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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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Model of the Retroelement Retrotransposition Cycle.A–G represent individual steps in the retrotransposition cycle: A. The first step requires the transcription of the RNA, processing and export to cytoplasm. B.and C. L1 protein translation needs to occur and both SINE and LINE RNPs form in the cytoplasm. L1 ORF1 and ORF2 proteins are represented by small and large circles, respectively. The SRP9 and SRP14 proteins are represented by pentagons. D. The RNA and proteins reach the nucleus in an unknown manner. In the nucleus: E. To prepare for insertion, the DNA is cleaved by the L1 ORF2p endonuclease. The L1 endonuclease cleaves at AT-rich sequences with the consensus 5′-TTAAAA-3′/3′-AA↑TTTT-5′. At this stage the “A-tail” of the L1 or Alu transcript is thought to interact with the cleaved DNA. It is proposed that reverse transcription occurs through a process referred to as target primed reverse transcription (TPRT). The L1 ORF2p reverse transcriptase generates the first strand of DNA. It is unknown whether or not SINE RNA can be involved in a template switch or compete for L1 factors at this step (indicated by the “?”). F. Completion of the retrotransposition requires second-strand synthesis, a second nick caused by an unknown source, and ligation of the 3′ end of the cDNA to the genome. At least some of these steps could involve endogenous cellular activities. DNA repair processes are likely to be involved in the final steps. G. The end product results in the generation of an insert with the hallmark direct repeats.
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pgen-1000458-g001: Model of the Retroelement Retrotransposition Cycle.A–G represent individual steps in the retrotransposition cycle: A. The first step requires the transcription of the RNA, processing and export to cytoplasm. B.and C. L1 protein translation needs to occur and both SINE and LINE RNPs form in the cytoplasm. L1 ORF1 and ORF2 proteins are represented by small and large circles, respectively. The SRP9 and SRP14 proteins are represented by pentagons. D. The RNA and proteins reach the nucleus in an unknown manner. In the nucleus: E. To prepare for insertion, the DNA is cleaved by the L1 ORF2p endonuclease. The L1 endonuclease cleaves at AT-rich sequences with the consensus 5′-TTAAAA-3′/3′-AA↑TTTT-5′. At this stage the “A-tail” of the L1 or Alu transcript is thought to interact with the cleaved DNA. It is proposed that reverse transcription occurs through a process referred to as target primed reverse transcription (TPRT). The L1 ORF2p reverse transcriptase generates the first strand of DNA. It is unknown whether or not SINE RNA can be involved in a template switch or compete for L1 factors at this step (indicated by the “?”). F. Completion of the retrotransposition requires second-strand synthesis, a second nick caused by an unknown source, and ligation of the 3′ end of the cDNA to the genome. At least some of these steps could involve endogenous cellular activities. DNA repair processes are likely to be involved in the final steps. G. The end product results in the generation of an insert with the hallmark direct repeats.

Mentions: Retroelements are mobile elements that amplify through an RNA intermediate in a process known as retrotransposition [14]. There are limited data on the details of the mechanism of LINE retrotransposition, and even less for SINE retrotransposition. The process begins with the generation of RNA (Figure 1A). Active L1 elements express two proteins from a bicistronic mRNA: ORF1p[15] and ORF2p (Figure 1B and C). Both L1 proteins are needed for L1 retrotransposition [16]. In contrast to L1, ORF2p expression is sufficient for SINE retrotransposition [4],[9],[17], while ORF1p may enhance the process [17]. ORF1p possesses nucleic acid chaperone activity [18],[19], an essential property for L1 retrotransposition [19],[20]. ORF2p is a multifunctional protein with endonuclease and reverse transcriptase activities [21],[22]. Both proteins are proposed to interact in cis [10],[11] with the L1 RNA to form a cytoplasmic RNP complex interacting with polyribosomes [20],[23]. SINE RNA is predominantly found in the cytoplasm as an RNP complex [12],[24],[25] (Figure 1C) and uses L1 protein(s) in trans for its mobilization. The endonuclease of the L1 ORF2p generates the first nick within the L1 endonuclease recognition sequence generating single stranded DNA that primes the reverse transcription [22],[26]. Both L1 and Alu are proposed to undergo integration through a target-primed reverse transcription (TPRT) reaction [27].


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)

Model of the Retroelement Retrotransposition Cycle.A–G represent individual steps in the retrotransposition cycle: A. The first step requires the transcription of the RNA, processing and export to cytoplasm. B.and C. L1 protein translation needs to occur and both SINE and LINE RNPs form in the cytoplasm. L1 ORF1 and ORF2 proteins are represented by small and large circles, respectively. The SRP9 and SRP14 proteins are represented by pentagons. D. The RNA and proteins reach the nucleus in an unknown manner. In the nucleus: E. To prepare for insertion, the DNA is cleaved by the L1 ORF2p endonuclease. The L1 endonuclease cleaves at AT-rich sequences with the consensus 5′-TTAAAA-3′/3′-AA↑TTTT-5′. At this stage the “A-tail” of the L1 or Alu transcript is thought to interact with the cleaved DNA. It is proposed that reverse transcription occurs through a process referred to as target primed reverse transcription (TPRT). The L1 ORF2p reverse transcriptase generates the first strand of DNA. It is unknown whether or not SINE RNA can be involved in a template switch or compete for L1 factors at this step (indicated by the “?”). F. Completion of the retrotransposition requires second-strand synthesis, a second nick caused by an unknown source, and ligation of the 3′ end of the cDNA to the genome. At least some of these steps could involve endogenous cellular activities. DNA repair processes are likely to be involved in the final steps. G. The end product results in the generation of an insert with the hallmark direct repeats.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2666806&req=5

pgen-1000458-g001: Model of the Retroelement Retrotransposition Cycle.A–G represent individual steps in the retrotransposition cycle: A. The first step requires the transcription of the RNA, processing and export to cytoplasm. B.and C. L1 protein translation needs to occur and both SINE and LINE RNPs form in the cytoplasm. L1 ORF1 and ORF2 proteins are represented by small and large circles, respectively. The SRP9 and SRP14 proteins are represented by pentagons. D. The RNA and proteins reach the nucleus in an unknown manner. In the nucleus: E. To prepare for insertion, the DNA is cleaved by the L1 ORF2p endonuclease. The L1 endonuclease cleaves at AT-rich sequences with the consensus 5′-TTAAAA-3′/3′-AA↑TTTT-5′. At this stage the “A-tail” of the L1 or Alu transcript is thought to interact with the cleaved DNA. It is proposed that reverse transcription occurs through a process referred to as target primed reverse transcription (TPRT). The L1 ORF2p reverse transcriptase generates the first strand of DNA. It is unknown whether or not SINE RNA can be involved in a template switch or compete for L1 factors at this step (indicated by the “?”). F. Completion of the retrotransposition requires second-strand synthesis, a second nick caused by an unknown source, and ligation of the 3′ end of the cDNA to the genome. At least some of these steps could involve endogenous cellular activities. DNA repair processes are likely to be involved in the final steps. G. The end product results in the generation of an insert with the hallmark direct repeats.
Mentions: Retroelements are mobile elements that amplify through an RNA intermediate in a process known as retrotransposition [14]. There are limited data on the details of the mechanism of LINE retrotransposition, and even less for SINE retrotransposition. The process begins with the generation of RNA (Figure 1A). Active L1 elements express two proteins from a bicistronic mRNA: ORF1p[15] and ORF2p (Figure 1B and C). Both L1 proteins are needed for L1 retrotransposition [16]. In contrast to L1, ORF2p expression is sufficient for SINE retrotransposition [4],[9],[17], while ORF1p may enhance the process [17]. ORF1p possesses nucleic acid chaperone activity [18],[19], an essential property for L1 retrotransposition [19],[20]. ORF2p is a multifunctional protein with endonuclease and reverse transcriptase activities [21],[22]. Both proteins are proposed to interact in cis [10],[11] with the L1 RNA to form a cytoplasmic RNP complex interacting with polyribosomes [20],[23]. SINE RNA is predominantly found in the cytoplasm as an RNP complex [12],[24],[25] (Figure 1C) and uses L1 protein(s) in trans for its mobilization. The endonuclease of the L1 ORF2p generates the first nick within the L1 endonuclease recognition sequence generating single stranded DNA that primes the reverse transcription [22],[26]. Both L1 and Alu are proposed to undergo integration through a target-primed reverse transcription (TPRT) reaction [27].

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