JOHNS HOPKINS UNIVERSITY, THE
"LINE-1, or L1, elements, are the most prolific and prominent mobile elements in mammalian genomes. In the human and mouse genomes, active L1s and their relics account for 17-20% of the genome mass. By providing activities necessary for mobility of Alu and other elements, they indirectly account for another 10- 12% of these genomes. L1 retrotransposons move through a duplicative ""copy and paste"" mechanism. In this renewal proposal, we attack a number of unanswered questions concerning the timing and cell type specificity of retrotransposition, the mechanism of L1 insertion into the genome, and the role of siRNA produced by the bidirectional L1 promoter in the control of retrotransposition. In Specific Aim 1, we seek to determine the fraction of total retrotransposition events that occur in male germ cells, female germ cells, and in the early stages of embryonic development. We also wish to determine whether retrotransposition decreases with age, is suppressed after a number of generations, and is increased by defects in methylation or DMA double strand break repair. In Specific Aim 2 we develop further evidence of the role of siRNA derived from the L1 bidirectional promoter. We also characterize the small RNA sequences derived from L1 RNA, and determine whether specific knockdown of L1 transcript levels alters the phenotype of transformed cells. In Specific Aim 3, we will test a novel mechanism of L1 integration. Postulated features of this mechanism are 1) 5' truncation is due to 5' degradation of L1 RNA, 2) template jumping of reverse transcriptase is a key feature of the mechanism, and 3) synthesis of DMA second strand is carried out by a DNA polymerase activity of L1 reverse transcriptase. In Specific Aim 3A we determine which RNA polymerase transcribes L1 and how the RNA is processed (capped and polyadenylated). In Specific Aim 3B we determine the processes involved in L1 RNA degradation, in particular, whether deadenylation, decapping and 5' degradation are involved and whether a significant fraction of L1 RNA is rapidly 5' degraded. These studies will increase greatly our knowledge of the biology of L1 retrotransposition. In addition, they will provide important new information towards the effort to use L1 as a random insertional mutagen in mammals.