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. 2006 Jan 15;20(2):210-24.
doi: 10.1101/gad.1380406.

Unconventional translation of mammalian LINE-1 retrotransposons

Reid S Alisch  1 , Jose L Garcia-Perez, Alysson R Muotri, Fred H Gage, John V Moran
Affiliations

Unconventional translation of mammalian LINE-1 retrotransposons

Reid S Alisch et al. Genes Dev. .

Abstract

Long Interspersed Element-1 (LINE-1 or L1) retrotransposons encode proteins required for their mobility (ORF1p and ORF2p), yet little is known about how L1 mRNA is translated. Here, we show that ORF2 translation generally initiates from the first in-frame methionine codon of ORF2, and that both ORF1 and the inter-ORF spacer are dispensable for ORF2 translation. Remarkably, changing the ORF2 AUG codon to any other coding triplet is compatible with retrotransposition. However, introducing a premature termination codon in ORF1 or a thermostable hairpin in the inter-ORF spacer reduces ORF2p translation or L1 retrotransposition to approximately 5% of wild-type levels. Similar data obtained from "natural" and codon optimized "synthetic" mouse L1s lead us to propose that ORF2 is translated by an unconventional termination/reinitiation mechanism.

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Figures

Figure 1.
Figure 1.
The 3′ end of ORF1 and the inter-ORF spacer are dispensable for L1 retrotransposition. (A) Schematic of an RC human L1: The cartoon depicts the structure of JM102/L1.3. The yellow and blue rectangles represent ORF1 and ORF2, respectively. EN, RT, and C represent the approximate positions of the endonuclease, reverse transcriptase, and cysteine-rich domains of ORF2, respectively. The green rectangle indicates the CMV immediate early promoter used to drive L1 expression. The blue lollipop indicates the SV40 polyadenylation sequence downstream of the L1 3′ UTR (gray rectangle). The 3′ UTR of the L1 is tagged with a retrotransposition indicator cassette (mneoI). The indicator cassette consists of a backward copy of the neomycin phosphotransferase gene that contains its own promoter (red arrow) and polyadenylation signal (red lollipop). The neomycin phosphotransferase gene also is interrupted by intron 2 from the γ-globin gene, which is in the same transcriptional orientation of the L1. SD and SA represent the splice donor and splice acceptor sequences of the intron, respectively (Freeman et al. 1994). This arrangement ensures that G418-resistant foci will arise only if the primary L1 RNA transcript is spliced and then undergoes retrotransposition (Moran et al. 1996). The approximate positions of two mutations that render the L1 retrotransposition-defective (JM111 and RA105) are indicated below the schematic (Moran et al. 1996). The sequence of the 63-base inter-ORF spacer is magnified above the schematic. Red lettering indicates stop codons at the end of ORF1 and in the inter-ORF spacer. The red underlining signifies an AUG codon in the inter-ORF spacer that can, in principle, initiate the translation of a short ORF (green box) of six codons. Green lettering indicates the first in-frame AUG codon in ORF2. (B) Mutations in the inter-ORF spacer have little effect on retrotransposition. Constructs containing a 30-base deletion of the 3′ end of ORF1 (RJ159) or nonoverlapping partial deletions of the inter-ORF spacer (RA-BCD, Δ19 bases; RA-CD, Δ17 bases; RA-D, Δ13 bases; RA-E, Δ14 bases) are indicated by the rectangles (see the Supplemental Material). Insertion mutations in the inter-ORF spacer are indicated below the schematic. Blue numbering indicates the relative retrotransposition efficiencies of each construct. (C) ORF1 and ORF2 need to be separated by a stop codon for efficient retrotransposition. Representative data showing the relative retrotransposition efficiencies of the wild-type construct (RA101), a mutant containing a partial deletion of the inter-ORF spacer (RA-CD), a mutant lacking the inter-ORF spacer (RA1TAA2), and mutants containing an in-frame fusion between ORF1 and ORF2 (RA-CDNostop and GP1AAA2, respectively) are indicated next to the cartoons of each construct. The relative retrotransposition efficiency is indicated and is compared with the relative retrotransposition efficiency of the wild-type control (RA101).
Figure 2.
Figure 2.
ORF2p translation starts at the first in-frame AUG codon of ORF2. The schematic depicts cartoons of each of the mutant constructs tested in the retrotransposition assay. The red asterisks signify the two stop codons in the inter-ORF spacer. The relative position of each mutation is shown below the cartoons. The construct names are indicated in the left column, and representative data from the cultured cell retrotransposition assay are indicated at the right of the figure. The relative retrotransposition efficiency is indicated in the parenthesis and is compared with the relative retrotransposition efficiency of the wild-type control (RA101). RA105 is a retrotransposition-defective L1 containing a missense mutation in the RT active site (Moran et al. 1996; Wei et al. 2001). JM111/L1.3 is a retrotransposition-defective L1 containing a pair of missense mutations in ORF1 (Moran et al. 1996; Wei et al. 2001).
Figure 3.
Figure 3.
The ORF2 AUG is dispensable for retrotransposition. (A) Mutating the AUG codon to either AUA or CCC is still compatible with retrotransposition. The schematic depicts cartoons of each of the mutant constructs tested in the retrotransposition assay. The respective mutations (AUG to AUA in RA102; AUG to CCC in RA103; AUGACAGGA to CCCUAAUAA in RAPXX) are indicated below the cartoon. Construct names are indicated in the left column, and representative data from the cultured cell retrotransposition assay are indicated at the right of the figure. The relative retrotransposition efficiency is indicated and is expressed as compared with the relative retrotransposition efficiency of the wild-type control (RA101). (B) Exogenous sources of RT are not promoting retrotransposition of the mutant constructs. Double mutants containing either the M1P or M1X mutation in conjunction with an RT active site mutation (RA103/RT- or RA111/RT-) were assayed for retrotransposition. Both mutants were retrotransposition defective, indicating that ORF2p was translated in the original M1P and M1X mutants. (C) The ORF2 AUG codon can be substituted with any coding triplet. The ORF2 AUG was mutated individually so that it could encode the other 19 amino acids. Each of the resultant constructs (X-axis) retrotransposed at 10%-70% of wild-type levels (Y-axis). By comparison, mutating the AUG to each stop codon (Opal, Ochre, and Amber) reduced retrotransposition by ∼50-fold. The percent of retrotransposition is shown compared with the wild-type element RA101 (i.e., M1M in the bar graph). The error bars indicate the standard deviation, which was calculated from at least six independent experiments for each construct.
Figure 4.
Figure 4.
AUG-independent translation is not peculiar to human L1 elements. (A) AUG-independent translation of a synthetic mouse L1. A schematic of a synthetic mouse L1 is shown at the top of the figure (Han and Boeke 2004). The synthetic mouse L1 contains its own promoter (gray rectangles with arrows) as well as a heterologous cytomegalovirus immediate early promoter (green rectangle). It also contains the SV40 polyadenylation sequence (blue lollipop) downstream of its 3′ UTR (gray box). The sequence of the 40-nt inter-ORF spacer is magnified below the schematic. The red lettering indicates stop codons at the end of ORF1 and in the inter-ORF spacer. The red asterisk subsequently is used to indicate the relative position of the stop codon in the inter-ORF spacer. The 5′ end of ORF2 contains a 21-nt extension (coding for seven amino acids) when compared with a human RC-L1. The green lettering indicates the first in-frame AUG codon in ORF2. The relative position of each mutation is shown below the cartoons. The construct names are indicated in the left column, and representative data from the cultured cell retrotransposition assay are indicated at the right of the figure. The relative retrotransposition efficiency of each mutant is reported relative to the retrotransposition efficiency of the wild-type control (pCEPL1SM). pCEPL1SM N21A is a retrotransposition-defective L1 containing a missense mutation of an amino acid critical for L1 endonuclease function (Feng et al. 1996). (B) AUG-independent translation of a natural mouse L1. A schematic of a natural mouse L1 (pCEPTGf21) is shown at the top of the figure (Goodier et al. 2001). Labeling is the same as in A. A PacI restriction site (underlined) was introduced in the inter-ORF spacer of the TGf21 element (pCEPTGf21Pac) to generate a stop codon between ORF1 and ORF2 (red letter and red asterisks). The PacI site does not significantly affect retrotransposition and makes the expression context of the resultant construct (pCEPTGf21Pac) similar to the synthetic mouse L1 in A. The retrotransposition efficiencies of the M1P and M1X mutants are reported with respect to the retrotransposition efficiency of the corresponding control (pCEPTGf21Pac).
Figure 5.
Figure 5.
ORF2 AUG-independent translation is not peculiar to human or transformed cells. A representative group of mutant constructs were assayed for retrotransposition in CHO (CHO-1) cells (A) and rat neural progenitor cells (B). In B, the percentage of EGFP-positive cells and standard deviation are indicated in the figure. In all instances, the results were similar to those obtained in HeLa cells.
Figure 6.
Figure 6.
An independent assay to monitor ORF2p translation. (A) Schematic of the trans-complementation assay. To assay for trans-complementation, wild-type and mutant L1 constructs lacking the mneoI indicator cassette (i.e., driver elements; cartoon on the right) were cotransfected into HeLa cells with a construct consisting of the L1 5′ UTR, ORF1, and the mneoI indicator cassette (ORF1mneoI). We previously demonstrated that ORF1mneoI RNA is a preferential substrate for authentic trans-complementation (Wei et al. 2001). G418-resistant foci only will arise if ORF1mneoI RNA is trans-mobilized by the ORF2 protein provided by the driver L1 lacking the indicator cassette. (B) Positive correlation between results obtained with the cis-based retrotransposition and trans-complementation assays. Column 1 of the inset table indicates the name of each construct. Column 2 provides a brief description of each construct. Column 3 indicates the retrotransposition efficiency of each construct obtained in the cis-based retrotransposition assay. Column 4 indicates the trans-complementation efficiency of each construct. A positive correlation is seen between the results gained from the two assays. Because we are detecting fewer G418-resistant foci in the trans-complementation assay, we encounter a larger standard error when compared with the cis-based retrotransposition assay.
Figure 7.
Figure 7.
A nonspecific upstream ORF facilitates ORF2p translation. The names and schematics of wild-type and mutant driver elements used in the trans-complementation assay are indicated at the left of the figure. The relative trans-complementation efficiency of each construct is compared with its respective wild-type control (RA101NN) and is indicated at the right of the figure.
Figure 8.
Figure 8.
ORF2p translation appears to occur by an unconventional termination reinitiation mechanism. (A) A thermostable hairpin in the inter-ORF spacer inhibits ORF2 translation. A 95-base thermostable hairpin (ΔG = -80 kcal/mol) lacking AUG codons that is known to efficiently inhibit the translocation of scanning ribosomes (Yueh and Schneider 1996) was inserted into the inter-ORF spacer of either a wild-type (RA101Schloop) or a mutant (RA103Schloop) construct. Construct names are indicated in the left column, and representative data from the cultured cell retrotransposition assay are indicated at the right of the figure. The relative retrotransposition efficiency is indicated and is expressed as compared with the relative retrotransposition efficiency of the wild-type control (RA101). (B) An out-of-frame AUG codon in the inter-ORF spacer does not inhibit ORF2p translation initiation from the natural AUG codon. An out-of-frame AUG codon (green lettering) that would direct the synthesis of an irrelevant polypeptide with respect to L1 retrotransposition (reading frames are indicated by the green, purple, and gold rectangles, respectively) was placed in either the inter-ORF spacer (GP101NaugGNostop or GP101AaugVG, respectively) or downstream of the natural ORF2p initiation codon (GP101DAaugVG). Neither modification had a significant effect on L1 retrotransposition. Placement of an out-of-frame AUG codon upstream of the M1P mutant (GP103AaugVG) inhibited retrotransposition to ∼13% the level of its respective control (RA103); however, introducing an out-of-frame AUG codon downstream of the mutant (GP103DAaugVG) had no significant effect on retrotransposition. (C) Suppression of a nonsense mutation at the ORF2p initiation codon by a single base insertion. Insertion of a guanosine residue after the UAA stop codon (denoted by the gold lettering in RA101UAAg) restored retrotransposition to 39% of wild-type levels. Reintroducing a stop codon at the ORF2p initiation codon (denoted by the gold lettering in RA101UAuAg) reduced retrotransposition to 3% of the level of the wild-type control (RA101).
Figure 9.
Figure 9.
A model for ORF2p translation. The curved line represents the polyadenylated, bicistronic L1 mRNA. The gray line indicates the 5′ UTR. The green line indicates ORF1 coding sequences. The blue line indicates ORF2 coding sequences. The gray ovals indicate the 40S and 60S subunits of the ribosome, respectively. The green and blue circles indicate ORF1p and ORF2p, respectively. Upon reaching the ORF1 stop codon, ORF1p is released from the ribosome and the ribosome is dissociated. The 40S subunit remains associated with L1 RNA and scans through the inter-ORF spacer until it reaches the first in-frame AUG in ORF2. The ribosome then is reassembled to initiate ORF2p translation. We speculate that ORF2p (or perhaps ORF1p) binding to L1 RNA inhibits ORF2 translation. This would enable ORF1p to be made at greater quantities than ORF2p so that it can coat the transcript. Remarkably, our data indicate that ORF2p translation requires a nonspecific, translatable upstream ORF and that ORF2p translation can initiate from a non-AUG codon. Other details of the model are summarized in the text.
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References

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