Figure 6. Schematic representation of initial process of TPRT in the R1 element. (Top) First, R1-EN cleaves the non-coding bottom strand of 28S rDNA in the A–C junction. Boxes represent the 14 bp of TSD. (Middle) Then, the target DNA is partially denatured, allowing the UGU on the RNA template to base-pair with the loose target DNA. The template RNA is indicated by a gray line. In this model, the read-through 28S rRNA sequence is base-paired with the DNA target in longer region. During this process, RT of R1Bm may recognize the 30UTR and base-paired region (open arrow), and place the RNA template at the accurate position for initiation of reverse transcription. (Bottom) Next, reverse transcription starts from the position next to the UGU sequence of template RNA, using the 30-OH of A residue as primer. requirement of 30 downstream sequence derived from 28S gene for the precise, efﬁcient integration into its target DNA. Luan and Eickbush reported that addition of downstream target sequence to the R2 element resulted in the accurate start of reverse transcription from the precise R2-28S gene junction (10,14). The R2 insertion site is located just 74 bp upstream of the R1 site (9), but R1 and R2 are distantly related from the phylogenetic study (6). It is of some interest that these distantly related LINEs showed the similar requirements of downstream 28S sequence for the precise integration. Phylogenetically, R1 element belongs to the ‘R1 clade’ (6). Most R1 clade non-LTR retrotransposons are target-speciﬁc and show the diversiﬁed target speciﬁcities (27,28), suggesting that they have evolved to change their target sequences from the common ancestors. Using in vivo retrotransposition assay, we previously characterized another R1 clade element, SART1, which integrates into the telomeric repeats (TTAGG)n (17,20). The comparison between R1 and SART1 revealed some distinct features in the initial processes of reverse transcription. The full-length SART1 element with poly (A) at the 30 terminal initiates its reverse transcription Nucleic Acids Research, 2005, Vol. 33, No. 6 2001 from the poly (A) tract (17). However, the complete R1Bm unit with 30UTR started its reverse transcription from various positions of template RNA (Figure 3B), suggesting that reverse transcriptase of R1 recognizes its 30UTR in a less stringent manner than that of SART1. SART1 was shown to recognize some motifs within the 30UTR and the loss of the 30 terminal poly (A) tract severely decreased its efﬁciency for integration (20), while R1 elements do not contain A-rich tract at its 30 junctions. As shown in Figure 5, the loss of 30UTR decreased the retrotransposition activity of R1Bm, compared to WT con- struct with 30UTR. This indicates that RT encoded in ORF2 of R1Bm also recognizes some structure in 30UTR, but it seems insufﬁcient for specifying the start position of reverse transcription. In case of WT R1Bm retrotransposition, many copies started reverse transcription from UG or UGU (Figure 3B), which are presumably base-paired with the 30 end of the bottom strand of target DNA (Figure 6). The similar short base-paring between the template RNA and the target DNA was also presumed in the SART1 element, which lacks 30-half of 30UTR or poly (A) tract (20). Thus, the short inter- action between RNA and DNA seem to play some roles in promoting the initial process of TPRT in R1 and SART1. To ensure the accurate and effective retrotransposition, R1Bm needed the downstream 28S sequences in addition to 30UTR, such as 30-half of 30UTR or poly (A) in SART1 element. In this context, the read-through 28S rRNA sequence of R1Bm may enable the long base-paring between RNA and DNA, and contribute to increase the accuracy for reverse transcription and the retrotransposition efﬁciency (Figure 5B). R1 elements are oriented in the same direction as the ribosomal gene tran- scription, and in Drosophila melanogaster, endogenous R1 transcripts include upstream and downstream 28S sequences (12), so it is suggested that R1Bm is co-transcribed with the 28S genes. We suggest that constructs most reﬂecting the endogenous R1Bm are +14 nt/+50 nt and the annealing system between read-through RNA and the target DNA may be actu- ally used in endogenous R1Bm. It is of some interest that addition of 5 nt poly (A) tract at the end of R1Bm resulted in a severe loss of retrotransposition activity (Figure 5B), suggesting that the spatial distance between the R1 30UTR and the downstream 28S rRNA sequence is somehow recog- nized by RT of R1Bm (Figure 6). The spatial arrangement might be disturbed by insertion of 5 nt of A tract. Retrotransposition by R1 also showed the characteristic feature of LINE at the 50 boundary; frequent 50 truncation (Figure 4B), due to the incomplete reverse transcription of RNA template into cDNA. Some 50 border of endogenous R1Bm contains duplication of R1 sequence, but no 28S sequence deletion or 50 truncations are found (9). However, R1Bm construct in this assay showed various 50 junction struc- ture and severe aberrations, such as the deletion of 28S sequence by 17 bp (Figure 4). This may be due to the replace- ment of upstream 28S sequences and R1-50UTR to polyhedrin 50UTR and GST sequence. The inclusion of 28S sequence to its 50 termini of the construct achieved intact 50 junction forma- tion in R2 (26,29), so it may be the same for R1. This implic- ates that the free ends generated during this process are repaired by the cellular proteins. It was unexpected that the retrotransposition into the site 180 bp upstream of R1 target site was observed for R1