Title: Self-Interaction, Nucleic Acid Binding, and Nucleic Acid

80 Pages·2011·0.85 MB·English

MCB Accepts, published online ahead of print on 21 November 2011
Mol. Cell. Biol. doi:10.1128/MCB.06162-11
Copyright © 2011, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.
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2 Self-Interaction, Nucleic Acid Binding, and Nucleic Acid Chaperone Activities Are D
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3 Unexpectedly Retained in the Unique ORF1p of Zebrafish LINE d
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5 Running title: Conserved Activities in ZfL2-1 ORF1p b
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7 Mitsuhiro Nakamura, Masaki Kajikawa*, and Norihiro Okada* u
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9 Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, g
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10 4259-B-21 Nagatsuta-cho, Midori-ku, Yokohama, Kanagawa 226-8501, Japan
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12 *Corresponding authors. M Kajikawa and N Okada, Graduate School of Bioscience and
13 Biotechnology, Tokyo Institute of Technology, 4259-B-21 Nagatsuta-cho, Midori-ku, D
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14 Yokohama, Kanagawa 226-8501, Japan. Tel.: [phone]; Fax: [phone]; d
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15 E-mails: [email protected] and [email protected] h
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16 b
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17 Materials and Methods; 1710 words g
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18 The Combined word count for the introduction, Results, and Discussion; 4648 words u
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20 ABSTRUCT
21 Long interspersed elements (LINEs) are mobile elements that comprise a large D
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22 proportion of many eukaryotic genomes. Although some LINE-encoded ORF1 proteins d
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23 (ORF1ps) were suggested to be required for LINE mobilization through binding to their h
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24 RNA, their general role is not known. The ZfL2-1 ORF1p, which belongs to the b
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25 esterase-type ORF1p, is especially interesting because it has no known RNA-binding g
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26 domain. Here we demonstrate that ZfL2-1 ORF1p has all the canonical activities associated u
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27 with known ORF1ps including self-interaction, nucleic acid binding, and nucleic acid 2
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28 chaperone activities. In particular, we showed that its chaperone activity is reversible, g
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29 suggesting that the chaperone activities of many other ORF1ps are also reversible. From
30 this discovery, we propose that LINE ORF1ps play a general role in LINE integration by
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31 forming a complex with LINE RNA and rearranging its conformation. (133 words)
32 D
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33 INTRODUCTION
34 Long interspersed elements (LINEs), or non-long terminal repeat (non-LTR) D
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35 retrotransposons, are transposable elements that comprise a large proportion of many d
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36 eukaryotic genomes. Mobilization or amplification of LINEs causes various alterations in h
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37 their host genomes, thus having profound effects on eukaryotic genome evolution (7, 12). b
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38 LINEs mobilize by a mechanism called retrotransposition. In LINE retrotransposition, a g
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39 LINE-encoded endonuclease (EN) nicks a target site of the host genomic DNA by which a u
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40 3′ hydroxyl group is generated. The 3′ hydroxyl group is then used as a primer from which 2
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41 a LINE-encoded reverse transcriptase (RT) initiates reverse transcription of the LINE RNA. g
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42 This reaction, which is called target-primed reverse transcription (TPRT), is characteristic
43 of LINE retrotransposition (1, 16). After TPRT, the newly synthesized LINE DNA is
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44 integrated into the genomic DNA with the help of the host DNA repair system(s), although
45 the mechanism of this integration is not well understood (32). D
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46 LINEs are divided into 12 or more clades based on phylogenetic analysis of the RTs d
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47 they encode (15, 18). The clades are classified into two major groups that differ in structure. h
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48 One group encodes a single multidomain protein that is responsible for TPRT and contains b
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49 a restriction-like endonuclease (RLE) and an RT domain. LINEs of this group are all g
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50 integrated into a specific site of the host genome DNA defined by their RLE (4). The other u
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51 group encodes two proteins, called open reading frame 1 and 2 proteins (ORF1p and 2
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52 ORF2p). ORF2p, which is responsible for TPRT, contains an apurinic/apyrimidinic g
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53 endonuclease (APE) and an RT domain (6). APE-type LINEs are usually dispersed in the
54 host genomes because most APEs do not have strict specificity for their target sequence.
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55 ORF1p is also required for retrotransposition, although its role is not well understood (24,
56 29, 33). D
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57 ORF1ps in APE-type LINEs do not contain any domains conserved in common, d
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58 and their amino acid sequences are frequently quite different among LINEs of different h
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59 clades and even among those classified into one clade. This contrasts with the fact that the b
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60 APE and RT domains of ORF2p are well conserved among APE-type LINEs. Research g
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61 regarding the function of ORF1p is predominantly conducted using ORF1p encoded by the u
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62 mammalian LINE, L1, which is classified into the L1 clade. L1 ORF1p is composed of 2
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63 three distinct structural domains, an N-terminal coiled-coil (CC), a middle noncanonical g
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64 RNA-recognition-motif (RRM), and a C-terminal domain (CTD) (10, 13, 22) (Fig. 1A).
65 Biochemical analyses showed that L1 ORF1p forms a trimer via the CC domain and
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66 requires the RRM and CTD for nucleic acid binding (13, 19, 22). Consistent with these
67 findings, L1 ORF1p forms a large ribonucleoprotein particle (RNP) with L1 RNA in D
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68 cultured cells (3, 14). These data suggest that L1 ORF1p is necessary for RNP formation, d
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69 although the role of the RNP in retrotransposition remains unclear. The RRM domain is h
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70 present in many other ORF1ps encoded by LINEs of various clades, suggesting that ORF1p b
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71 having the RRM domain has a common feature for RNP formation (13). Consistent with g
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72 this notion, the ORF1p encoded by the fruit fly LINE, I factor (which encodes the RRM u
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73 domain in the I clade), forms a multimer and binds nucleic acids in vitro, and the ORF1p 2
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74 encoded by the silk worm LINE, SART1 (which encodes the RRM domain in the R1 clade), g
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75 forms an RNP in cultured cells (2, 23) (Fig. 1A). Besides the RRM domain, intriguingly, a
76 zinc knuckle motif at the C-terminus of SART1 ORF1p is essential for packaging of the
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77 SART1 RNA into the RNP (23). Zinc knuckles are also found in many other ORF1ps in
78 which they are frequently located downstream of the RRM, indicating that the zinc knuckle D
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79 and RRM may cooperatively bind LINE RNA. d
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80 Nucleic acid chaperones are defined as proteins that catalyze rearrangement of h
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81 nucleic acids into a conformation that has the maximum number of base pairs (15). Nucleic b
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82 acid chaperones can catalyze two reactions in vitro: nucleic acid annealing and strand g
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83 exchange. Nucleic acid annealing is the reaction in which two single-stranded nucleic acids u
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84 complementary to each other are annealed, whereas strand exchange is the reaction in 2
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85 which the replacement of strands occurs between a double-stranded nucleic acid and a g
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86 single-stranded nucleic acid. Recently, nucleic acid chaperones that can catalyze only one
87 of these reactions were found, suggesting that their catalytic mechanism can be dissected
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88 into at least two distinct manners (28). However, their molecular basis is not well
89 understood. The mouse L1 ORF1p catalyzes both reactions, and mutations abolishing its D
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90 nucleic acid chaperone activity dramatically decrease the frequency of L1 retrotransposition d
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91 (20, 21). This indicates that the nucleic acid chaperone activity is required for L1 h
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92 retrotransposition, although its precise role has not been elucidated (21). It has not been b
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93 determined, however, whether the ORF1ps other than the one encoded by L1 have nucleic g
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94 acid chaperone activity, except for the I factor–encoded ORF1p, which catalyzes nucleic u
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95 acid annealing but has not been tested for strand exchange (2). 2
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96 We previously isolated two retrotransposition-competent LINEs from the zebrafish g
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97 genome, one of which is ZfL2-1 (31). ZfL2-1 is an APE-type LINE and belongs to the L2
98 clade. ORF1ps encoded by the L2-clade LINEs can be divided into at least three types
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