Nucleic Acids Research, 2005, Vol. 33, No. 6 1993–2002 doi:10.1093/nar/gki347 Functional roles of 30-terminal structures of template RNA during in vivo retrotransposition of non-LTR retrotransposon, R1Bm Tomohiro Anzai, Mizuko Osanai, Mitsuhiro Hamada and Haruhiko Fujiwara* Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Bioscience Building 501, Kashiwa, 277-8562, Japan Received as resubmission March 3, 2005; Revised March 9, 2005; Accepted March 21, 2005 ABSTRACT R1Bm is a non-LTR retrotransposon found specific- ally 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 30 end. To study how R1Bm initiates reverse transcription from the poly (A)-less template RNA, we established an in vivo retrotrans- position system using recombinant baculovirus, and characterized retrotransposition activities of R1Bm. Target-primed reverse transcription (TPRT) of R1Bm occurred from the cleavage site generated by endo- nuclease (EN). The 147 bp of 30-untranslated region (30UTR) 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 50-UG-30 or 50-UGU-30 at their 30 ends, which are presumably base-paired with 30 end of the EN-digested 28S rDNA target sequence, 50-AGTAGATAGGGACA-30. When the downstream sequence of 28S rDNA target was added to the 30 end of R1 unit, reverse transcription started exactly from the 30 end of 30UTR and retro- transposition efficiency increased. These results indicate that 30-terminal structure of template RNA including read-through region interacts with its tar- get rDNA sequences of R1Bm, which plays important roles in initial process of TPRT in vivo. INTRODUCTION Non-long terminal repeat (non-LTR) retrotransposons, also called long interspersed nuclear elements (LINEs) or poly (A) elements, are the most abundant family among mobile elements. LINEs have been identiﬁed in all major groups of eukaryotes, with the exception of the bdelloid rotifers (1). In humans, up to 21% of the genome is comprised of LINEs (2), and the involvement of LINEs in the gene evolution and gen- ome reconstruction had been proposed (3–5). LINEs can be classiﬁed into two subtypes based on their structures and modes of retrotransposition (6). The early- branched subtype, such as R2 element speciﬁcally integrated in 28S rDNA of arthropods, comprises a single open reading frame (ORF) that encodes reverse transcriptase (RT) in the middle and a restriction enzyme-like endonuclease (EN) near its C-terminal end (7,8). Biochemical analyses of R2Bm of the silkworm led to the current model for LINE retrotransposition. R2Bm protein ﬁrst recognizes the 30UTR sequence of R2 RNA, and makes a speciﬁc nick on the target DNA by its endonuclease (9,10). Then, the exposed 30 hydroxyl is utilized to initiate reverse transcription of the RNA template (11). Thus, the processes of nicking, reverse transcription and integ- ration are basically coupled, which are termed target-primed reverse transcription (TPRT). It is suggested that the rare transcript of R2 element is provided by co-transcription with the 28S rDNA target (12,13), and an additional 28S rRNA sequence downstream of the R2 sequence reduced the efﬁciency of the reaction, but increased the rate of accurate junctions seen in vivo (14). The recently-branched subtype of LINEs has two ORFs: ORF1 and ORF2. The ORF2 encodes catalytic domains responsible for LINE retrotransposition; apurinic/apyrimidinic endonucleases (APE)-like endonuclease at the N-terminus (15) and reverse transcriptase at the central region. Retrotransposi- tion of this class is mainly studied for human L1 element using an assay to monitor retrotransposition in cultured human HeLa cells (16) and SART1 of the silkworm using the Autographa californica nuclear polyhedrosis virus (AcNPV) expression system (17). Although L1 and SART1 both have 30UTR and poly (A) tail at their 30 end, they show different requirement for the RNA sequence to initiate the reverse transcription. The presence of 30 UTR is dispensable for L1 retrotransposition, *To whom correspondence should be addressed. Tel: +81 4 7136 3659; Fax: +81 4 7136 3660; Email: haruh@k.u-tokyo.ac.jp ª The Author 2005. Published by Oxford University Press. All rights reserved. The online version of this article has been published under an open access model. 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