Human L1 retrotransposition: cis preference versus trans complementation

Abstract

Long interspersed nuclear elements (LINEs or L1s) comprise approximately 17% of human DNA; however, only about 60 of the approximately 400,000 L1s are mobile. Using a retrotransposition assay in cultured human cells, we demonstrate that L1-encoded proteins predominantly mobilize the RNA that encodes them. At much lower levels, L1-encoded proteins can act in trans to promote retrotransposition of mutant L1s and other cellular mRNAs, creating processed pseudogenes. Mutant L1 RNAs are mobilized at 0.2 to 0.9% of the retrotransposition frequency of wild-type L1s, whereas cellular RNAs are mobilized at much lower frequencies (ca. 0.01 to 0.05% of wild-type levels). Thus, we conclude that L1-encoded proteins demonstrate a profound cis preference for their encoding RNA. This mechanism could enable L1 to remain retrotransposition competent in the presence of the overwhelming number of nonfunctional L1s present in human DNA.

FIG. 1

Rationale of the assay. Retrotransposition-defective L1s (RD-L1s) containing the mneoI indicator cassette were cotransfected into HeLa cells with retrotransposition-competent L1s (RC-L1s) lacking the cassette, and retrotransposition was determined as described in Materials and Methods. Explanations for the possible experimental outcomes are noted.

FIG. 2

Controls used in this study. (A) trans complementation of β-galactosidase enzymatic activity in HeLa cells. Plasmids with the α or Ω regions of the β-galactosidase gene that contained a nuclear localization signal (nls) were transfected into HeLa cells individually or together, and β-galactosidase activity was monitored 3 days posttransfection (35). Mock transfection (no DNA) and a wild-type β-galactosidase gene (CMV β-gal; Clontech; GenBank accession no. U02451) served as negative and positive controls, respectively. (B) Mutants used in this study. Mutations in ORF1 or the endonuclease or RT domains of L1.3mneoI are indicated. The wild-type amino acids that were mutated are underlined. The arrows indicate the mutant amino acid sequence changes (e.g., ARR was changed to AAA). (C) RNA expression of representative L1 constructs. Structures of the hyg (hygromycin resistance gene) and L1 probes. Sizes of the full-length input and protected bands are indicated at the top of the figure. RNase protection assays were carried out of total RNAs prepared from HeLa cells transfected with the indicated plasmids. Probes that have undergone the RNase protection assay with (lanes 8 and 10) or without (lanes 9 and 11) the addition of RNase are shown. A longer exposure of the pCEP4-derived hyg transcripts, which serves as an internal control, is shown in the bottom panel. Consistent with earlier studies, we were unable to detect the expression of endogenous L1 transcripts in HeLa cells (43).

FIG. 3

L1s retrotranspose in cis. (A) Results of the retrotransposition assay. RD-L1s containing the mneoI indicator cassette were cotransfected into 2 × 10⁵ HeLa cells with an RC-L1 lacking the cassette (JM101/L1.3 Δneo). G418^r foci were fixed and stained with Giemsa for visualization. Samples cotransfected with JM101/L1.3 Δneo and representative mutants in ORF1 (JM111/L1.3), the endonuclease or RT domains of ORF2 (JM116/L1.3 or JM105/L1.3), or a double mutant (JM124/L1.3) are shown. Cells transfected with JM101/L1.3, as well as 1/10 (2 × 10⁴) and 1/100 (2 × 10³) dilutions of transfected cells are indicated as positive controls. Cells transfected with JM105/L1.3 are shown as a negative control. (B) The coexpression of RD-L1s does not inhibit RC-L1 retrotransposition. A RC-L1 containing the mneoI indicator cassette (JM101/L1.3) was cotransfected into 2 × 10⁴ HeLa cells with RD-L1s lacking the cassette, and retrotransposition was determined as described above. An experiment using a 1:9 (RC-L1 to RD-L1) molar ratio of transfected DNAs is shown. Cells transfected with JM101/L1.3 and an empty expression vector (CEP4) yielded G418^r foci at roughly the same levels as cells that were cotransfected with JM101/L1.3 and RD-L1s lacking the indicator cassette (i.e., there was less than a 20% difference between respective samples). JM105/L1.3 was used as a negative control. Notably, RD-L1s whose transcription is driven from either the CMV promoter or the CMV promoter and L1 5′ UTR are complemented to similar extents (not shown).

FIG. 4

G418^r foci must arise by trans complementation. (A) The low-level rescue of RD-L1s cannot be accounted for by DNA recombination. An allele of JM101/L1.3 that lacked both the 5′ UTR and the CMV promoter (ΔΔJM101/L1.3) was transfected into HeLa cells alone or with a wild-type allele of L1.3 that lacked the mneoI indicator cassette, and retrotransposition was assayed as described in Fig. 3. The rationale for this experiment is described in the text. JM101/L1.3 and JM105/L1.3 were used as appropriate positive and negative controls. (B) Constructs used in the study. The structure of L1.3 ORF1mneoI is shown, and the rationale for the experiment is described in the text. (C) The resultant G418^r foci have the predicted structure. PCR experiments using the oligonucleotides depicted in Fig. 4B (indicated by converging arrows) revealed that the retrotransposed mneoI cassette lacked the intron and was linked physically to L1.3 ORF1. The details of the experiment are provided in the text.

FIG. 5

The RC-L1 proteins can generate processed pseudogenes. The structures of three pAI1bmneoI processed pseudogenes and the accession numbers of the empty sites prior to insertion of the pseudogenes are indicated. Vertical upward arrows indicate the precise insertion sites. The poly(A) tail length in each insertion is indicated in subscript; notably, polyadenylation occurred precisely at the SV40 pA site present in the CEP4 vector (36, 37). The target site duplications flanking each insertion are underlined. The black boxes represent pAI1b sequences, while the gray boxes in clones 2 and 3 represent L1 sequences that lie immediately upstream of the cDNA. The gray box between the inverted L1s indicates the pCEP4 derived plasmid sequences (see the text for additional details).