A Novel Long Non-Coding RNA in the hTERT Promoter Region Regulates hTERT Expression

Abstract

A novel antisense transcript was identified in the human telomerase reverse transcriptase (hTERT) promoter region, suggesting that the hTERT promoter is bidirectional. This transcript, named hTERT antisense promoter-associated (hTAPAS) RNA, is a 1.6 kb long non-coding RNA. hTAPAS transcription is initiated 167 nucleotides upstream of the hTERT transcription start site and is present in both the nucleus and the cytoplasm. Surprisingly, we observed that a large fraction of the hTERT polyadenylated RNA is localized in the nucleus, suggesting this might be an additional means of regulating the cellular abundance of hTERT protein. Both hTAPAS and hTERT are expressed in immortalized B-cells and human embryonic stem cells but are not detected in normal somatic cells. hTAPAS expression inversely correlates with hTERT expression in different types of cancer samples. Moreover, hTAPAS expression is not promoted by an hTERT promoter mutation (-124 C>T). Antisense-oligonucleotide mediated knockdown of hTAPAS results in an increase in hTERT expression. Conversely, ectopic overexpression of hTAPAS down regulates hTERT expression, suggesting a negative role in hTERT gene regulation. These observations provide insights into hTAPAS as a novel player that negatively regulates hTERT expression and may be involved in telomere length homeostasis.

Keywords: TERT; bidirectional transcription; cancer; lncRNA.

Figure 1

An antisense RNA, named human TAPAS RNA, is expressed in the human telomerase reverse transcriptase (hTERT) promoter region. Normalized and stranded RNA sequencing (RNA-seq) (Bedgraph) transcription coverage for hTERT (green) and hTAPAS (blue) expression, as well as corresponding cap analysis gene expression (CAGE) start sites, are depicted for the human B-cell line GM12878. The schematic below denotes Reverse Transcription PCR (RT-PCR) validation of hTAPAS transcript from human embryonic kidney (HEK-293) cells by tiling arrays. The first two exons of hTERT and an approximately 1.6 kb long hTAPAS gene, located 167 nts upstream of the hTERT transcriptional start site are depicted. Arrowheads between hTERT and hTAPAS represent sites of point mutations in the hTERT promoter, located 124 or 146 nucleotides upstream of the hTERT translational start site.

Figure 2

Sub-cellular localization of hTERT and hTAPAS RNAs (A) Normalized and stranded RNA-seq (Bedgraph) transcription coverage for hTERT (green) and hTAPAS (blue) expression are depicted for the polyadenylated (polyA+) and non-polyadenylated (polyA-) transcripts in the nuclear and cytoplasmic fractions, from the human B-cell cell line (GM12878). All plus strand (blue) and minus strand (green) track signals are depicted on a log-scale (0–420). Transcription coverage for corresponding transcripts of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (red) are depicted as a control, on a log scale (0–715562). Represented data are from the ENCODE Consortium. (B) Abundance of hTAPAS, hTERT and GAPDH (mRNA and pre-mRNA) transcripts in the nucleus and cytoplasm cellular fractions determined by quantitative Reverse Transcription PCR (qRT-PCR) after sub-cellular fractionation of HEK-293 cells.

Figure 3

hTERT and hTAPAS expression are inversely correlated in primary tumors. RNA-seq data samples from TCGA, which have detectable expression levels for either hTERT or hTAPAS were analyzed in the eight different cancers (melanomas, gliomas, glioblastomas, hepatocellular carcinomas, bladder, lymphoma, prostate and breast cancers). A majority of these tumor samples express either hTERT or hTAPAS, but not both. The heat-maps depict corresponding expression levels of hTAPAS and hTERT among individual patient samples from the eight different cancers. Expression is represented as log-transformed Fragments Per Kilobase of transcript per Million mapped reads (FPKM) values (white indicates undetectable expression).

Figure 4

hTAPAS knockdown in HEK-293 cells results in increased hTERT expression. hTERT and hTAPAS expression was determined by qRT-PCR in HEK-293 cells transfected with mock, scrambled oligos, and antisense oligonucleotides (ASO) targeted to hTAPAS. Use of ASO resulted in a 4-fold knock down of hTAPAS expression, relative to scrambled. hTERT expression was analyzed using primers spanning its exons 13–14 or exons 7–8 (in the Reverse Transcriptase domain).

Figure 5

Presence of the hTERT promoter mutation does not alter hTAPAS expression. (A) Schematic representation of an empty control vector (1), and hTAPAS insert with a wild-type (2) or mutant (3) hTERT promoter region, in a luciferase reporter vector. (B) Fold-change in luciferase activity observed for the different constructs relative to empty vector. (C) Fold-change in hTAPAS or (D) endogenous hTERT expression levels observed for the different constructs relative to empty vector.

Figure 6

TAPAS is a viral integration hotspot in chicken and human tumors. The schematic represents the hTERT promoter region, including the transcription start sites (black arrows) for hTERT and TAPAS, in the chicken (top) and human (bottom) genomes. This region is depicted as a viral integration hotspot for the avian leukosis virus (ALV) and hepatitis B virus (HBV), respectively in tumors. Red arrowheads represent the integration sites for ALV or HBV in this region.