Abstract
LINE-1 (L1) retroelements emerged in mammalian genomes over 80 million years ago with a few dominant subfamilies amplifying over discrete time periods that led to distinct human and mouse L1 lineages. We evaluated the functional conservation of L1 sequences by comparing retrotransposition rates of chimeric human-rodent L1 constructs to their parental L1 counterparts. Although amino acid conservation varies from ∼35% to 63% for the L1 ORF1p and ORF2p, most human and mouse L1 sequences can be functionally exchanged. Replacing either ORF1 or ORF2 to create chimeric human-mouse L1 elements did not adversely affect retrotransposition. The mouse ORF2p retains retrotransposition-competency to support both Alu and L1 mobilization when any of the domain sequences we evaluated were substituted with human counterparts. However, the substitution of portions of the mouse cys-domain into the human ORF2p reduces both L1 retrotransposition and Alu trans-mobilization by 200–1000 fold. The observed loss of ORF2p function is independent of the endonuclease or reverse transcriptase activities of ORF2p and RNA interaction required for reverse transcription. In addition, the loss of function is physically separate from the cysteine-rich motif sequence previously shown to be required for RNP formation. Our data suggest an additional role of the less characterized carboxy-terminus of the L1 ORF2 protein by demonstrating that this domain, in addition to mediating RNP interaction(s), provides an independent and required function for the retroelement amplification process. Our experiments show a functional modularity of most of the LINE sequences. However, divergent evolution of interactions within L1 has led to non-reciprocal incompatibilities between human and mouse ORF2 cys-domain sequences.
Highlights
The activity of LINE-1 (L1) elements has contributed both directly and indirectly to almost a third of the human genome mass [1]
Because RNA processing and secondary structure could possibly affect retrotransposition rate, we chose to minimize the impact of the differences between the L1 sequences contributing to effects on RNA levels by working with codon optimized human and mouse coding sequences driven by the CMV promoter
We find that optimization reduces some of the processing of the full length L1 RNA and that our tagged minimal human L1 construct generates about twice as much full length L1 mRNA compared to the wildtype L1.3 element (Figure 1B)
Summary
The activity of LINE-1 (L1) elements has contributed both directly and indirectly to almost a third of the human genome mass [1]. Evidence of LINE retroelement activity dates as far back as 100 million years ago (mya) [1,2,3]. L1 continues to be active in the vast majority of mammalian species tested to date, with a few exceptions [4,5]. Human and rodent lineages diverged approximately 80 million years ago, with each lineage harboring unique L1 subfamilies [6,7]. Current activity of L1 and its non-autonomous partners, Alu and SVA, account for about 0.3% of new human germ-line diseases [11]. Since the split between eutherians and marsupials, a single L1 clade continues to amplify with separate single dominant lineages of L1 families in primates and rodents [13]
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