Characterization of a synthetic human LINE-1 retrotransposon ORFeus-Hs

Abstract

Long interspersed elements, type 1(LINE-1, L1) are the most abundant and only active autonomous retrotransposons in the human genome. Native L1 elements are inefficiently expressed because of a transcription elongation defect thought to be caused by high adenosine content in L1 sequences. Previously, we constructed a highly active synthetic mouse L1 element (ORFeus-Mm), partially by reducing the nucleotide composition bias. As a result, the transcript abundance of ORFeus-Mm was greatly increased, and its retrotransposition frequency was > 200-fold higher than its native counterpart. In this paper, we report a synthetic human L1 element (ORFeus-Hs) synthesized using a similar strategy. The adenosine content of the L1 open reading frames (ORFs) was reduced from 40% to 27% by changing 25% of the bases in the ORFs, without altering the amino acid sequence. By studying a series of native/synthetic chimeric elements, we observed increased levels of full-length L1 RNA and ORF1 protein and retrotransposition frequency, mostly proportional to increased fraction of synthetic sequence. Overall, the fully synthetic ORFeus-Hs has > 40-fold more RNA but is at most only ~threefold more active than its native counterpart (L1RP); however, its absolute retrotransposition activity is similar to ORFeus-Mm. Owing to the elevated expression of the L1 RNA/protein and its high retrotransposition ability, ORFeus-Hs and its chimeric derivatives will be useful tools for mechanistic L1 studies and mammalian genome manipulation.

Figure 1

Schematic representation of native, synthetic and chimeric human L1 elements. Three sets of such constructs differing from each other at the promoter region are illustrated: the first set carries a cytomegalovirus (CMV) promoter and a Kozak (K) signal, the second set has a dual CMV-L1 5' untranslated region (UTR) promoter, and the third has a 5' UTR promoter only. All elements are cloned in a pCEP-Puro vector backbone. AMP^R = ampicillin resistance gene; B, E, P = restriction sites EcoRI, BamHI and PmlI, respectively, at the junctions in various chimeras; EBNA-1 = Epstein-Barr nuclear antigen 1 gene permitting extrachromosomal replication; Intron = human gamma globin intron; Marker = either enhanced green fluorescent protein or neomycin marker; ORF = open reading frame; Puro^R = puromycin resistance gene; SV40pA = Simian virus 40 polyadenylation signal. Blue = native sequence; purple = synthetic sequence.

Figure 2

Total RNA analysis of L1 expression. Expression levels of native, partially synthetic, and completely synthetic ORFeus-Hs were compared in 293T cells. The vectors used were: pLD223, pWA172, pWA170, pWA176, pWA163, pWA174, pLD224, pLD227, pLD225, pWA165. Top, L1 mRNA expression; note both spliced and unspliced transcripts. Bottom, RNA expression of loading control, ARPP0.

Figure 3

Relative increases in RNA, protein and retrotransposition frequency. (A) Relative increases in RNA, protein and retrotransposition frequency in the constructs containing CMV only. Chimera B = pWA176; chimera E = pWA170; chimera P = pWA172; native = pLD223; synthetic = pWA163. (B) Relative increases in RNA, protein and retrotransposition frequency in the constructs containing both CMV and 5' UTR promoters. Chimera E = pLD227; chimera B = pLD225; chimera P = pLD224; native = pWA174; synthetic = pWA165. Values of pLD223 and pWA174 were assigned as control in each group of constructs. Data are mean of a minimum of three independent experiments plus standard error. Blue = RNA; gray = relative retrotransposition frequency; purple = ORF1p.

Figure 4

Analysis of ORF1 protein expression and relative ORF2 RT activity. (A) The same constructs as in Figure 2 were analyzed. The vectors used were: pLD223, pWA172, pWA170, pWA176, pWA163, pWA174, pLD224, pLD227, pLD225, pWA165. Top, protein expression of ORF1. Bottom, protein expression of the tubulin loading control. (B) L1 element amplification protocol (LEAP): assay was performed using ribonucleoproteins (RNPs) prepared from cells transfected with pWA174 (L1_RP) and pWA165 (ORFeus-Hs). The numbers 0, 0.1, 0.2, 0.5, 1, 5 and 10 indicate the amount (μg) of total protein of the RNP prep added to each LEAP reaction. The arrows indicate the mobility of LEAP PCR product. SuperScript III represents a positive control in which 100 U of SuperScript III reverse transcriptase (Invitrogen, Carlsbad, CA, USA) was added to the LEAP reaction.