Most insects have telomeres that consist of pentanucleotide (TTAGG) telomeric repeats, which are synthesized by telomerase. However, all species in Diptera so far examined and several species in other orders of insect have lost the (TTAGG)n repeats, suggesting that some of them recruit telomerase-independent telomere maintenance. The silkworm, Bombyx mori, retains the TTAGG motifs in the chromosomal ends but expresses quite a low level of telomerase activity in all stages of various tissues. Just proximal to a 6-8-kb stretch of the TTAGG repeats in B. mori, more than 1000 copies of non-LTR retrotransposons, designated TRAS and SART families, occur among the telomeric repeats and accumulate. TRAS and SART are abundantly transcribed and actively retrotransposed into TTAGG telomeric repeats in a highly sequence-specific manner. They have three possible mechanisms to ensure specific integration into the telomeric repeats. This article focuses on the telomere structure and telomere-specific non-LTR retrotransposons in B. mori and discusses the mechanisms for telomere maintenance in this insect.

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Non-long-terminal-repeat (non-LTR) retrotransposons amplify their copies by reverse transcribing mRNA from the 3 end, but the initial processes of reverse transcription are still unclear. We have shown that a telomere-specific non-LTR retrotransposon of the silkworm, SART1, requires the 3 untranslated region (3 UTR) for retrotransposition. With an in vivo retrotransposition assay, we identified several novel motifs within the 3 UTR involved in precise and efficient reverse transcription. Of 461 nucleotides (nt) of the 3 UTR, the central region, from nt 163 to nt 295, was essential for SART1 retrotransposition. Of five putative stem-loops formed in RNA for the SART1 3 UTR, the second stem-loop (nt 159 to 221) is included in this region. Loss of the 3 region (nt 296 to 461) in the 3 UTR and the poly(A) tract resulted in decreased and inaccurate reverse transcription, which starts mostly from several telomeric repeat-like GGUU sequences just downstream of the second stem-loop. These results suggest that short telomeric repeat-like sequences in the 3 UTR anneal to the bottom strand of (TTAGG) n repeats. We also demonstrated that the mRNA for green fluorescent protein (GFP) could be retrotransposed into telomeric repeats when the GFP coding region is fused with the SART1 3 UTR and SART1 open reading frame proteins are supplied in trans.Non-long-terminal-repeat (non-LTR) retrotransposons are endogenous mobile genetic elements that are widespread among the genome of eukaryotes. Non-LTR retrotransposons multiply their copies through reverse transcription of RNA intermediates with a self-encoding reverse transcriptase (RT). Non-LTR retrotransposons, some of which are called long interspersed nuclear elements (LINEs) in vertebrates, contribute to genome structure and evolution through their replication process (5,8,10,21). In humans, LINEs have accumulated up to 21% of the genome, and they are the major source of insertional mutagenesis. LINEs shape the mammalian genome through exon shuffling, mobilization of short interspersed nuclear elements, and processed pseudogene formation (4,6,10,18,20). However, we have little knowledge of the molecular basis underlying these genomic events because the retrotransposition mechanisms of non-LTR retrotransposons are insufficiently understood, compared with those of other retroelements, such as LTR retrotransposons and retroviruses.Non-LTR retrotransposons encode an RT domain and an endonuclease (EN) domain. Pioneering studies of the Bombyx non-LTR element R2 showed that the EN domain nicks the bottom strand of target DNA and that the RT domain uses the 3Ј-hydroxyl end of the nicked DNA as a primer for reverse transcribing non-LTR element RNA (12). This reverse transcription initiation process is termed target-primed reverse transcription (TPRT) and is inherent to all non-LTR retrotransposons. During TPRT, reverse transcription is initiated at the 3Ј end of non-LTR elements. Most genomic copies of non-LTR retrotransposons are 5Ј truncated, presumably because of the arrest of reverse ...

R1Bm is a non-LTR retrotransposon found specifically within 28S rRNA genes of the silkworm. Different from other non-LTR retrotransposons encoding two open reading frames (ORFs), R1Bm structurally lacks a poly (A) tract at its 3′ end. To study how R1Bm initiates reverse transcription from the poly (A)-less template RNA, we established an in vivo retrotransposition system using recombinant baculovirus, and characterized retrotransposition activities of R1Bm. Target-primed reverse transcription (TPRT) of R1Bm occurred from the cleavage site generated by endonuclease (EN). The 147 bp of 3′-untranslated region (3′UTR) was essential for efficient retrotransposition of R1Bm. Even using the complete R1Bm element, however, reverse transcription started from various sites of the template RNA mostly with 5′-UG-3′ or 5′-UGU-3′ at their 3′ ends, which are presumably base-paired with 3′ end of the EN-digested 28S rDNA target sequence, 5′-AGTAGATAGGGACA-3′. When the downstream sequence of 28S rDNA target was added to the 3′ end of R1 unit, reverse transcription started exactly from the 3′ end of 3′UTR and retrotransposition efficiency increased. These results indicate that 3′-terminal structure of template RNA including read-through region interacts with its target rDNA sequences of R1Bm, which plays important roles in initial process of TPRT in vivo.

R1Bm is a long interspersed element (LINE) inserted into a specific sequence within 28S rDNA of the silkworm genome. Of two open reading frames (ORFs) of R1Bm, ORF2 encodes a reverse transcriptase (RT) and an endonuclease (EN) domain which digests specifically both top and bottom strand of the target sequence in 28S rDNA. To elucidate the sequence specificity of EN domain of R1Bm (R1Bm EN), we examined the cleavage tendency for the target sequences, and found that 5′-A(G/C)(A/T)!(A/G)T-3′ is the consensus sequence (! = cleavage site). We also determined the crystal structure of R1Bm EN at 2.0 Å resolution. Its structure was basically similar to AP endonuclease family, but had a special β-hairpin at the edge of the DNA binding surface, which is a common feature among EN of LINEs. Point-mutations on the DNA binding surface of R1Bm EN significantly decreased the cleavage activities, but did not affect the sequence recognition in most residues. However, two mutants Y98A and N180A had altered cleavage patterns, suggesting an important role of these residues (Y98 and N180) for the sequence recognition of R1Bm EN. In addition, Y98A mutant showed another cleavage pattern, that implies de novo design of novel sequence-specific EN.