Telomeric RNA Expression: Length Matters

A commentary on TERRA expression levels do not correlate with telomere length and radiation sensitivity in human cancer cell lines We read with great interest the Research Article by Smirnova et al. (1) in which sets of isogenic subclones isolated from human cancer cell lines were used to investigate the impact of telomere length on telomeric repeat-containing RNA (TERRA) expression. The first conclusion from this study, i.e., that telomere length does not impact on TERRA expression, was based on results obtained from a combination of Southern (telomeres) and northern (TERRA) blots. Using the same 32 P-α[dCTP]-labeled telo-meric probe, the authors, on one hand, measured telomere length by evaluating the molecular weight range in which TTAGGG signals were detected, and, on the other hand, measured TERRA expression levels by quantifying radioactive signals coming from hybridization of the probe with UUAGGG repeats. We believe, however, that the latter approach is misleading when used to quantify TERRA expression in cells with distinct telomere lengths for two main reasons. First, TERRA transcript length is naturally increased upon elongation of its template DNA, the telomeric repeats [(2); Figures 1A,B], and therefore northern blots measure UUAGGG content rather than the number of TERRA molecules. Second, the transfer efficiency of TERRA onto nylon membranes is drastically reduced upon increase in molecule length. Hence, if alkaline treatment is omitted after electro-phoresis, long TERRA molecules simply do not transfer (Figures 1B,C). A drawback of alkaline treatment however explaining why this is usually not applied for northern blots is that it releases RNA from the gel (Figures 1B,C), making quantifications rather difficult. Smirnova et al. (1) do not appear to have included an alkaline treatment step as, despite high differences in telomere length, notably between HeLa parental cells (7 kb) and cells overexpress-ing telomerase (50 kb), TERRA profiles were similar (Figure 1 of their article). Most probably, a dot-blot analysis of total RNA would have revealed much higher levels of UUAGGG repeats in HeLa cells overexpressing telomerase. However, here too, data must be carefully interpreted as, despite higher UUAGGG repeat content, the number of TERRA molecules may still be lower compared to the isogenic cell line with shorter telomeres. This is easily explained as follows: if telomere length increases from 7 to 50 kb, and provided that UUAGGG tract length mirrors that of the TTAGGG tract, UUAGGG repeats may be up to sevenfold more abundant in cells with longer telomeres. …