Quantitation of telomerase components and hTERT mRNA splicing patterns in immortal human cells

Abstract

Telomerase is a reverse transcriptase that adds telomeric repeats to chromosomal ends. In most normal human somatic cells, telomerase is repressed and telomeres progressively shorten, leading to limited proliferative life-span. Telomerase reactivation is associated with cellular immortalization and is a frequent event during tumorigenesis. The telomerase ribonucleoprotein complex consists of two essential components, a catalytic protein subunit [human telomerase reverse transcriptase (hTERT)] and a template RNA (hTR). hTR is constitutively expressed, while hTERT is almost universally absent in telomerase-negative cells. Although repression of telomerase is transcriptional in telomerase-negative cells, post-transcriptional and assembly processes are likely to play important roles in regulating telomerase activity in those that are telomerase-positive. The telomerase transcript can also be alternatively spliced into a variety of non-functional forms. To establish the quantitative relationships between telomerase activity and its various components, we determined the numbers of molecules of hTR and hTERT mRNA, and the levels of alternatively spliced hTERT mRNA variants in normal, in vitro immortalized and cancer cell lines. We report here that there is surprisingly little variation in the proportion of alternatively spliced forms of hTERT in different cell lines. The only variation observed occurred when a change in splicing to non-functional forms appeared in response to conditions that repress telomerase activity in IDH4 cells. We also found that most telomerase-positive cell lines only contain a few molecules of potentially functional hTERT mRNA, and there is a correlation between telomerase activity and the levels of both hTR and hTERT +alpha+beta mRNA.

Figure 1

Genomic organization of the hTERT gene. RT motifs (T, 1&2, A, B′, C, D and E) (9) and alternate splicing sites (insertions 1–4 and deletions α and β) are marked (32,33). Filled boxes are translated regions; shaded boxes are 5′- and 3′-UTR. In cases of alternative splicing, an asterisk marks the location of termination codon for the insertions. A cross marks the location of the termination codon for the variant containing deletion β. The location of the PCR primers used to determine splicing patterns is shown at the bottom.

Figure 2

Telomerase activity levels of the cell types included in this study were determined by the TRAP assay as described in Materials and Methods.

Figure 3

Determination of hTR levels by competitive RT–PCR. Results of the quantitation step are shown, in which the amount of competitor has been adjusted to be equivalent to the endogenous transcript. The PCR product corresponding to the competitor RNA is 20 bp longer than that of hTR. The equivalent intensity of the two bands in each sample demonstrates that an accurate quantitation of the level of hTR has been obtained. Results are summarized in Table 3.

Figure 4

Determination of the mRNA levels of the +α+β and three alternate splicing variants of hTERT. The products corresponding to hTERT –α, hTERT –β, hTERT –α–β are 36, 182 and 218 bp shorter than that of hTERT +α+β, respectively. Distribution among the alternate splicing variants is summarized in Table 4.

Figure 5

(A and C) Data for all cell types marked with an asterisk in Table 1 for which number of hTR molecules per cell were determined. (B) Data for all telomerase-positive cell types in Table 1. In each panel, the open circle denotes DU145 data. The standard error of the slope was calculated by the formula: S.E. = {S.D. of Y × [(1 – r²)/(n – 2)]/S.D.of X}, and was determined after excluding the data from DU145.

Figure 5

(A and C) Data for all cell types marked with an asterisk in Table 1 for which number of hTR molecules per cell were determined. (B) Data for all telomerase-positive cell types in Table 1. In each panel, the open circle denotes DU145 data. The standard error of the slope was calculated by the formula: S.E. = {S.D. of Y × [(1 – r²)/(n – 2)]/S.D.of X}, and was determined after excluding the data from DU145.

Figure 5

(A and C) Data for all cell types marked with an asterisk in Table 1 for which number of hTR molecules per cell were determined. (B) Data for all telomerase-positive cell types in Table 1. In each panel, the open circle denotes DU145 data. The standard error of the slope was calculated by the formula: S.E. = {S.D. of Y × [(1 – r²)/(n – 2)]/S.D.of X}, and was determined after excluding the data from DU145.