Telomeric Repeat-Containing RNA Increases in Aged Human Cells

Telomeric repeat-containing RNA (TERRA), transcribed from subtelomeric regions towards telomeric ends, poses challenges in deciphering its complete sequences. Utilizing TERRA-capture RNA-seq and Oxford Nanopore direct RNA sequencing to acquire full-length TERRA, we annotate TERRA transcription regions in the human T2T-CHM13 reference genome. TERRA transcripts encompass hundreds to over a thousand nucleotides of telomeric repeats, predominantly originating from 61-29-37 bp repeat promoters enriched with H3K4me3, RNA pol II, CTCF, and R-loops. We develop a bioinformatics tool, TERRA-QUANT, for quantifying TERRA using RNA-seq datasets and find that TERRA increases with age in blood, brain, and fibroblasts. TERRA upregulation in aged leukocytes is confirmed by RT-qPCR. Single-cell RNA-seq analysis demonstrates TERRA expression across various cell types, with upregulation observed in neurons during human embryonic stem cell differentiation. Additionally, TERRA levels are elevated in brain cells in the early stage of Alzheimer's disease. Our study provides evidence linking TERRA to human aging and diseases. Overall design: TERRA was enriched using a C-rich telomeric antisense probe and subsequently subjected to Illumina pair-end or Nanopore RNA direct sequencing. For Illumina sequencing, first-strand cDNA was synthesized with a random hexamer (random group) or a C-rich telomeric primer (specific group), followed by library construction. In the case of Nanopore sequencing, the TERRA-enriched sample was polyadenylated and ligated to an adaptor for direct RNA sequencing.