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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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The RNA Polymerase Dictates the Retrotransposition Kinetics of Alu.A. Schematic of pol II-driven ORF1 and Alu vectors. The ORF1mneo construct was selected as a representative of retropseudogene activity. The constructs use the CMV promoter (CMVp, black box) to generate pol II transcripts. The full mneoI indicator cassette from the L1 vector, consisting of the neomycin interrupted by an inverted intron (hatched box), its SV40 promoter (SV40p) and complete polyadenylation signal (pA signal) is located downstream of the L1 ORF1 (ORF1mneo) or a consensus AluYa5 (AluYa5mneo “pol II Alu”) (arrow indicates where the Alu “normal A-tail” would have been located). B. Spliced RNA pol II generated transcripts of tagged ORF1 and Alu are readily available by 24 hours. Poly-A selected RNA extracts from different post-transfection time points (24, 48 and 72 h) were evaluated by Northern blot analysis using an RNA strand specific probe to the neomycin resistance gene. The unspliced (open arrowhead) and spliced (black arrow) transcripts from the pol II-vectors AluYa5mneo and ORF1mneo are shown. β-actin is indicated by an *. C. The tagged ORF1 transcript mimics tagged L1 insertion kinetics. Retrotransposition assays were performed using the ORF1mneo vector supplemented with an L1 (black) or ORF2p expression (gray) vector. Cells were treated with d4t plus G418 at 24 and 48 h post-transfection. Bars represent the relative % mean G418R colonies±standard deviation shown as error bars for each construct (n = 3). The 72 h data were used to define 100%. The mean of the observed G418 resistant colonies is shown in parentheses above each column. Only one colony (1) was observed at the 24 h time point. D. Transcription from a pol II promoter alters the retrotransposition requirements of a tagged Alu element. The retrotransposition capability of the pol II-driven Alu (AluYa5mneo) supplemented with ORF1p and ORF2p expression vectors was evaluated in HeLa cells. Cells were treated with d4t plus G418 at 24 and 48 h post-transfection. The 72 h data were used to define 100%. Bars represent the relative % mean G418R colonies±standard deviation shown as error bars for each construct (n = 6). The total number of G418 resistant colonies for all experiments combined is shown in parentheses indicated by a “t”. No colonies were ever observed at the 24 h time point. E. Transcription and retrotransposition kinetics of pol II driven ORF1 and Alu. HeLa cells were transiently transfected with ORF1mneo (top panel) or AluYa5mneo (lower panel) and either harvested for RNA quantitation (left y axis, black square) or treated with d4t plus G418 treatment for colony quantitation (right y axis, gray circles) at the indicated time points post-transfection (x axis). RNA was quantitated relative to β-actin as control. The data demonstrate that the generation of spliced pol II and pol III Alu transcripts are equivalent; however pol II Alu inserts are not detected at 24 h.
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pgen-1000458-g006: The RNA Polymerase Dictates the Retrotransposition Kinetics of Alu.A. Schematic of pol II-driven ORF1 and Alu vectors. The ORF1mneo construct was selected as a representative of retropseudogene activity. The constructs use the CMV promoter (CMVp, black box) to generate pol II transcripts. The full mneoI indicator cassette from the L1 vector, consisting of the neomycin interrupted by an inverted intron (hatched box), its SV40 promoter (SV40p) and complete polyadenylation signal (pA signal) is located downstream of the L1 ORF1 (ORF1mneo) or a consensus AluYa5 (AluYa5mneo “pol II Alu”) (arrow indicates where the Alu “normal A-tail” would have been located). B. Spliced RNA pol II generated transcripts of tagged ORF1 and Alu are readily available by 24 hours. Poly-A selected RNA extracts from different post-transfection time points (24, 48 and 72 h) were evaluated by Northern blot analysis using an RNA strand specific probe to the neomycin resistance gene. The unspliced (open arrowhead) and spliced (black arrow) transcripts from the pol II-vectors AluYa5mneo and ORF1mneo are shown. β-actin is indicated by an *. C. The tagged ORF1 transcript mimics tagged L1 insertion kinetics. Retrotransposition assays were performed using the ORF1mneo vector supplemented with an L1 (black) or ORF2p expression (gray) vector. Cells were treated with d4t plus G418 at 24 and 48 h post-transfection. Bars represent the relative % mean G418R colonies±standard deviation shown as error bars for each construct (n = 3). The 72 h data were used to define 100%. The mean of the observed G418 resistant colonies is shown in parentheses above each column. Only one colony (1) was observed at the 24 h time point. D. Transcription from a pol II promoter alters the retrotransposition requirements of a tagged Alu element. The retrotransposition capability of the pol II-driven Alu (AluYa5mneo) supplemented with ORF1p and ORF2p expression vectors was evaluated in HeLa cells. Cells were treated with d4t plus G418 at 24 and 48 h post-transfection. The 72 h data were used to define 100%. Bars represent the relative % mean G418R colonies±standard deviation shown as error bars for each construct (n = 6). The total number of G418 resistant colonies for all experiments combined is shown in parentheses indicated by a “t”. No colonies were ever observed at the 24 h time point. E. Transcription and retrotransposition kinetics of pol II driven ORF1 and Alu. HeLa cells were transiently transfected with ORF1mneo (top panel) or AluYa5mneo (lower panel) and either harvested for RNA quantitation (left y axis, black square) or treated with d4t plus G418 treatment for colony quantitation (right y axis, gray circles) at the indicated time points post-transfection (x axis). RNA was quantitated relative to β-actin as control. The data demonstrate that the generation of spliced pol II and pol III Alu transcripts are equivalent; however pol II Alu inserts are not detected at 24 h.

Mentions: To better understand the RNA polymerase influence on retrotransposition, we also evaluated the time requirement of two pol II-driven (CMV) constructs: ORF1mneo and pol II Alu (Figure 6A). We selected ORF1mneo because it generates a transcript of L1 ORF1, which has previously been used to reflect retropseudogene activity [10]. The ORF1mneo vector can retrotranspose when a source of ORF2p is supplied in trans [10]. The pol II Alu (pCMVYa5mneo) contains an Alu tagged with the “mneo” cassette from the L1-tagged construct [61], which contains pol III terminators (4 Ts) that would generate truncated transcripts if the internal pol III A and B boxes in the Alu sequence are used for transcription. The “normal A-tail” at the end of the Alu sequence and 5′ of the neo cassette (Figure 6A) was not included in order to prevent potential internal priming for TPRT in the cDNA extension step (Figure 1E), which would circumvent inclusion of the neo reporter gene in the retrotransposed copy. Thus, only the Alu body sequence was utilized in the construct. Just like the L1 construct, the A-tail used in the TPRT step is generated from the transcript polyadenylation by the RNA polymerase II from the SV40pA signal at the 3′ end of the neo cassette (Figure 6A). Spliced and unspliced transcripts were detected from both constructs by 24 h (Figure 6B). The tagged ORF1p transcript driven by an ORF2p generated one single insert at 24 hours (Figure 6C), while the total number of colonies generated were 136 and 226 for 48 h and 72 h respectively. It is possible that the endogenous L1 expression in HeLa cells [6] affected the timing. However, our data on Alu retrotransposition indicates that effects from endogenous L1 expression under our experimental conditions are negligible (Figure 4). Most likely, the single G418R colony observed at 24 hours is due to a rare event that escaped d4t inhibition. A quantitative time course evaluation of the spliced RNA product in cells transiently transfected with ORF1mneo and AluYa5mneo further indicates that the availability of spliced product is not limiting retrotransposition timing (Figure 6E).


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)

The RNA Polymerase Dictates the Retrotransposition Kinetics of Alu.A. Schematic of pol II-driven ORF1 and Alu vectors. The ORF1mneo construct was selected as a representative of retropseudogene activity. The constructs use the CMV promoter (CMVp, black box) to generate pol II transcripts. The full mneoI indicator cassette from the L1 vector, consisting of the neomycin interrupted by an inverted intron (hatched box), its SV40 promoter (SV40p) and complete polyadenylation signal (pA signal) is located downstream of the L1 ORF1 (ORF1mneo) or a consensus AluYa5 (AluYa5mneo “pol II Alu”) (arrow indicates where the Alu “normal A-tail” would have been located). B. Spliced RNA pol II generated transcripts of tagged ORF1 and Alu are readily available by 24 hours. Poly-A selected RNA extracts from different post-transfection time points (24, 48 and 72 h) were evaluated by Northern blot analysis using an RNA strand specific probe to the neomycin resistance gene. The unspliced (open arrowhead) and spliced (black arrow) transcripts from the pol II-vectors AluYa5mneo and ORF1mneo are shown. β-actin is indicated by an *. C. The tagged ORF1 transcript mimics tagged L1 insertion kinetics. Retrotransposition assays were performed using the ORF1mneo vector supplemented with an L1 (black) or ORF2p expression (gray) vector. Cells were treated with d4t plus G418 at 24 and 48 h post-transfection. Bars represent the relative % mean G418R colonies±standard deviation shown as error bars for each construct (n = 3). The 72 h data were used to define 100%. The mean of the observed G418 resistant colonies is shown in parentheses above each column. Only one colony (1) was observed at the 24 h time point. D. Transcription from a pol II promoter alters the retrotransposition requirements of a tagged Alu element. The retrotransposition capability of the pol II-driven Alu (AluYa5mneo) supplemented with ORF1p and ORF2p expression vectors was evaluated in HeLa cells. Cells were treated with d4t plus G418 at 24 and 48 h post-transfection. The 72 h data were used to define 100%. Bars represent the relative % mean G418R colonies±standard deviation shown as error bars for each construct (n = 6). The total number of G418 resistant colonies for all experiments combined is shown in parentheses indicated by a “t”. No colonies were ever observed at the 24 h time point. E. Transcription and retrotransposition kinetics of pol II driven ORF1 and Alu. HeLa cells were transiently transfected with ORF1mneo (top panel) or AluYa5mneo (lower panel) and either harvested for RNA quantitation (left y axis, black square) or treated with d4t plus G418 treatment for colony quantitation (right y axis, gray circles) at the indicated time points post-transfection (x axis). RNA was quantitated relative to β-actin as control. The data demonstrate that the generation of spliced pol II and pol III Alu transcripts are equivalent; however pol II Alu inserts are not detected at 24 h.
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Related In: Results  -  Collection

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

pgen-1000458-g006: The RNA Polymerase Dictates the Retrotransposition Kinetics of Alu.A. Schematic of pol II-driven ORF1 and Alu vectors. The ORF1mneo construct was selected as a representative of retropseudogene activity. The constructs use the CMV promoter (CMVp, black box) to generate pol II transcripts. The full mneoI indicator cassette from the L1 vector, consisting of the neomycin interrupted by an inverted intron (hatched box), its SV40 promoter (SV40p) and complete polyadenylation signal (pA signal) is located downstream of the L1 ORF1 (ORF1mneo) or a consensus AluYa5 (AluYa5mneo “pol II Alu”) (arrow indicates where the Alu “normal A-tail” would have been located). B. Spliced RNA pol II generated transcripts of tagged ORF1 and Alu are readily available by 24 hours. Poly-A selected RNA extracts from different post-transfection time points (24, 48 and 72 h) were evaluated by Northern blot analysis using an RNA strand specific probe to the neomycin resistance gene. The unspliced (open arrowhead) and spliced (black arrow) transcripts from the pol II-vectors AluYa5mneo and ORF1mneo are shown. β-actin is indicated by an *. C. The tagged ORF1 transcript mimics tagged L1 insertion kinetics. Retrotransposition assays were performed using the ORF1mneo vector supplemented with an L1 (black) or ORF2p expression (gray) vector. Cells were treated with d4t plus G418 at 24 and 48 h post-transfection. Bars represent the relative % mean G418R colonies±standard deviation shown as error bars for each construct (n = 3). The 72 h data were used to define 100%. The mean of the observed G418 resistant colonies is shown in parentheses above each column. Only one colony (1) was observed at the 24 h time point. D. Transcription from a pol II promoter alters the retrotransposition requirements of a tagged Alu element. The retrotransposition capability of the pol II-driven Alu (AluYa5mneo) supplemented with ORF1p and ORF2p expression vectors was evaluated in HeLa cells. Cells were treated with d4t plus G418 at 24 and 48 h post-transfection. The 72 h data were used to define 100%. Bars represent the relative % mean G418R colonies±standard deviation shown as error bars for each construct (n = 6). The total number of G418 resistant colonies for all experiments combined is shown in parentheses indicated by a “t”. No colonies were ever observed at the 24 h time point. E. Transcription and retrotransposition kinetics of pol II driven ORF1 and Alu. HeLa cells were transiently transfected with ORF1mneo (top panel) or AluYa5mneo (lower panel) and either harvested for RNA quantitation (left y axis, black square) or treated with d4t plus G418 treatment for colony quantitation (right y axis, gray circles) at the indicated time points post-transfection (x axis). RNA was quantitated relative to β-actin as control. The data demonstrate that the generation of spliced pol II and pol III Alu transcripts are equivalent; however pol II Alu inserts are not detected at 24 h.
Mentions: To better understand the RNA polymerase influence on retrotransposition, we also evaluated the time requirement of two pol II-driven (CMV) constructs: ORF1mneo and pol II Alu (Figure 6A). We selected ORF1mneo because it generates a transcript of L1 ORF1, which has previously been used to reflect retropseudogene activity [10]. The ORF1mneo vector can retrotranspose when a source of ORF2p is supplied in trans [10]. The pol II Alu (pCMVYa5mneo) contains an Alu tagged with the “mneo” cassette from the L1-tagged construct [61], which contains pol III terminators (4 Ts) that would generate truncated transcripts if the internal pol III A and B boxes in the Alu sequence are used for transcription. The “normal A-tail” at the end of the Alu sequence and 5′ of the neo cassette (Figure 6A) was not included in order to prevent potential internal priming for TPRT in the cDNA extension step (Figure 1E), which would circumvent inclusion of the neo reporter gene in the retrotransposed copy. Thus, only the Alu body sequence was utilized in the construct. Just like the L1 construct, the A-tail used in the TPRT step is generated from the transcript polyadenylation by the RNA polymerase II from the SV40pA signal at the 3′ end of the neo cassette (Figure 6A). Spliced and unspliced transcripts were detected from both constructs by 24 h (Figure 6B). The tagged ORF1p transcript driven by an ORF2p generated one single insert at 24 hours (Figure 6C), while the total number of colonies generated were 136 and 226 for 48 h and 72 h respectively. It is possible that the endogenous L1 expression in HeLa cells [6] affected the timing. However, our data on Alu retrotransposition indicates that effects from endogenous L1 expression under our experimental conditions are negligible (Figure 4). Most likely, the single G418R colony observed at 24 hours is due to a rare event that escaped d4t inhibition. A quantitative time course evaluation of the spliced RNA product in cells transiently transfected with ORF1mneo and AluYa5mneo further indicates that the availability of spliced product is not limiting retrotransposition timing (Figure 6E).

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