Limits...
The RNA polymerase dictates ORF1 requirement and timing of LINE and SINE retrotransposition.

Kroutter EN, Belancio VP, Wagstaff BJ, Roy-Engel AM - PLoS Genet. (2009)

Bottom Line: The additional time requirement by L1 cannot be directly attributed to differences in transcription, transcript length, splicing processes, ORF2 protein production, or the ability of functional ORF2p to reach the nucleus.We postulate that the observed differences in retrotransposition kinetics of these elements are dictated by the type of RNA polymerase generating the transcript.We present a model that highlights the critical differences of LINE and SINE transcripts that likely define their retrotransposition timing.

View Article: PubMed Central - PubMed

Affiliation: Tulane Cancer Center SL-66, Tulane University Health Sciences Center, New Orleans, Louisiana, United States of America.

ABSTRACT
Mobile elements comprise close to one half of the mass of the human genome. Only LINE-1 (L1), an autonomous non-Long Terminal Repeat (LTR) retrotransposon, and its non-autonomous partners-such as the retropseudogenes, SVA, and the SINE, Alu-are currently active human retroelements. Experimental evidence shows that Alu retrotransposition depends on L1 ORF2 protein, which has led to the presumption that LINEs and SINEs share the same basic insertional mechanism. Our data demonstrate clear differences in the time required to generate insertions between marked Alu and L1 elements. In our tissue culture system, the process of L1 insertion requires close to 48 hours. In contrast to the RNA pol II-driven L1, we find that pol III transcribed elements (Alu, the rodent SINE B2, and the 7SL, U6 and hY sequences) can generate inserts within 24 hours or less. Our analyses demonstrate that the observed retrotransposition timing does not dictate insertion rate and is independent of the type of reporter cassette utilized. The additional time requirement by L1 cannot be directly attributed to differences in transcription, transcript length, splicing processes, ORF2 protein production, or the ability of functional ORF2p to reach the nucleus. However, the insertion rate of a marked Alu transcript drastically drops when driven by an RNA pol II promoter (CMV) and the retrotransposition timing parallels that of L1. Furthermore, the "pol II Alu transcript" behaves like the processed pseudogenes in our retrotransposition assay, requiring supplementation with L1 ORF1p in addition to ORF2p. We postulate that the observed differences in retrotransposition kinetics of these elements are dictated by the type of RNA polymerase generating the transcript. We present a model that highlights the critical differences of LINE and SINE transcripts that likely define their retrotransposition timing.

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Alu and L1 Exhibit Different Retrotransposition Kinetics.A. Assay design. A schematic of the constructs used for the L1 and Alu tissue culture assay are shown on the top. RNA transcription is driven by a CMV promoter for the L1 construct or the internal pol III Alu promoter. The restriction sites used in the construction of the other pol III driven vectors are shown. The L1 construct contains a full-length retrocompetent L1 element with its ORF1 and ORF2. The L1 vector is tagged with the mneoI indicator cassette containing an inverted neomycin resistance gene (neo, light gray box) disrupted by an intron [16]. The Alu vector contains a neoTET cassette with a tetrahymena self-splicing intron interrupting the neo gene [4]. In both constructs, the introns will only splice out from a transcript generated by the L1 or Alu promoter. The spliced RNA is reverse transcribed, followed by integration of the cDNA into the genome. The new insert contains a functional neomycin gene. G418 resistance will be obtained only if retrotransposition occurs. B. Schematic of treatment timeline. HeLa cells were seeded and transfected the next day with the appropriate constructs. After the three hour incubation with the transfection cocktail (3h*) the first set of cells was treated with d4t and G418 containing media (0 h). Note that at this time point the plasmid DNA has already been in contact with the cells for 3 h. The second set of cells was treated after 3 hours (3 h), and so forth until completing all the time points (shown as arrows above). Cells were stained after 2 weeks of growth under selection. C. Alu inserts are detected at 24 h, while L1 requires at least 48 hours to generate inserts. HeLa cells were transiently transfected with L1mneo (black bar) or AluYa5neoTET+ORF2p expression vector (gray bar) and d4t plus G418 treatment started 3, 6, 18, 24, and 48 h post-transfection (x axis). Inset shows representative G418R foci results of the retrotransposition assay. Bars represent the relative % mean G418R colonies±standard deviation shown as error bars for each construct. The 48 h data were used to define 100%. The mean of the observed G418 resistant colonies is shown in parentheses above each column.
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pgen-1000458-g002: Alu and L1 Exhibit Different Retrotransposition Kinetics.A. Assay design. A schematic of the constructs used for the L1 and Alu tissue culture assay are shown on the top. RNA transcription is driven by a CMV promoter for the L1 construct or the internal pol III Alu promoter. The restriction sites used in the construction of the other pol III driven vectors are shown. The L1 construct contains a full-length retrocompetent L1 element with its ORF1 and ORF2. The L1 vector is tagged with the mneoI indicator cassette containing an inverted neomycin resistance gene (neo, light gray box) disrupted by an intron [16]. The Alu vector contains a neoTET cassette with a tetrahymena self-splicing intron interrupting the neo gene [4]. In both constructs, the introns will only splice out from a transcript generated by the L1 or Alu promoter. The spliced RNA is reverse transcribed, followed by integration of the cDNA into the genome. The new insert contains a functional neomycin gene. G418 resistance will be obtained only if retrotransposition occurs. B. Schematic of treatment timeline. HeLa cells were seeded and transfected the next day with the appropriate constructs. After the three hour incubation with the transfection cocktail (3h*) the first set of cells was treated with d4t and G418 containing media (0 h). Note that at this time point the plasmid DNA has already been in contact with the cells for 3 h. The second set of cells was treated after 3 hours (3 h), and so forth until completing all the time points (shown as arrows above). Cells were stained after 2 weeks of growth under selection. C. Alu inserts are detected at 24 h, while L1 requires at least 48 hours to generate inserts. HeLa cells were transiently transfected with L1mneo (black bar) or AluYa5neoTET+ORF2p expression vector (gray bar) and d4t plus G418 treatment started 3, 6, 18, 24, and 48 h post-transfection (x axis). Inset shows representative G418R foci results of the retrotransposition assay. Bars represent the relative % mean G418R colonies±standard deviation shown as error bars for each construct. The 48 h data were used to define 100%. The mean of the observed G418 resistant colonies is shown in parentheses above each column.

Mentions: Reverse transcriptase (RT) domains of multiple sources can be grouped into a family of shared sequence homology [NCBI cdd pfam00078.12] [46], including the RT of the human immunodeficiency virus and L1 ORF2 protein. Endogenous RT activity is inhibited by two antiretroviral agents nevirapine and efavirenz [47]. L1 retrotransposition in a culture assay system can be suppressed by the addition of a variety of HIV RT inhibitors [48],[49]. This system utilizes a tagged vector designed to allow expression of the reporter gene only when the retroelement goes through its reverse transcriptase-dependent amplification process (Figure 2A). Therefore, only the newly inserted element will express the reporter gene (e.g. neo).


The RNA polymerase dictates ORF1 requirement and timing of LINE and SINE retrotransposition.

Kroutter EN, Belancio VP, Wagstaff BJ, Roy-Engel AM - PLoS Genet. (2009)

Alu and L1 Exhibit Different Retrotransposition Kinetics.A. Assay design. A schematic of the constructs used for the L1 and Alu tissue culture assay are shown on the top. RNA transcription is driven by a CMV promoter for the L1 construct or the internal pol III Alu promoter. The restriction sites used in the construction of the other pol III driven vectors are shown. The L1 construct contains a full-length retrocompetent L1 element with its ORF1 and ORF2. The L1 vector is tagged with the mneoI indicator cassette containing an inverted neomycin resistance gene (neo, light gray box) disrupted by an intron [16]. The Alu vector contains a neoTET cassette with a tetrahymena self-splicing intron interrupting the neo gene [4]. In both constructs, the introns will only splice out from a transcript generated by the L1 or Alu promoter. The spliced RNA is reverse transcribed, followed by integration of the cDNA into the genome. The new insert contains a functional neomycin gene. G418 resistance will be obtained only if retrotransposition occurs. B. Schematic of treatment timeline. HeLa cells were seeded and transfected the next day with the appropriate constructs. After the three hour incubation with the transfection cocktail (3h*) the first set of cells was treated with d4t and G418 containing media (0 h). Note that at this time point the plasmid DNA has already been in contact with the cells for 3 h. The second set of cells was treated after 3 hours (3 h), and so forth until completing all the time points (shown as arrows above). Cells were stained after 2 weeks of growth under selection. C. Alu inserts are detected at 24 h, while L1 requires at least 48 hours to generate inserts. HeLa cells were transiently transfected with L1mneo (black bar) or AluYa5neoTET+ORF2p expression vector (gray bar) and d4t plus G418 treatment started 3, 6, 18, 24, and 48 h post-transfection (x axis). Inset shows representative G418R foci results of the retrotransposition assay. Bars represent the relative % mean G418R colonies±standard deviation shown as error bars for each construct. The 48 h data were used to define 100%. The mean of the observed G418 resistant colonies is shown in parentheses above each column.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2666806&req=5

pgen-1000458-g002: Alu and L1 Exhibit Different Retrotransposition Kinetics.A. Assay design. A schematic of the constructs used for the L1 and Alu tissue culture assay are shown on the top. RNA transcription is driven by a CMV promoter for the L1 construct or the internal pol III Alu promoter. The restriction sites used in the construction of the other pol III driven vectors are shown. The L1 construct contains a full-length retrocompetent L1 element with its ORF1 and ORF2. The L1 vector is tagged with the mneoI indicator cassette containing an inverted neomycin resistance gene (neo, light gray box) disrupted by an intron [16]. The Alu vector contains a neoTET cassette with a tetrahymena self-splicing intron interrupting the neo gene [4]. In both constructs, the introns will only splice out from a transcript generated by the L1 or Alu promoter. The spliced RNA is reverse transcribed, followed by integration of the cDNA into the genome. The new insert contains a functional neomycin gene. G418 resistance will be obtained only if retrotransposition occurs. B. Schematic of treatment timeline. HeLa cells were seeded and transfected the next day with the appropriate constructs. After the three hour incubation with the transfection cocktail (3h*) the first set of cells was treated with d4t and G418 containing media (0 h). Note that at this time point the plasmid DNA has already been in contact with the cells for 3 h. The second set of cells was treated after 3 hours (3 h), and so forth until completing all the time points (shown as arrows above). Cells were stained after 2 weeks of growth under selection. C. Alu inserts are detected at 24 h, while L1 requires at least 48 hours to generate inserts. HeLa cells were transiently transfected with L1mneo (black bar) or AluYa5neoTET+ORF2p expression vector (gray bar) and d4t plus G418 treatment started 3, 6, 18, 24, and 48 h post-transfection (x axis). Inset shows representative G418R foci results of the retrotransposition assay. Bars represent the relative % mean G418R colonies±standard deviation shown as error bars for each construct. The 48 h data were used to define 100%. The mean of the observed G418 resistant colonies is shown in parentheses above each column.
Mentions: Reverse transcriptase (RT) domains of multiple sources can be grouped into a family of shared sequence homology [NCBI cdd pfam00078.12] [46], including the RT of the human immunodeficiency virus and L1 ORF2 protein. Endogenous RT activity is inhibited by two antiretroviral agents nevirapine and efavirenz [47]. L1 retrotransposition in a culture assay system can be suppressed by the addition of a variety of HIV RT inhibitors [48],[49]. This system utilizes a tagged vector designed to allow expression of the reporter gene only when the retroelement goes through its reverse transcriptase-dependent amplification process (Figure 2A). Therefore, only the newly inserted element will express the reporter gene (e.g. neo).

Bottom Line: The additional time requirement by L1 cannot be directly attributed to differences in transcription, transcript length, splicing processes, ORF2 protein production, or the ability of functional ORF2p to reach the nucleus.We postulate that the observed differences in retrotransposition kinetics of these elements are dictated by the type of RNA polymerase generating the transcript.We present a model that highlights the critical differences of LINE and SINE transcripts that likely define their retrotransposition timing.

View Article: PubMed Central - PubMed

Affiliation: Tulane Cancer Center SL-66, Tulane University Health Sciences Center, New Orleans, Louisiana, United States of America.

ABSTRACT
Mobile elements comprise close to one half of the mass of the human genome. Only LINE-1 (L1), an autonomous non-Long Terminal Repeat (LTR) retrotransposon, and its non-autonomous partners-such as the retropseudogenes, SVA, and the SINE, Alu-are currently active human retroelements. Experimental evidence shows that Alu retrotransposition depends on L1 ORF2 protein, which has led to the presumption that LINEs and SINEs share the same basic insertional mechanism. Our data demonstrate clear differences in the time required to generate insertions between marked Alu and L1 elements. In our tissue culture system, the process of L1 insertion requires close to 48 hours. In contrast to the RNA pol II-driven L1, we find that pol III transcribed elements (Alu, the rodent SINE B2, and the 7SL, U6 and hY sequences) can generate inserts within 24 hours or less. Our analyses demonstrate that the observed retrotransposition timing does not dictate insertion rate and is independent of the type of reporter cassette utilized. The additional time requirement by L1 cannot be directly attributed to differences in transcription, transcript length, splicing processes, ORF2 protein production, or the ability of functional ORF2p to reach the nucleus. However, the insertion rate of a marked Alu transcript drastically drops when driven by an RNA pol II promoter (CMV) and the retrotransposition timing parallels that of L1. Furthermore, the "pol II Alu transcript" behaves like the processed pseudogenes in our retrotransposition assay, requiring supplementation with L1 ORF1p in addition to ORF2p. We postulate that the observed differences in retrotransposition kinetics of these elements are dictated by the type of RNA polymerase generating the transcript. We present a model that highlights the critical differences of LINE and SINE transcripts that likely define their retrotransposition timing.

Show MeSH