Functional roles of 3′-terminal structures of template RNA during in vivo retrotransposition of non-LTR retrotransposon, R1Bm
Functional roles of 3′-terminal structures of template RNA during in vivo retrotransposition of non-LTR retrotransposon, R1Bm
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DOI：
10.1093/nar/gki347
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发表时间：
2005-01-01
影响因子：
14.9
通讯作者：
Fujiwara, H
中科院分区：
文献类型：
--
作者：
Anzai, T;Osanai, M;Fujiwara, H
R1Bm是在家蚕28S rRNA基因中特异发现的非ltr反转录转座子。与其他编码两个开放阅读框（orf）的非ltr反转录转座子不同，R1Bm在其3'端结构上缺乏poly (a)束。为了研究R1Bm如何从poly (A)-less模板RNA启动逆转录，我们利用重组杆状病毒建立了体内逆转录转位系统，并对R1Bm的逆转录转位活性进行了表征。R1Bm的靶引反转录（target -引反转录，TPRT）发生在由核酸内切酶（enase）产生的裂解位点。147 bp的3‘非翻译区（3’ utr）对R1Bm的有效反转录至关重要。然而，即使使用完整的R1Bm元件，逆转录也从模板RNA的各个位点开始，主要是在它们的3‘端有5’-UG-3‘或5’-UG-3‘，它们可能与en酶切的28S rDNA靶序列5’-AGTAGATAGGGACA-3‘的3’端碱基配对。当将28S rDNA靶标的下游序列添加到R1单元的3‘端时，逆转录恰好从3’ utr的3'端开始，反转录转座效率提高。这些结果表明，模板RNA的3'端结构包括read-through区与其靶rDNA R1Bm序列相互作用，在体内TPRT初始过程中发挥重要作用。
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.