Figure 1.
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.
Figure 2.
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.
Figure 3.
Availability of Spliced L1 RNA Is Not a Limiting Factor.
Transcription and retrotransposition kinetics were evaluated for the different constructs. HeLa cells were transiently transfected with L1mneo, AluYa5neoTET+ORF2p or L1neoTET (with the same self-splicing neo cassette used for the Alu construct). Cells were 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 (details in Materials and Methods). Note that the colony numbers reflect the actual cumulative inserts that occurred from transfection to the d4t treatment time point. The data demonstrate that all constructs generate the spliced tagged transcripts at early time points in a similar manner; however the observed inserts between Alu and L1 differ at 24 h.
Figure 4.
Detection of L1 ORF2p Trans-mobilization Activity at 24 h.
HeLa cells were transiently transfected with AluYa5neoTET plus L1 no tag or empty vector (control). The d4t and G418 treatment was started at 24, 32, 36, 48 and 72 h post-transfection (x axis). Bars represent the relative % mean G418R colonies±standard deviation shown as error bars for each construct. The 72 h data were used to define 100%. The mean of the observed G418 resistant colonies is shown in parentheses above each column. Note that for the control only 2 and 1 colonies were observed at 42 and 72 hours, respectively. The data demonstrate that functional ORF2p generated by the L1 no tag “wildtype” vector must reach the nucleus by 24 h for Alu retrotransposition to occur.
Figure 5.
RNA Pol III Transcripts Share Similar Retrotransposition Kinetics.
Other pol III genes known to generate retrotransposed copies parallel Alu kinetics by generating inserts within 24 h. HeLa cells were transiently transfected with the ORF2p expression vector plus the tagged vector of the rodent SINE B2 (vertical lines), and the 7SL (light gray), U6 (dark gray), hY1 (black), hY3 (white), hY4 (dotted) or hY5 (slanted-lines) RNA genes. The above Inset shows representative G418R foci results of the B2 retrotransposition assay. Cells were treated with d4t+G418 at 0, 24, 48 and 72 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 = 3).
Figure 6.
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.
Figure 7.
L1 Retrotransposition Rate Does Not Correlate with the Timing of Insertion.
The retrotransposition capability of the L1 construct with the highest retrotransposition rate reported (L1m syn) [52] was evaluated. HeLa cells were transiently transfected and treated with d4t plus G418 at different time points post-transfection or harvested for RNA extractions. Bars represent the mean G418R colonies±standard deviation. The total number of G418 resistant colonies of the early time points is shown in parentheses. The top panel shows the northern blot analysis of the tagged L1m syn transcripts with the time points indicated above, where the arrow indicates the spliced product. The lower panel shows the β-actin transcripts (*).
Table 1.
Relative retrotransposition rate of the different tagged constructs in HeLa cells under the same transfection conditions.
Figure 8.
Model of SINE and LINE Cellular Interactions Potentially Contributing to Differences in Retrotransposition Kinetics.
We present a model of how pol II (L1 and mRNA) and pol III transcript interactions with their respective cellular components in the cytoplasm may influence the retrotransposition timing of LINEs and SINEs. Structures are not drawn to scale. Transcription and processing are not the limiting steps for L1 and Alu, as both pol II and pol III spliced tagged transcripts are present in the cytoplasm as early as 3 h post-transfection. However, Alu requires about 24 h to generate an insert, while L1 requires about 48 h (*). Pol II transcript: LINE RNA and pol II-driven mRNAs reach the cytoplasm after processing and modifications. The cytoplasmic pol II transcript has been spliced, polyadenylated and capped at its 5′ end (shown as a black dot at the end of the RNA). The cap allows the recognition by several proteins involved in translation forming a large protein complex interacting with the transcript. Capping also allows for the association with the PABP-1, elongation factors and the circularization of the mRNA (not shown). This large multi-protein complex interacts with the translation machinery to generate the needed ORF1 and ORF2 proteins. The generated proteins will preferentially bind to the L1 RNA that encoded them (cis-preference). Multiple ORF1p molecules (yellow circles) and possibly ORF2p (red circle) bind the L1 transcript. We propose that the formation of the L1 RNP complex will allow the L1 RNA to separate from the translation machinery and evade the normal degradation pathway en route to the nucleus. This process of detachment from the ribosomal complex and avoidance of the RNA decay pathways may increase the time requirement for L1 retrotransposition. These extra steps probably contribute to the extended time requirement for L1 to complete the retrotransposition process. The L1 RNA (likely as an RNP with ORF1 and ORF2) reaches the nucleus and generates a new insert (represented as a white box in the DNA). In our assay system, the insertion process of L1 elements requires about 48 hours for completion. Cellular RNAs (e.g., mRNAs and the pol II Alu RNA) can occasionally use the L1 proteins to mediate their mobility in trans. However, retropseudogene (pol II mRNA) inserts are not efficiently generated in our experimental system and require ORF1p. We propose that the spurious interaction with ORF1p allows these mRNAs to be shunted to the nucleus to go through the retrotransposition process. In addition, it is likely that ORF1p also contributes to the retrotransposition process in the nucleus. The ORF1mneo transcript generates inserts more efficiently than the other tested pol II transcripts, possibly because of close proximity to ORF1p in cis. Overall, the efficiency of a pol II transcript to generate inserts in tissue culture is likely correlated with its ability to interact with ORF1p. The role of ORF2p in the cytoplasm is unclear. Pol III transcript: SINE RNA reaches the cytoplasm with little or no processing and interacts with specific proteins. The cytoplasmic SINE RNP is stable and compact. In the case of Alu, the transcript forms a specific structure which binds the SRP9 (green circle) and SRP14 (blue circle) proteins. It is hypothesized that these proteins may target SINE RNA to the ribosomes, generating transient close proximity to nascent L1 proteins that might be essential for SINEs to efficiently use L1 in trans for retrotransposition. Although SINE RNPs may be targeted to the ribosomes, they are not functional components of the translational complex, making this interaction likely transitory. Whether pol III RNA gains access to the L1 retrotransposition machinery in the cytoplasm or in the nucleus remains undetermined. Because SINE transcripts are not translated or functional components of the translational complex, the SINE RNPs are likely “free” to sequester the L1 proteins and immediately proceed with the retrotransposition cycle. We propose that the pol III SINE probably reaches the nucleus in a more efficient manner than the pol II transcripts, such as the L1 RNA, which must first dissociate from the translational complex and avoid the normal mRNA degradation pathway. In our system, the insertion process of an Alu requires 24 hours or less for completion (* represented as a white box in the DNA).