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Activation of an endogenous retrovirus-associated long non-coding RNA in human adenocarcinoma.

Gibb EA, Warren RL, Wilson GW, Brown SD, Robertson GA, Morin GB, Holt RA - Genome Med (2015)

Bottom Line: Compared to protein-coding genes, lncRNA genes are often associated with transposable elements, particularly with endogenous retroviral elements (ERVs).ERVs can have potentially deleterious effects on genome structure and function, so these elements are typically silenced in normal somatic tissues, albeit with varying efficiency.Genome-wide, MER48 insertions are associated with nine lncRNAs, but none of the MER48-associated lncRNAs other than EVADR were consistently expressed in adenocarcinomas, demonstrating the specific activation of EVADR.

View Article: PubMed Central - PubMed

Affiliation: Genome Sciences Centre, British Columbia Cancer Agency, 675 West 10th Ave, Vancouver, British Columbia V5Z 1L3 Canada ; Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia V6T 1Z4 Canada.

ABSTRACT

Background: Long non-coding RNAs (lncRNAs) are emerging as molecules that significantly impact many cellular processes and have been associated with almost every human cancer. Compared to protein-coding genes, lncRNA genes are often associated with transposable elements, particularly with endogenous retroviral elements (ERVs). ERVs can have potentially deleterious effects on genome structure and function, so these elements are typically silenced in normal somatic tissues, albeit with varying efficiency. The aberrant regulation of ERVs associated with lncRNAs (ERV-lncRNAs), coupled with the diverse range of lncRNA functions, creates significant potential for ERV-lncRNAs to impact cancer biology.

Methods: We used RNA-seq analysis to identify and profile the expression of a novel lncRNA in six large cohorts, including over 7,500 samples from The Cancer Genome Atlas (TCGA).

Results: We identified the tumor-specific expression of a novel lncRNA that we have named Endogenous retroViral-associated ADenocarcinoma RNA or 'EVADR', by analyzing RNA-seq data derived from colorectal tumors and matched normal control tissues. Subsequent analysis of TCGA RNA-seq data revealed the striking association of EVADR with adenocarcinomas, which are tumors of glandular origin. Moderate to high levels of EVADR were detected in 25 to 53% of colon, rectal, lung, pancreas and stomach adenocarcinomas (mean = 30 to 144 FPKM), and EVADR expression correlated with decreased patient survival (Cox regression; hazard ratio = 1.47, 95% confidence interval = 1.06 to 2.04, P = 0.02). In tumor sites of non-glandular origin, EVADR expression was detectable at only very low levels and in less than 10% of patients. For EVADR, a MER48 ERV element provides an active promoter to drive its transcription. Genome-wide, MER48 insertions are associated with nine lncRNAs, but none of the MER48-associated lncRNAs other than EVADR were consistently expressed in adenocarcinomas, demonstrating the specific activation of EVADR. The sequence and structure of the EVADR locus is highly conserved among Old World monkeys and apes but not New World monkeys or prosimians, where the MER48 insertion is absent. Conservation of the EVADR locus suggests a functional role for this novel lncRNA in humans and our closest primate relatives.

Conclusions: Our results describe the specific activation of a highly conserved ERV-lncRNA in numerous cancers of glandular origin, a finding with diagnostic, prognostic and therapeutic implications.

No MeSH data available.


Related in: MedlinePlus

The lncRNAEVADRis partially derived from a MER48 ERV element. (a) Gene structure indicating the MER48 element overlapping with the 5′ termini of EVADR (red); lncRNA exons are shown in blue, predicted poly(A) signal in yellow and the promoter by a bent arrowhead. (b) Partial sequences of 10 clones from 5′ RNA ligase-mediated RACE analysis of MER48 initiated EVADR transcripts aligned to human genomic DNA. The predicted TATA box is indicated by a line, the minor transcriptional start sites by an asterisk, and the predominant initiating nucleotide is bolded and indicated by a bent arrow. The RNA adaptor sequences are light grey and in lower case. (c) Promoter deletion experimental design showing truncations of the MER48 element. The MER48 LTR is indicated by a red arrow, the luciferase ORF by green rectangles, and EVADR is indicated by blue rectangles. (d) Results of the promoter analysis in K562 cells. (e) Results of the promoter analysis in SW480 cells. *Adjusted P < 0.05; **adjusted P < 0.005; two-sample t-test with Bonferroni correction.
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Fig4: The lncRNAEVADRis partially derived from a MER48 ERV element. (a) Gene structure indicating the MER48 element overlapping with the 5′ termini of EVADR (red); lncRNA exons are shown in blue, predicted poly(A) signal in yellow and the promoter by a bent arrowhead. (b) Partial sequences of 10 clones from 5′ RNA ligase-mediated RACE analysis of MER48 initiated EVADR transcripts aligned to human genomic DNA. The predicted TATA box is indicated by a line, the minor transcriptional start sites by an asterisk, and the predominant initiating nucleotide is bolded and indicated by a bent arrow. The RNA adaptor sequences are light grey and in lower case. (c) Promoter deletion experimental design showing truncations of the MER48 element. The MER48 LTR is indicated by a red arrow, the luciferase ORF by green rectangles, and EVADR is indicated by blue rectangles. (d) Results of the promoter analysis in K562 cells. (e) Results of the promoter analysis in SW480 cells. *Adjusted P < 0.05; **adjusted P < 0.005; two-sample t-test with Bonferroni correction.

Mentions: Detailed sequence analysis of the EVADR genomic locus revealed sequence identity with a MER48 LTR, which is an endogenous retroviral element of the ERV1 family (Figure 4a) [54]. The MER48 LTR contributes 127 nucleotides to the primary sequence of the 5′ exon of EVADR, and also encodes numerous transcription factor binding sites and a putative TATA box, suggesting a possible role for these regulatory sequences in the transcriptional activation of EVADR in adenocarcinoma. As K562 cells strongly express EVADR, we selected these cells for 5′-RACE [55] and mapped three distinct 5′ transcript termini to the EVADR MER48 LTR, each downstream of the predicted TATA box (Figure 4b). These data refine the length of the predominant EVADR transcript from the predicted value of 397 nucleotides to the confirmed value of 394 nucleotides. To experimentally test the capacity of the MER48 LTR in driving downstream transcription, we generated a series of truncated MER48 constructs and measured promoter activity, in triplicate, using a dual luciferase assay (Figure 4c). While full-length MER48 is active in both K562 and SW480 cells (MER1F; K562, 73.7 ± 5.1 RLU (mean ± SD); SW480 8.14 ± 0.40 RLU), each subsequent 5′ truncation dramatically reduced luciferase activity (MER2F; K562, mean 1.45 ± 0.14 RLU; SW480, 1.26 ± 0.08 RLU), with negligible promoter activity observed for MER48 sequence overlapping with the 5′ exon of EVADR (MER3F; K562, 0.08 ± 0.01 RLU; SW480, 0.10 ± 0.01 RLU) (Figure 4d,e). Surprisingly, we observed strong luciferase activity when the EVADR MER48 LTR was in the opposite orientation (MER_FLIP; K562, 198 ± 15.4 RLU; SW480, 23.6 ± 2.4 RLU), indicating the MER48 LTR can function as a bidirectional promoter (Figure 4d,e; MER_FLIP). Despite the high transcriptional activity observed from the MER_FLIP plasmid constructs, we found no evidence supporting bidirectional transcription in vivo (Figure S6 in Additional file 1). These results demonstrate that the MER48 LTR not only contributes to the 5′ exon of EVADR, but also provides a promoter sequence capable of driving the transcription of this lncRNA.Figure 4


Activation of an endogenous retrovirus-associated long non-coding RNA in human adenocarcinoma.

Gibb EA, Warren RL, Wilson GW, Brown SD, Robertson GA, Morin GB, Holt RA - Genome Med (2015)

The lncRNAEVADRis partially derived from a MER48 ERV element. (a) Gene structure indicating the MER48 element overlapping with the 5′ termini of EVADR (red); lncRNA exons are shown in blue, predicted poly(A) signal in yellow and the promoter by a bent arrowhead. (b) Partial sequences of 10 clones from 5′ RNA ligase-mediated RACE analysis of MER48 initiated EVADR transcripts aligned to human genomic DNA. The predicted TATA box is indicated by a line, the minor transcriptional start sites by an asterisk, and the predominant initiating nucleotide is bolded and indicated by a bent arrow. The RNA adaptor sequences are light grey and in lower case. (c) Promoter deletion experimental design showing truncations of the MER48 element. The MER48 LTR is indicated by a red arrow, the luciferase ORF by green rectangles, and EVADR is indicated by blue rectangles. (d) Results of the promoter analysis in K562 cells. (e) Results of the promoter analysis in SW480 cells. *Adjusted P < 0.05; **adjusted P < 0.005; two-sample t-test with Bonferroni correction.
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Fig4: The lncRNAEVADRis partially derived from a MER48 ERV element. (a) Gene structure indicating the MER48 element overlapping with the 5′ termini of EVADR (red); lncRNA exons are shown in blue, predicted poly(A) signal in yellow and the promoter by a bent arrowhead. (b) Partial sequences of 10 clones from 5′ RNA ligase-mediated RACE analysis of MER48 initiated EVADR transcripts aligned to human genomic DNA. The predicted TATA box is indicated by a line, the minor transcriptional start sites by an asterisk, and the predominant initiating nucleotide is bolded and indicated by a bent arrow. The RNA adaptor sequences are light grey and in lower case. (c) Promoter deletion experimental design showing truncations of the MER48 element. The MER48 LTR is indicated by a red arrow, the luciferase ORF by green rectangles, and EVADR is indicated by blue rectangles. (d) Results of the promoter analysis in K562 cells. (e) Results of the promoter analysis in SW480 cells. *Adjusted P < 0.05; **adjusted P < 0.005; two-sample t-test with Bonferroni correction.
Mentions: Detailed sequence analysis of the EVADR genomic locus revealed sequence identity with a MER48 LTR, which is an endogenous retroviral element of the ERV1 family (Figure 4a) [54]. The MER48 LTR contributes 127 nucleotides to the primary sequence of the 5′ exon of EVADR, and also encodes numerous transcription factor binding sites and a putative TATA box, suggesting a possible role for these regulatory sequences in the transcriptional activation of EVADR in adenocarcinoma. As K562 cells strongly express EVADR, we selected these cells for 5′-RACE [55] and mapped three distinct 5′ transcript termini to the EVADR MER48 LTR, each downstream of the predicted TATA box (Figure 4b). These data refine the length of the predominant EVADR transcript from the predicted value of 397 nucleotides to the confirmed value of 394 nucleotides. To experimentally test the capacity of the MER48 LTR in driving downstream transcription, we generated a series of truncated MER48 constructs and measured promoter activity, in triplicate, using a dual luciferase assay (Figure 4c). While full-length MER48 is active in both K562 and SW480 cells (MER1F; K562, 73.7 ± 5.1 RLU (mean ± SD); SW480 8.14 ± 0.40 RLU), each subsequent 5′ truncation dramatically reduced luciferase activity (MER2F; K562, mean 1.45 ± 0.14 RLU; SW480, 1.26 ± 0.08 RLU), with negligible promoter activity observed for MER48 sequence overlapping with the 5′ exon of EVADR (MER3F; K562, 0.08 ± 0.01 RLU; SW480, 0.10 ± 0.01 RLU) (Figure 4d,e). Surprisingly, we observed strong luciferase activity when the EVADR MER48 LTR was in the opposite orientation (MER_FLIP; K562, 198 ± 15.4 RLU; SW480, 23.6 ± 2.4 RLU), indicating the MER48 LTR can function as a bidirectional promoter (Figure 4d,e; MER_FLIP). Despite the high transcriptional activity observed from the MER_FLIP plasmid constructs, we found no evidence supporting bidirectional transcription in vivo (Figure S6 in Additional file 1). These results demonstrate that the MER48 LTR not only contributes to the 5′ exon of EVADR, but also provides a promoter sequence capable of driving the transcription of this lncRNA.Figure 4

Bottom Line: Compared to protein-coding genes, lncRNA genes are often associated with transposable elements, particularly with endogenous retroviral elements (ERVs).ERVs can have potentially deleterious effects on genome structure and function, so these elements are typically silenced in normal somatic tissues, albeit with varying efficiency.Genome-wide, MER48 insertions are associated with nine lncRNAs, but none of the MER48-associated lncRNAs other than EVADR were consistently expressed in adenocarcinomas, demonstrating the specific activation of EVADR.

View Article: PubMed Central - PubMed

Affiliation: Genome Sciences Centre, British Columbia Cancer Agency, 675 West 10th Ave, Vancouver, British Columbia V5Z 1L3 Canada ; Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia V6T 1Z4 Canada.

ABSTRACT

Background: Long non-coding RNAs (lncRNAs) are emerging as molecules that significantly impact many cellular processes and have been associated with almost every human cancer. Compared to protein-coding genes, lncRNA genes are often associated with transposable elements, particularly with endogenous retroviral elements (ERVs). ERVs can have potentially deleterious effects on genome structure and function, so these elements are typically silenced in normal somatic tissues, albeit with varying efficiency. The aberrant regulation of ERVs associated with lncRNAs (ERV-lncRNAs), coupled with the diverse range of lncRNA functions, creates significant potential for ERV-lncRNAs to impact cancer biology.

Methods: We used RNA-seq analysis to identify and profile the expression of a novel lncRNA in six large cohorts, including over 7,500 samples from The Cancer Genome Atlas (TCGA).

Results: We identified the tumor-specific expression of a novel lncRNA that we have named Endogenous retroViral-associated ADenocarcinoma RNA or 'EVADR', by analyzing RNA-seq data derived from colorectal tumors and matched normal control tissues. Subsequent analysis of TCGA RNA-seq data revealed the striking association of EVADR with adenocarcinomas, which are tumors of glandular origin. Moderate to high levels of EVADR were detected in 25 to 53% of colon, rectal, lung, pancreas and stomach adenocarcinomas (mean = 30 to 144 FPKM), and EVADR expression correlated with decreased patient survival (Cox regression; hazard ratio = 1.47, 95% confidence interval = 1.06 to 2.04, P = 0.02). In tumor sites of non-glandular origin, EVADR expression was detectable at only very low levels and in less than 10% of patients. For EVADR, a MER48 ERV element provides an active promoter to drive its transcription. Genome-wide, MER48 insertions are associated with nine lncRNAs, but none of the MER48-associated lncRNAs other than EVADR were consistently expressed in adenocarcinomas, demonstrating the specific activation of EVADR. The sequence and structure of the EVADR locus is highly conserved among Old World monkeys and apes but not New World monkeys or prosimians, where the MER48 insertion is absent. Conservation of the EVADR locus suggests a functional role for this novel lncRNA in humans and our closest primate relatives.

Conclusions: Our results describe the specific activation of a highly conserved ERV-lncRNA in numerous cancers of glandular origin, a finding with diagnostic, prognostic and therapeutic implications.

No MeSH data available.


Related in: MedlinePlus