cells of the mouse brain and its expression declined with age. To understand the role of telomerase in non-mitotic, fully differentiated cells such neurons we here examined the expression of the other component, TERC, in mouse brain. Surprisingly, by first using bioinformatics analysis, we identified an alternative TERC gene (alTERC) in the mouse genome. Using further experimental approaches we described the presence of a functional alTERC in the mouse brain and spleen, in cultures of motor neurons- like cells and neuroblastoma tumor cells. The alTERC is similar (87%) to mouse TERC (mTERC) with a deletion of 18 bp in the TERC conserved region 4 (CR4). This alTERC gene is expressed and its product interacts with the endogenous mTERT protein and with an exogenous human TERT protein (hTERT) to form an active enzyme. Overexpression of the alTERC and the mTERC genes, in mouse motor neurons like cells, increased the activity of TERT without affecting its protein level. Under oxidative stress conditions, alTERC significantly increased the survival of motor neurons cells without altering the level of TERT protein or its activity.The results suggest that the expression of the alTERC gene in the mouse brain provides an additional way for regulating telomerase activity under normal and stress conditions and confers protection to neuronal cells from oxidative stress.

Keywords: Gerotarget; alternative TERC; mouse brain; oxidative stress; telomerase; telomerase RNA component.

Figure 1. Identification of additional TERC gene in mouse genome using BLAT analysis

A. BLAT search was performed using the UCSC Genome Browser and the mTERC sequence as a query. Identification of 18 hits, 17 short sequences (20-104 nt) and one long sequence of 365nt (designated alTERC). B. Global pairwise alignment to the known mTERC (397nt) revealed 80.7% similarity, deletion of 18nt in the CR4. Arrows represent the location of the designed primers for the amplification by PCR: Fp1 and Rp2 (set 1) for mTERC and Fp2 and Rp2 (set 2) for alTERC. C. Comparison of the MSA consensus sequence of TERC to alTERC and to randomly generated sequences of the same length and nucleotide composition.

Figure 2. alTERC gene is transcribed to RNA in vivo in mouse organs and in vitro in mouse cell lines

A. RNA was extracted from adult mouse brain, n = 3, cDNA was generated and subjected to PCR analysis using the set 1 primers for mTERC and set 2 primers for alTERC. Two bands of ~220 bp for mTERC and ~150bp for alTERC were observed. NTC- control, no cDNA. (A is a representative picture of 3 independent experiments). B. The PCR reaction described in A was carried out in the presence of radioactive nucleotide (dCTP [αp³²] and the reaction products were analysed by 14% polyacrylamide gel electrophoresis following autoradiography. Two bands of ~230 bp and ~210 bp were observed with set 1 primers (Fpr1) for mTERC and one band of ~150 bp with set primers 2 (Fpr2) for alTERC were detected. C. RNA was extracted from mouse NSC-34 motor neurons like cells followed by cDNA production in the presence or absence of DNase or RNase and subjected to PCR amplification as described in A using the set1 and set 2 primers. D. RNA was extracted from mouse organs (brain and spleen) or from mouse neuroblastoma cell line (N2a) and subjected to sqPCR analysis using the set 1 and 2 primers for TERC and alTERC and GAPDH primers as control. A Representative picture of 3 independent experiments. E. The results of experiments described in D were quantified by densitometric analysis with the EZQuant software, calculated relatively to GAPDH and the alTERC/TERC expression ratio was estimated. The data are means ± SD of 3 independent experiments.

Figure 3. alTERC interacts with mTERT and hTERT

A. Mouse TERT protein, derived from NSC-34 protein extract, was immunoprecipitated with anti-TERT antibody or unspecific IgG antibody. The immunocomplexes were subjected to Western blot analysis using anti-TERT antibody or B. subjected to RNA purification procedure followed by cDNA preparation and PCR with the mTERC or alTERC specific primers. C. Proteins extract derived from NSC-34^GFP-hTERT transduced cells and from NSC-34 cells were analyzed by Western blot with anti-hTERT antibody, in the transduced cells in addition to the endogenous mTERT the GFP-hTERT is also detected. D.Proteins extracts derived from the NSC-34^GFP-hTERT transduced cells was subjected to immunoprecipitation assay with anti- GFP or anti-IgG antibodies or precipitated with the protein A sepharose beads only as controlled. RNA was purified from the immune complexes (P) or from the supernatant (S), or from the transduced cells (RNA), cDNA was prepared and analysed by PCR using the alTERC and mTERC specific primers. Fig 3 is a representative picture of 3 independent experiments.

Figure 4. Stable overexpression of mTERC and alTERC increased telomerase activity in NSC-34 cells

A. mTERC and alTERC were cloned into a retroviral vector and stable transduction of NSC-34 cells was performed. The expression of mTERC and alTERC in the transduced cells and in the control untransduced (UTr) or transduced with the empty vector (NV) cells were detected by PCR using the appropriate mTERC and alTERC primers. NTC- control without cDNA. B. TERT protein was detected by Western blot analysis with anti-TERT antibody and C. quantification of TERT protein relatively to the control β-actin protein was performed by densitometric analysis using the EZQuant software. The data are means ± SD of 3 independent experiments. D. Telomerase activity was measured by TRAP assay and, E. quantified by densitometric analysis using the EZQuant software. The results are % of the control NV and are means±SD, t Test, p < 0.05.

Figure 5. alTERC protects NSC-34 cells from oxidative stress without affecting TERT expression

A. The alTERC or mTERC overexpressing NSC-34 cells were exposed to increasing concentrations of H₂O₂ in a FCS depleted medium for 4 hrs and then replaced by fresh FCS containing medium for 24 hrs following by cell cytotoxicity measurement using XTT assay. Cell survival as % from control H₂O₂ -untreated cells was determined. The results are means ±SD, symbols: UTr, untransduced NSC-34 cells; NV, NSC-34 cells transduced with the empty vector; alTERC, NSC-34 cells overexpressed alTERC; TERC, NSC-34 cells overexpressed mTERC. B. Protein extracts were prepared from the cells treated as described in A and subjected to Western Blot analysis with anti TERT antibody and anti β- actin antibody (as control) or to telomerase activity using TRAP assay. Quantification of TERT level or telomerase activity was performed as described in “Materials and Methods” section and the data are presented as % of the control NV-transduced cells. The results are means ±SD of 3 independent experiments, * p < 0.05.

Figure 6. Predicted secondary structure of alTERC

TERC MSA was manually modified to resemble to alTERC sequence (see supl 3) and the Alofold software was used for the secondary structure prediction of the alTERC in comparison to that of the predicted mTERC.