"Copy and paste" transposable elements in the human genome.

Since the early 1980's we have known about the major role that transposable elements play in the biology of a wide variety of organisms. In Drosophila, most spontaneous mutations result from new insertions of transposable elements. In yeast, about 1% of all mutations are due to insertions ofmobile elements. In humans, the effects of transposition events are harder to estimate, but the best data indicate that they account for between 1 in 500 and 1 in 1,000 mutations. In prokaryotes, most of transposition occurs through DNA itself. Transposon DNA is copied and reinserted into the genome at a new location. In eukaryotes, the process is one of retrotransposition in which the element is transcribed into RNA, reverse transcribed into cDNA, and the double-stranded cDNA is reinserted into the genome. In computer lingo the process is "copy and paste," as the original sequence remains intact. When a retrotransposon inserts into the genome, it is flanked by short direct repeats (target site duplications) of the endogenous sequence. These have been seen in nearly all retrotransposition events. There are two general structures ofeukaryotic retrotransposons, Class I and Class II elements (1). Class I elements have long terminal repeats at their two ends. They also have 1-3 protein-coding regions, one ofwhich makes a reverse transcriptase used by the element for retrotransposition. Examples of these elements which are 4-6 kb in length are copia ofDrosophila, Ty of yeast, and intracisternal A particle of mice. Retroviruses look very much like Class I elements, but they contain an envelope protein gene to help them move from cell to cell while only an exceptional Class I element contains an envelope gene. Evolutionary biologists have developed a "tree" for transposable elements based on reverse transcriptase sequences (2). From this "tree," retroviruses appear to have had a very recent origin, while Class II LI elements (discussed below) appear near the root and are very old. Full-length Class II elements are 5-7 kb in size; they lack long terminal repeats, have 3' poly A tails, and have one to two open reading frames (ORFs), one of which encodes a reverse transcriptase. Examples of these elements are the I factor of Drosophila and LI elements of mammals. LI elements are present in the genome of all mammals: in humans, they are present in about 100,000 copies and account for about 5% of genomic DNA. Most of the LI copies are 5'-truncated so that only about 3,500 are full-length, 6-kb elements. A number of facts suggested that some LI elements were retrotransposons. They were present in many copies. They had a 3' poly A tail and two ORFs in their consensus sequence. They had an internal promoter for transcription, and they also had a region of ORF2 that could potentially encode a polypeptide with reverse transcriptase homology. Five years ago, our group found two patients with hemophilia A in whom de novo insertion of a truncated LI element into the Factor VIII gene on the X chromosome produced the disease (3). In 199 1, the full-length precursor to one ofthose insertions was isolated (4). It contains the two ORFs predicted by the LI consensus sequence. ORF1 makes a protein of unknown function in a transient expression assay (5) while ORF2 encodes a reverse transcriptase activity in a yeast assay system (6). This LI element is present in two copies on chromosome 22 in all humans studied to date and it is flanked by a target site duplication, indicating that it originated as the product ofa retrotransposition event. Over the past two years, three separate insertions of Alu elements have been reported, all of which caused genetic disease (7-9). Alu elements presumably expanded to their present number ofabout 300,000 in the human genome by reinsertion of reverse transcripts. A similar process is thought to account for processed pseudogenes, i.e., the dispersion of mRNAs. The cognoscenti are betting that the reverse transcriptase for these events is provided by active L1 elements. Evidence suggests that LI transcripts are sequestered in cytoplasmic particles within which LI cDNA is produced (10). Alu RNAs and mRNAs could be trapped in LI particles, reverse transcribed by LI reverse transcriptase, and then be escorted back to the genome using LI machinery for reinsertion. Although this scenario is speculative at present, it is attractive because it provides these wandering RNAs with the "goods" of a proven retrotransposon for reentering the genome. Recently, two new twists to the LI retrotransposition story have emerged. First, a somatic insertion was found in an adenomatous polyposis coli (APC) tumor suppressor gene (I I). This insertion of roughly 500 bp of the 3' sequence of an LI element disrupted the coding region of an APC gene in the tumor, but was not present in the normal colonic epithelium of the patient. Thus, in this instance the retrotransposition was a somatic event which occurred in a dedifferentiating cell. Second, the paper by Narita et al. in this issue of The Journal describes an inherited LI insertion into the dystrophin gene causing Duchenne muscular dystrophy ( 12). This insertion is of special interest because it lacks a target site duplication; instead, two nucleotides of the dystrophin sequence are deleted and one nucleotide is added at the point of insertion. This observation suggests that a minority of transposon insertions are processes whereby breaks in the DNA are repaired, in contrast to the active reintegration envisaged for most transposition events. Many interesting questions about LI retrotransposition are yet to be answered. For instance, we wonder whether these sequences are merely parasitic DNAs that we (as mammals) have been able to tolerate for a hundred million years or are they, like the mitochondria, symbiotes that provide some necessary biological function? If so, what might that function be? At present, the only known biochemical property ofL I is a reverse transcriptase activity. Does this reverse transcriptase play some role in the ova or sperm? In fact, is LI normally expressed in germline cells and at what level? Does L I really form a particle like retroviruses and can the particle trap other RNAs and reverse transcribe them? Work cited above along with that of others suggests that LI transcription and expression may be induced in some cancers. Is this unusual expression a consequence ofgeneral dedifferentiation? Could abnormal L I expression be used as a tumor cell