Mechanism Of Insertion

Understanding the mechanism of Alu insertion is critical to understanding how they influence genetic instability. A typical Alu element, shown schematically in Fig. 1, contains an internal, RNA polymerase III promoter that allows transcription through the element and into the flanking sequence. Alu elements have a dimer structure, with both halves having an ancestral derivation from the 7SL RNA gene and have no capacity to code for proteins. At the 3' end of each Alu element is an A-rich sequence of variable length and the elements are generally flanked by short, direct repeats of 5-20 bp in length that are formed from duplications of the target site during insertion. It has been shown that the 3' A-rich region is routinely lengthened by the insertion process (4,5), and rapidly shrinks after insertion (6).

It is thought that the RNA polymerase III-transcribed Alu RNA must associate in some way with at least the ORF2 product of L1, which codes for endonuclease and reverse transcriptase activities. Thus, Alu is considered a nonautonomous mobile element, which is dependent on the autonomous L1 elements. The endonuclease then cleaves the genomic DNA at a loose consensus sequence, and the genomic site primes reverse transcription of the Alu RNA, using the 3' oligo-A region of the Alu RNA as a template. It has been hypothesized that Alu elements with longer A-tails are more active in the retrotransposition process (6). The typical integration is then finished off by the formation of a second-strand nick and integration of the end of the cDNA (Fig. 2A). However, significant portions of the events appear to complete the integration process by recombining with another upstream Alu element (7) (Fig. 2B). This would cause sequences between the point of insertion and the upstream Alu element to be deleted. It also seems likely that many Alu elements may not complete the insertion process. Thus, the initiation of the insertion may create genomic nicks that contribute to mutation and recombination processes in the cell, but show no evidence of Alu insertion.

Although Alu amplification is clearly dependent on L1 elements for their retroposition, there are definite differences in their amplification process. The most notable difference is that, using a tagged Alu reporter system, Alu insertion was found to not require exogenous L1 ORF1 expression (5).

Fig. 2. Integration of Alu elements into genomic sites. The basic mechanism of Alu insertion is thought to involve a target-primed reverse transcription (TPRT) in which the endonuclease provided by L1 elements creates a nick in the genomic DNA at a consensus sequence resembling TTTT'AA. The nicked strand with the T residues can then prime reverse transcription from the 3' A-rich region of theAlu RNA. The second-strand integration process is poorly understood. The typical integration is schematized in (A) with a second nick occurring from an unknown source, probably allowing integration of the 3' end of the cDNA using some sort of microhomology-driven priming. (B) An alternative mechanism that occurs occasionally where the 5' end finds a homology with another Alu element upstream. (C) It is possible that the integration process may abort prematurely, possibly with the intervention of DNA repair processes, leading to DNA damage. (D) Alu elements may undergo unequal recombination with other Alu elements nearby. We hypothesize that nicking by L1 endonuclease at consensus sites adjacent to Alu elements may facilitate this recombination process.

Fig. 2. Integration of Alu elements into genomic sites. The basic mechanism of Alu insertion is thought to involve a target-primed reverse transcription (TPRT) in which the endonuclease provided by L1 elements creates a nick in the genomic DNA at a consensus sequence resembling TTTT'AA. The nicked strand with the T residues can then prime reverse transcription from the 3' A-rich region of theAlu RNA. The second-strand integration process is poorly understood. The typical integration is schematized in (A) with a second nick occurring from an unknown source, probably allowing integration of the 3' end of the cDNA using some sort of microhomology-driven priming. (B) An alternative mechanism that occurs occasionally where the 5' end finds a homology with another Alu element upstream. (C) It is possible that the integration process may abort prematurely, possibly with the intervention of DNA repair processes, leading to DNA damage. (D) Alu elements may undergo unequal recombination with other Alu elements nearby. We hypothesize that nicking by L1 endonuclease at consensus sites adjacent to Alu elements may facilitate this recombination process.

Was this article helpful?

0 0

Post a comment