Essential Motifs in the 3′ Untranslated Region Required for Retrotransposition and the Precise Start of Reverse Transcription in Non-Long-Terminal-Repeat Retrotransposon SART1

Abstract

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 ...

Eukaryotic Translational Coupling in UAAUG Stop-Start Codons for the Bicistronic RNA Translation of the Non-Long Terminal Repeat Retrotransposon SART1

“…It indicates that ORF2 is less efficiently translated than ORF1 by nature. In our in vivo retrotransposition assay system, retrotransposition can be detected by PCR with a SART1 internal primer (SART1-S6311) and a telomeric repeat-specific primer (CCTAAϩT) (26,29,36). We detected the PCR products, which represented the retrotransposition of SART1 into the telomeric repeats in Sf9 cells, when construct I was infected (Fig.…”

“…2A, 4). RNA including both ORF1 and ORF2 was presumed to be transcribed from the AcNPV polyhedrin promoter, based on the observation of our previous report (29). Because the SART1 5Ј UTR was replaced by the GST-His tag sequence in the AcNPV-expressed SART1, the length of SART1 RNA expressed by AcNPV was 127 nt shorter than the native SART1.…”

“…As described above, SART1 ORF2 is translated separately from ORF1, which suggests that ORF1p and ORF2p can be supplied for retrotransposition independently. To investigate this possibility, we performed the in vivo retrotransposition assay reported previously (26,29,36). We used four constructs (Fig.…”

“…Thus, the functional ORF2p of SART1 is translated in the AcNPV expression system, which will be useful for studying the translational control in non-LTR retrotransposons. In addition, we previously reported that the SART1 RNA transcribed from the polyhedrin promoter showed a band which corresponds to the full-length SART1 and the downstream polyhedrin 3Ј region (29). There were no clear bands below the full-length transcripts.…”

“…In addition, only a single band corresponding to a full-length SART1 unit was observed in several Bombyx tissues by Northern hybridization (35). Recently, we constructed an in vivo transposition assay system of SART1 in Sf9, the moth Spodoptera frugiperda cell line, using the expression of Autographa californica nuclear polyhedrosis virus (AcNPV) (26,29,36). Both the silkworm, B. mori, and the moth, S. frugiperda, belong to the same order, Lepidoptera.…”