TERRA transcripts and promoters from telomeric and interstitial sites

The transcription of human telomeres gives rise to a family of long noncoding RNAs, named TERRA. We previously showed that TERRA transcription is driven by CpG island promoters that are composed of stretches of three types of repeats. Using the human genome assembly that was available at that time, putative promoter sequences were localized at several subtelomeres. In this work, using the T2T-CHM13v2.0 human reference genome, we found that 39 out of 46 subtelomeres contain TERRA promoters and grouped them in classes depending on their organization. We then discovered 106 intrachromosomal TERRA-like promoters, adjacent to interstitial telomeric sequences (ITSs) or far away from them. Forty-seven of these promoters are flanked and may regulate the transcription of coding genes, ncRNAs or pseudogenes. Comparative sequence analysis showed that interstitial and subtelomeric promoters belong to a previously undescribed family of segmental duplications deriving from common ancestral sequences. RT-PCR experiments in seven cell lines demonstrated that TERRA transcripts can be synthesized from ITSs. TERRA expression was always low in primary fibroblasts and HeLa cells, while highly variable in the other two telomerase-positive (HT1080 and HEK293) and in the three telomerase-negative ALT cell lines (GM847, U2OS, and VA13). The analysis of RNA-seq data from U2OS, HeLa, and HEK293 cells showed that 205 ITSs were transcribed in at least one cell line. The fraction of transcribed ITSs and the level of their transcription increased with the length of the telomeric repeat stretch. Given the large number of transcribed ITSs, we propose that these loci contribute significantly to the production of the TERRA pool.