The L1 Retrotransposon–A Heavy Load on the Human Genome
JD Boeke
We have learned an enormous amount recently abut the mechanism
and biology of one of the major components of the human genome, the L1 (LINE)
retrotransposon. This remarkable element is likely responsible, directly
or indirectly, for about one third of our genome by weight; its reverse
transcriptase ORF (ORF2) is the most abundant ORF in the human genome. The
full-length L1 element encodes two large proteins and a 6 kb long RNA.
ORF2 contains enzymatic functions required for retrotransposition; ORF1 encodes
an RNA binding protein whose precise role is less clear cut. Recent gains
in understanding how this element works result from several technical
breakthroughs. We and our colleagues developed and exploited a new assay
for L1 retrotransposition in human tissue culture cells1 and also, we
identified and characterized a critical new functional domain of L1 ORF2, the
endonuclease2,3.
The precise mechanism of retrotransposition is under
investigation, but is likely to result from a series of target-primed reverse
transcriptase events (TRPT). We have observed a TPRT-like reaction in
vitro in a system containing recombinant ORF2 protein, L1 3′ end RNA, and a
target plasmid. The events occur at a low efficiency, but analysis of the
reaction products indicates that they resemble the transposon/target DNA
junctions observed in mammalian cells. The reaction is completely
dependent on ORF2 protein, L1 3′ end RNA (although other RNAs can also be used
successfully) and target DNA. However, we have also observed a rather
efficient reverse transcription reaction that works on L1 3′ end RNA that does
not require addition of an exogenous primer. Analysis of these reaction
products is in progress.
We are also sifting through the human genome for clues about the
mechanisms of insertion as well as about aberrant insertion events.
Interestingly, it appears that about 10-20% of the time, L1 may co-mobilize
flanking human DNA to the new location when it transposes4.
Such movement of human genomic sequences could be a potent force in genome
evolution, potentially shuffling genes in a variety of interesting ways5.
We see evidence of these events in the rapidly growing human genome database.
The regulation of L1 retrotransposition is poorly
understood. It is in a transposon’s interest to transpose in the germ
line. However, various data point to L1 activity in certain somatic
tissues, especially in diseases such as cancer. We are exploring the model
that DNA methylation is a major mechanism for regulating L1 retrotransposition.
1Moran JV et al. High-frequency
retrotransposition in cultured mammalian cells. Cell 87,917-927 (1996).
2Feng Q, Moran J, Kazazian H, & Boeke JD.
Human L1 retrotransposon encodes a conserved endonuclease required for
retrotransposition. Cell 87, 905-916 (1996).
3Cost GC & Boeke JD. Targeting of human
retrotransposon integration is directed by the specificity of the L1
endonuclease for regions of unusual DNA structure. Biochemistry 37,
18081-18093 (1998).
4Moran JV, DeBerardinis RJ & Kazazian HH
Jr. Exon shuffling by L1 retrotransposition. Science 283, 1530-1534
(1999).
5Boeke JD & Pickeral OK. Retroshuffling the
genomic deck. Nature 398, 108-111 (1999).