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A tumor-promoting mechanism mediated by retrotransposon-encoded reverse transcriptase is active in human transformed cell lines.

Sciamanna I, Gualtieri A, Cossetti C, Osimo EF, Ferracin M, Macchia G, Aricò E, Prosseda G, Vitullo P, Misteli T, Spadafora C - Oncotarget (2013)

Bottom Line: Decreasing RT activity in cancer cells, by either LINE-1-specific RNA interference, or by RT inhibitory drugs, was previously found to reduce proliferation and promote differentiation and to antagonize tumor growth in animal models.The RT-sensitive miRNAs and UCRs are significantly associated with Alu sequences.The results suggest that LINE-1-encoded RT governs the balance between single-stranded and double-stranded RNA production.

View Article: PubMed Central - PubMed

Affiliation: Istituto Superiore di Sanità, Viale Regina Elena 299, Rome, Italy.

ABSTRACT
LINE-1 elements make up the most abundant retrotransposon family in the human genome. Full-length LINE-1 elements encode a reverse transcriptase (RT) activity required for their own retrotranpsosition as well as that of non-autonomous Alu elements. LINE-1 are poorly expressed in normal cells and abundantly in cancer cells. Decreasing RT activity in cancer cells, by either LINE-1-specific RNA interference, or by RT inhibitory drugs, was previously found to reduce proliferation and promote differentiation and to antagonize tumor growth in animal models. Here we have investigated how RT exerts these global regulatory functions. We report that the RT inhibitor efavirenz (EFV) selectively downregulates proliferation of transformed cell lines, while exerting only mild effects on non-transformed cells; this differential sensitivity matches a differential RT abundance, which is high in the former and undetectable in the latter. Using CsCl density gradients, we selectively identify Alu and LINE-1 containing DNA:RNA hybrid molecules in cancer but not in normal cells. Remarkably, hybrid molecules fail to form in tumor cells treated with EFV under the same conditions that repress proliferation and induce the reprogramming of expression profiles of coding genes, microRNAs (miRNAs) and ultraconserved regions (UCRs). The RT-sensitive miRNAs and UCRs are significantly associated with Alu sequences. The results suggest that LINE-1-encoded RT governs the balance between single-stranded and double-stranded RNA production. In cancer cells the abundant RT reverse-transcribes retroelement-derived mRNAs forming RNA:DNA hybrids. We propose that this impairs the formation of double-stranded RNAs and the ensuing production of small regulatory RNAs, with a direct impact on gene expression. RT inhibition restores the 'normal' small RNA profile and the regulatory networks that depend on them. Thus, the retrotransposon-encoded RT drives a previously unrecognized mechanism crucial to the transformed state in tumor cells.

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Model for RT-mediated control of the transcriptome in cancer cellsA) Retroelements are distributed in the genome in tandem and inverted repeats (i.e. closely spaced elements with opposite orientation). The top left panel depicts four Alu elements in opposite orientation on complementary DNA strands. Below, RNA transcripts (sense, red; antisense, green) containing two Alu retroelements in opposite orientation can be generated either by the internal Alu promoters, or from one Alu promoter and a nearby gene promoter in antisense orientation (external black arrows) provided by the host genome. LINE-1 elements (right) are endowed with sense (SP) and antisense (ASP) promoters, supporting LINE-1 sense and antisense transcription. B) In normal cells, double-stranded RNA (dsRNA) forms either via intramolecular base paring of an RNA containing two oppositely orientated retroelements, or via base pairing between sense (S) RNA and antisense (AS) RNAs. dsRNAs are processed by Dicer into small regulatory RNAs. C) In cancer cells, the RT encoded by highly active LINE-1 elements reverse-transcribes the available RNA populations: RNA:cDNA hybrid molecules form efficiently (Figure 3B) to the detriment of dsRNA. D) EFV treatment of cancer cells inhibits the RT activity, reduces the formation of RNA:cDNA hybrid and restores the control of regulatory small RNAs, causing the epigenetic conversion to a normal phenotype.
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Figure 6: Model for RT-mediated control of the transcriptome in cancer cellsA) Retroelements are distributed in the genome in tandem and inverted repeats (i.e. closely spaced elements with opposite orientation). The top left panel depicts four Alu elements in opposite orientation on complementary DNA strands. Below, RNA transcripts (sense, red; antisense, green) containing two Alu retroelements in opposite orientation can be generated either by the internal Alu promoters, or from one Alu promoter and a nearby gene promoter in antisense orientation (external black arrows) provided by the host genome. LINE-1 elements (right) are endowed with sense (SP) and antisense (ASP) promoters, supporting LINE-1 sense and antisense transcription. B) In normal cells, double-stranded RNA (dsRNA) forms either via intramolecular base paring of an RNA containing two oppositely orientated retroelements, or via base pairing between sense (S) RNA and antisense (AS) RNAs. dsRNAs are processed by Dicer into small regulatory RNAs. C) In cancer cells, the RT encoded by highly active LINE-1 elements reverse-transcribes the available RNA populations: RNA:cDNA hybrid molecules form efficiently (Figure 3B) to the detriment of dsRNA. D) EFV treatment of cancer cells inhibits the RT activity, reduces the formation of RNA:cDNA hybrid and restores the control of regulatory small RNAs, causing the epigenetic conversion to a normal phenotype.

Mentions: The present findings suggest a model for the role of LINE-1-encoded RT in the transition from normal to tumorigenic transcriptome (Figure 6). The model builds on the evidence that, in normal cells, LINE-1 and Alu retrotransposons can generate dsRNAs [30] through several pathways: intramolecular base paring of mRNAs containing two oppositely oriented retroelements [9,39], or base pairing between sense (S) RNA and antisense (AS) transcripts - the latter originating from antisense promoters occasionally provided by the host genome at nearby loci [40] - or, for full-length LINE-1 elements, via transcription from sense (SP) and antisense (ASP) promoters located in their 5'-UTRs [12] (Fig. 6A). Retroelement-derived dsRNAs are cleaved by Dicer into small RNAs that have roles in regulation of the transcriptome [41] and in heterochromatin organization [42] (Fig. 6B).


A tumor-promoting mechanism mediated by retrotransposon-encoded reverse transcriptase is active in human transformed cell lines.

Sciamanna I, Gualtieri A, Cossetti C, Osimo EF, Ferracin M, Macchia G, Aricò E, Prosseda G, Vitullo P, Misteli T, Spadafora C - Oncotarget (2013)

Model for RT-mediated control of the transcriptome in cancer cellsA) Retroelements are distributed in the genome in tandem and inverted repeats (i.e. closely spaced elements with opposite orientation). The top left panel depicts four Alu elements in opposite orientation on complementary DNA strands. Below, RNA transcripts (sense, red; antisense, green) containing two Alu retroelements in opposite orientation can be generated either by the internal Alu promoters, or from one Alu promoter and a nearby gene promoter in antisense orientation (external black arrows) provided by the host genome. LINE-1 elements (right) are endowed with sense (SP) and antisense (ASP) promoters, supporting LINE-1 sense and antisense transcription. B) In normal cells, double-stranded RNA (dsRNA) forms either via intramolecular base paring of an RNA containing two oppositely orientated retroelements, or via base pairing between sense (S) RNA and antisense (AS) RNAs. dsRNAs are processed by Dicer into small regulatory RNAs. C) In cancer cells, the RT encoded by highly active LINE-1 elements reverse-transcribes the available RNA populations: RNA:cDNA hybrid molecules form efficiently (Figure 3B) to the detriment of dsRNA. D) EFV treatment of cancer cells inhibits the RT activity, reduces the formation of RNA:cDNA hybrid and restores the control of regulatory small RNAs, causing the epigenetic conversion to a normal phenotype.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3926826&req=5

Figure 6: Model for RT-mediated control of the transcriptome in cancer cellsA) Retroelements are distributed in the genome in tandem and inverted repeats (i.e. closely spaced elements with opposite orientation). The top left panel depicts four Alu elements in opposite orientation on complementary DNA strands. Below, RNA transcripts (sense, red; antisense, green) containing two Alu retroelements in opposite orientation can be generated either by the internal Alu promoters, or from one Alu promoter and a nearby gene promoter in antisense orientation (external black arrows) provided by the host genome. LINE-1 elements (right) are endowed with sense (SP) and antisense (ASP) promoters, supporting LINE-1 sense and antisense transcription. B) In normal cells, double-stranded RNA (dsRNA) forms either via intramolecular base paring of an RNA containing two oppositely orientated retroelements, or via base pairing between sense (S) RNA and antisense (AS) RNAs. dsRNAs are processed by Dicer into small regulatory RNAs. C) In cancer cells, the RT encoded by highly active LINE-1 elements reverse-transcribes the available RNA populations: RNA:cDNA hybrid molecules form efficiently (Figure 3B) to the detriment of dsRNA. D) EFV treatment of cancer cells inhibits the RT activity, reduces the formation of RNA:cDNA hybrid and restores the control of regulatory small RNAs, causing the epigenetic conversion to a normal phenotype.
Mentions: The present findings suggest a model for the role of LINE-1-encoded RT in the transition from normal to tumorigenic transcriptome (Figure 6). The model builds on the evidence that, in normal cells, LINE-1 and Alu retrotransposons can generate dsRNAs [30] through several pathways: intramolecular base paring of mRNAs containing two oppositely oriented retroelements [9,39], or base pairing between sense (S) RNA and antisense (AS) transcripts - the latter originating from antisense promoters occasionally provided by the host genome at nearby loci [40] - or, for full-length LINE-1 elements, via transcription from sense (SP) and antisense (ASP) promoters located in their 5'-UTRs [12] (Fig. 6A). Retroelement-derived dsRNAs are cleaved by Dicer into small RNAs that have roles in regulation of the transcriptome [41] and in heterochromatin organization [42] (Fig. 6B).

Bottom Line: Decreasing RT activity in cancer cells, by either LINE-1-specific RNA interference, or by RT inhibitory drugs, was previously found to reduce proliferation and promote differentiation and to antagonize tumor growth in animal models.The RT-sensitive miRNAs and UCRs are significantly associated with Alu sequences.The results suggest that LINE-1-encoded RT governs the balance between single-stranded and double-stranded RNA production.

View Article: PubMed Central - PubMed

Affiliation: Istituto Superiore di Sanità, Viale Regina Elena 299, Rome, Italy.

ABSTRACT
LINE-1 elements make up the most abundant retrotransposon family in the human genome. Full-length LINE-1 elements encode a reverse transcriptase (RT) activity required for their own retrotranpsosition as well as that of non-autonomous Alu elements. LINE-1 are poorly expressed in normal cells and abundantly in cancer cells. Decreasing RT activity in cancer cells, by either LINE-1-specific RNA interference, or by RT inhibitory drugs, was previously found to reduce proliferation and promote differentiation and to antagonize tumor growth in animal models. Here we have investigated how RT exerts these global regulatory functions. We report that the RT inhibitor efavirenz (EFV) selectively downregulates proliferation of transformed cell lines, while exerting only mild effects on non-transformed cells; this differential sensitivity matches a differential RT abundance, which is high in the former and undetectable in the latter. Using CsCl density gradients, we selectively identify Alu and LINE-1 containing DNA:RNA hybrid molecules in cancer but not in normal cells. Remarkably, hybrid molecules fail to form in tumor cells treated with EFV under the same conditions that repress proliferation and induce the reprogramming of expression profiles of coding genes, microRNAs (miRNAs) and ultraconserved regions (UCRs). The RT-sensitive miRNAs and UCRs are significantly associated with Alu sequences. The results suggest that LINE-1-encoded RT governs the balance between single-stranded and double-stranded RNA production. In cancer cells the abundant RT reverse-transcribes retroelement-derived mRNAs forming RNA:DNA hybrids. We propose that this impairs the formation of double-stranded RNAs and the ensuing production of small regulatory RNAs, with a direct impact on gene expression. RT inhibition restores the 'normal' small RNA profile and the regulatory networks that depend on them. Thus, the retrotransposon-encoded RT drives a previously unrecognized mechanism crucial to the transformed state in tumor cells.

Show MeSH
Related in: MedlinePlus