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• W2071660897 abstract "Long interspersed nuclear element 1 (LINE-1 or L1) is an interspersed repeated DNA found in mammalian genomes. L1 achieved its high copy number by retrotransposition, a process that requires the two L1-encoded proteins, ORF1p and ORF2p. The role of ORF1p in the retrotransposition cycle is incompletely understood, but it is known to bind single-stranded nucleic acids and act as a nucleic acid chaperone. This study assesses the nature and specificity of the interaction of ORF1p with RNA. Results of coimmunoprecipitation experiments demonstrate that ORF1p preferentially binds a single T1 nuclease digestion product of 38 nucleotides (nt) within the full-length mouse L1 transcript. The 38-nt fragment is localized within L1 RNA and found to be sufficient for binding by ORF1p but not necessary, because its complement is also efficiently coimmunoprecipitated, as are all sequences 38 nt or longer. Results of nitrocellulose filter-binding assays demonstrate that the binding of ORF1p to RNA does not require divalent cations but is sensitive to the concentration of monovalent cation. Both sense and antisense transcripts bind with apparent K Ds in the low nanomolar range. The results of both types of assay unambiguously support the conclusion that purified ORF1p from mouse L1 is a high-affinity, non-sequence-specific RNA binding protein. Long interspersed nuclear element 1 (LINE-1 or L1) is an interspersed repeated DNA found in mammalian genomes. L1 achieved its high copy number by retrotransposition, a process that requires the two L1-encoded proteins, ORF1p and ORF2p. The role of ORF1p in the retrotransposition cycle is incompletely understood, but it is known to bind single-stranded nucleic acids and act as a nucleic acid chaperone. This study assesses the nature and specificity of the interaction of ORF1p with RNA. Results of coimmunoprecipitation experiments demonstrate that ORF1p preferentially binds a single T1 nuclease digestion product of 38 nucleotides (nt) within the full-length mouse L1 transcript. The 38-nt fragment is localized within L1 RNA and found to be sufficient for binding by ORF1p but not necessary, because its complement is also efficiently coimmunoprecipitated, as are all sequences 38 nt or longer. Results of nitrocellulose filter-binding assays demonstrate that the binding of ORF1p to RNA does not require divalent cations but is sensitive to the concentration of monovalent cation. Both sense and antisense transcripts bind with apparent K Ds in the low nanomolar range. The results of both types of assay unambiguously support the conclusion that purified ORF1p from mouse L1 is a high-affinity, non-sequence-specific RNA binding protein. target DNA-primed reversed transcription open reading frame protein product of ORF1 ribonucleoprotein particle nucleotide(s) L1 is the most abundant long interspersed nuclear element (LINE) family in the mammalian genome. It belongs to one of the eleven ancient clades of non-long terminal repeat retrotransposons that are widely dispersed among eucaryotes (1Malik H.S. Burke W.D. Eickbush T.H. Mol. Biol. Evol. 1999; 16: 793-805Google Scholar) and propagate by a unique mechanism, target-DNA primed reverse transcription (TPRT)1 (2Luan D.D. Korman M.H. Jakubczak J.L. Eickbush T.H. Cell. 1993; 72: 595-605Google Scholar). Although there are more than 100,000 copies of L1 in the mouse genome, the majority of these are truncated, rearranged, or mutated and therefore incapable of further retrotransposition (reviewed in Ref. 3Hutchison III, C.A. Hardies S.C. Loeb D.D. Shehee W.R. Edgell M.H. Berg D.E. Howe M.M. Mobile DNA. American Society for Microbiology, Washington, D. C.1989: 593-617Google Scholar). Only about 3,000 of the present-day L1 elements are likely to be able to generate progeny (4Goodier J.L. Ostertag E.M. Du K. Kazazian H.H.J. Genome Res. 2001; 11: 1677-1685Google Scholar). Most of the transposition-competent mouse L1 elements belong to a subfamily known as TF, although elements belonging to the A and GF subfamilies are also active (4Goodier J.L. Ostertag E.M. Du K. Kazazian H.H.J. Genome Res. 2001; 11: 1677-1685Google Scholar, 5DeBerardinis R.J. Goodier J.L. Ostertag E.M. Kazazian Jr., H.H. Nat. Genet. 1998; 20: 288-290Google Scholar, 6Naas T.P. DeBerardinis R.J. Moran J.V. Ostertag E.M. Kingsmore S.F. Seldin M.F. Hayashizaki Y. Martin S.L. Kazazian Jr., H.H. EMBO J. 1998; 17: 590-597Google Scholar, 7Saxton J.A. Martin S.L. J. Mol. Biol. 1998; 280: 611-622Google Scholar, 8Hardies S.C. Wang L. Zhou L. Zhao Y. Casavant N.C. Huang S. Mol. Biol. Evol. 2000; 17: 616-628Google Scholar, 9Mears M.L. Hutchison III, C.A. J. Mol. Evol. 2001; 52: 51-62Google Scholar). Successful retrotransposition of L1 requires the proteins encoded by both of its two long open reading frames, ORF1 and ORF2 (5DeBerardinis R.J. Goodier J.L. Ostertag E.M. Kazazian Jr., H.H. Nat. Genet. 1998; 20: 288-290Google Scholar, 10Moran J.V. Holmes S.E. Naas T.P. DeBerardinis R.J. Boeke J.D. Kazazian Jr., H.H. Cell. 1996; 87: 917-927Google Scholar). The protein product of ORF2 (ORF2p) provides two enzymatic activities that are required for the TPRT reaction, endonuclease (11Feng Q. Moran J.V. Kazazian H.H.J. Boeke J.D. Cell. 1996; 87: 905-916Google Scholar) and reverse transcriptase (10Moran J.V. Holmes S.E. Naas T.P. DeBerardinis R.J. Boeke J.D. Kazazian Jr., H.H. Cell. 1996; 87: 917-927Google Scholar, 12Mathias S.L. Scott A.F. Kazazian Jr., H.H. Boeke J.D. Gabriel A. Science. 1991; 254: 1808-1810Google Scholar). The functions of the ORF1 protein product (ORF1p) in L1 retrotransposition are less clear but are likely to be structural rather than enzymatic (13Martin S.L. Mol. Cell. Biol. 1991; 11: 4804-4807Google Scholar, 14Martin S.L. Bushman F.D. Mol. Cell. Biol. 2001; 21: 467-475Google Scholar, 15Hohjoh H. Singer M.F. EMBO J. 1996; 15: 630-639Google Scholar, 16Hohjoh H. Singer M.F. EMBO J. 1997; 16: 6034-6043Google Scholar, 17Kolosha V.O. Martin S.L. Proc. Natl. Acad. Sci, U. S. A. 1997; 94: 10155-10160Google Scholar). Both proteins are requiredin cis (18Wei W. Gilbert N. Ooi S.L. Lawler J.F. Ostertag E.M. Kazazian H.H. Boeke J.D. Moran J.V. Mol. Cell. Biol. 2001; 21: 1429-1439Google Scholar). The ORF1p present in extracts of mouse embryonal carcinoma cells cofractionates with full-length L1 RNA as a ribonucleoprotein particle (RNP) (13Martin S.L. Mol. Cell. Biol. 1991; 11: 4804-4807Google Scholar). Mouse ORF1p, purified following expression inEscherichia coli, binds both single-stranded RNA and DNA (17Kolosha V.O. Martin S.L. Proc. Natl. Acad. Sci, U. S. A. 1997; 94: 10155-10160Google Scholar, 19Martin S.L. Li J. Weisz J.A. J. Mol. Biol. 2000; 304: 11-20Google Scholar) and functions as a nucleic acid chaperone in vitro(14Martin S.L. Bushman F.D. Mol. Cell. Biol. 2001; 21: 467-475Google Scholar). In addition, ORF1p forms multimers via a coiled-coil domain (17Kolosha V.O. Martin S.L. Proc. Natl. Acad. Sci, U. S. A. 1997; 94: 10155-10160Google Scholar,19Martin S.L. Li J. Weisz J.A. J. Mol. Biol. 2000; 304: 11-20Google Scholar). Both of these properties are likely to be important for RNP formation. ORF1p from human L1 shares the properties of protein-protein interaction and RNA binding with the mouse protein (15Hohjoh H. Singer M.F. EMBO J. 1996; 15: 630-639Google Scholar). In contrast to the mouse protein, however, the ORF1p enriched from human cell extracts appeared not to bind single-stranded DNA or random sequence RNA. Instead, results obtained using a coimmunoprecipitation assay indicate that the human L1 ORF1p binds preferentially to two regions of human L1 RNA (16Hohjoh H. Singer M.F. EMBO J. 1997; 16: 6034-6043Google Scholar). The experiments that led to these significantly different conclusions regarding the binding specificity of L1 ORF1p to RNA differed in several details, including sources of material, the methods of protein purification, and the biochemical assays utilized. Here we employ the coimmunoprecipitation assay of Hohjoh and Singer (16Hohjoh H. Singer M.F. EMBO J. 1997; 16: 6034-6043Google Scholar) and introduce a quantitative nitrocellulose filter binding assay to address the specificity of the interaction between mouse L1 ORF1p and RNA. Results obtained from both the coimmunoprecipitation assay and the filter binding assay confirm that the mouse ORF1 proteins from TFand A-type L1 elements bind to RNA with high-affinity in a sequence-independent manner. The non-sequence-specific binding of RNA to ORF1p has significant implications for the function(s) of this protein during L1 retrotransposition. The region encoding ORF1 was amplified by PCR using primers ORF1-start (5′-ATCCGAGCTCGATGGCGAAAGGCAAACG-3′) and ORF1-end (5′-GGGGAATTCGCTGTCTTCTTTTTGGTTTGTTGA-3′) with pVK15 (20Kolosha V.O. Martin S.L. J. Mol. Biol. 1995; 270: 2868-2873Google Scholar) and pTN201 (6Naas T.P. DeBerardinis R.J. Moran J.V. Ostertag E.M. Kingsmore S.F. Seldin M.F. Hayashizaki Y. Martin S.L. Kazazian Jr., H.H. EMBO J. 1998; 17: 590-597Google Scholar) as templates for the A-type and TF-type elements, respectively. The amplified fragments were digested withSacI and EcoRI and then cloned intoSacI-EcoRI digested pBlueBacHis2B (Invitrogen). These clones, pBacORF1A and pBacORFTF, were used to produce the A and TF forms of ORF1p after the infection of SF9 cells, respectively. pSV1A was constructed by PCR amplification of the L1Md-A2 element (21Loeb D.D. Padgett R.W. Hardies S.C. Shehee W.R. Comer M.B. Edgell M.H. Hutchison III, C.A. Mol. Cell. Biol. 1986; 6: 168-182Google Scholar) with primers 49 (5′-AGAACAGAATTCCAACTYTAACA-3′) and 5B (5′-CCTGTAAGCAGCAGAATG-3′). The product was cloned into pGEM-T-Easy (Promega). This plasmid was used as a template for in vitro transcription to produce transcripts F and G (Fig. 1). The double-stranded DNA oligonucleotide that corresponds to the 38-nt T1 nuclease product from L1 RNA (38 and c38 in 14) was cloned in both orientations into the blunted (T4 polymerase) ApaI site of pGEM-T-Easy after removing the vector's original EcoRI fragment. pJW5 contains the 38-nt fragment in the sense orientation but lost one nt in the upstream ApaI site relative to the antisense clone, pJW6. Digestion of these clones with NotI or NdeI produced templates for the transcription of the 76/77- or 110/111-nt MS and MA in Fig. 1. Note that the only sequence that differs between each sense and antisense pair is the region from L1; the appended vector sequences are constant. The DNA sequences of all cloned inserts were verified. Transcripts A–G and the 110/111- and 76/77-nt versions of MS and MA were generated by run-off in vitrotranscription using linearized plasmid DNA templates. Short transcripts, 43 nt each encompassing the 38-nt T1 nuclease-resistant fragment of mouse L1 (MS) and its complement (MC), were transcribed with T7 polymerase after annealing the T7 promoter primer (5′-TAATACGACTCACTATA-3′) to MS43 (5′-CATTGATATTAAGAGATATTAAGGAAAAGTAATTGTTGCTCCCTATAGTGAGTCGTATTA-3′) or MC (5′-GTAACTATAATTCTCTATAATTCCTTTTCATTAACAACCTCCCTATAG TGAGTCGTATTA-3′) oligonucleotides in transcription buffer. The 40-nt versions of the MS/MA region of L1 that were used for the filter binding assay were translated using fully double-stranded oligonucleotides as the transcription template to improve yields with T7 polymerase. MS40 (5′-TAATACGACTCACTATAGGCAACAATTACTTTTCCTTAATATCTCTTAACATCAATG-3′) and MA (5′-TAATACGACTCACTATAGGCATTGATGTTAAGAGATATTAAGGAAAAGTAATTGTTG-3′) were annealed to their perfect complements. The underlined region denotes the minimal T7 promoter plus two extra G residues to improve yields in the T7 transcription reaction (22Milligan J.F. Uhlenbeck O.C. Methods Enzymol. 1989; 180: 51-62Google Scholar). SP6 and T7 transcripts were synthesized in vitro using MEGAscript or MEGAshortscript transcription kits according to the manufacturer's recommendations (Ambion). Transcripts used in coimmunoprecipitation assays were synthesized with [32P]UTP and used after separating labeled transcripts from unincorporated nucleotides using Midi Select G25 columns (Eppendorf). For filter binding assays, in vitro transcripts were gel purified and then quantified using RiboGreen (Molecular Probes). 200 pmol were treated with calf alkaline phosphatase and end-labeled with [γ-32P]ATP using polynucleotide kinase. Unincorporated [32P]ATP was removed by gel filtration as above. Full-length ORF1p from mouse A-type and TF-type L1 elements were expressed in E. coli as GST-fusion proteins or in baculovirus-infected SF9 cells and then purified from the soluble fraction using non-denaturing conditions, as described elsewhere (14Martin S.L. Bushman F.D. Mol. Cell. Biol. 2001; 21: 467-475Google Scholar). Protein was incubated with32P-labeled in vitro transcribed RNA (100–350 ng) in 20–50 μl for 40–60 min at 30 °C in binding buffer (20 mm Hepes, pH 7.6, 100 mm NaCl, 4 mmMgCl2, 2 mm dithiothreitol, 5% glycerol, and 0.5 mm vanadium ribonucleotides). Some experiments used tRNA (100 ng/μl) carrier, although this reduced the efficiency of coimmunoprecipitation because tRNA competes for the ORF1 protein binding to [32P]RNA (17Kolosha V.O. Martin S.L. Proc. Natl. Acad. Sci, U. S. A. 1997; 94: 10155-10160Google Scholar). 100 units of T1 nuclease were added to the reaction mixture, and the incubation continued for 45 min at room temperature. The reaction mixture was diluted 10-fold with RIPB (20 mm Tris-HCl, pH 8.0, 100 mm NaCl, 1 mm EDTA, 0.2% Nonidet P-40, 0.3–0.5 mm vanadium ribonucleotide), then ∼4 μg of either ORF1 antibody or preimmune IgG were added, and the incubation was continued overnight at 4 °C. Protein A-Sepharose beads (Sigma) were added, and the incubation continued for an additional 2–3 h at 4 °C with gentle mixing. The beads were recovered by centrifugation at 2,000 × g for 3 min at 4 °C and then washed twice with phosphate-buffered saline containing 0.05% of Nonidet P-40, and twice in phosphate-buffered saline. Finally, immunoprecipitated [32P]RNA was removed from the pelleted beads by incubating in 100 μl of phosphate-buffered saline, 0.5% SDS, and 25 μg of proteinase K for 30 min at 37 °C, extracted with phenol-chloroform, then precipitated with ethanol in the presence of 10 μg of carrier yeast tRNA. Material in this pellet was separated on 7% polyacrylamide (20:1) sequencing gels (23Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989: 13.45-13.58Google Scholar) and analyzed by autoradiography. Increasing amounts of ORF1p were incubated with the indicated amounts of in vitrotranscribed 32P-labeled RNA for 30 min on ice in 80 μl of binding buffer (20 mm Hepes 7.5, 100 mm NaCl, 1 mm dithiothreitol, 5 mm EDTA, and 100 μg/ml bovine serum albumin). 25 μl of the binding reactions were filtered in triplicate (Invitrogen) through nitrocellulose and DE81 as described (24Wong I. Lohman T.M. Proc. Natl. Acad. Sci, U. S. A. 1993; 90: 5428-5432Google Scholar), and then washed with 100 μl of ice-cold binding buffer without dithiothreitol and bovine serum albumin. The 32P retained on nitrocellulose and DE81 was quantified by phosphorimaging analysis using ImageQuant software (Amersham Biosciences). Binding constants were calculated from graphs of protein concentrationversus fraction bound (nitrocellulose/DE81+nitrocellulose)(y) by fitting the equation y = ([Pf]/K Dapp)/(1+[Pf]/K Dapp) where [Pfree] ≅ [Ptotal] using KaleidaGraph software. Control experiments with each RNA determined that the amounts of32P retained by the nitrocellulose, and the DE81 did not change with 0, 1, 2, or 3 washes. In addition, no RNA was retained by the nitrocellulose when protein was first applied to the filters followed by transcript without allowing binding to occur in solution prior to filtration. Full-length, His-tagged ORF1p from both A-type and TF-type mouse L1 (Fig. 1) was expressed in baculovirus-infected SF9 cells. ORF1p was purified using non-denaturing conditions (Fig. 2) to eliminate the possibility that the nonspecific ORF1p-RNA interaction described previously (17Kolosha V.O. Martin S.L. Proc. Natl. Acad. Sci, U. S. A. 1997; 94: 10155-10160Google Scholar) was a consequence of misfolding of the protein upon renaturation out of urea. ORF1p was also purified from the soluble fraction of E. coli extracts as described (14Martin S.L. Bushman F.D. Mol. Cell. Biol. 2001; 21: 467-475Google Scholar). Mouse L1 ORF1p was tested for its ability to immunoisolate specific regions of mouse L1 RNA, as was demonstrated for human L1 ORF1p (16Hohjoh H. Singer M.F. EMBO J. 1997; 16: 6034-6043Google Scholar). Initially, nearly full-length sense-strand L1 RNA (transcript A, Fig. 1) was bound to our original preparation of A-type ORF1p from E. coli(17Kolosha V.O. Martin S.L. Proc. Natl. Acad. Sci, U. S. A. 1997; 94: 10155-10160Google Scholar, 25Martin S.L. Branciforte D. Mol. Cell. Biol. 1993; 13: 5383-5392Google Scholar). Following T1 nuclease treatment of the reaction, a 38-nt fragment was selectively coimmunoprecipitated with rabbit anti-mouse ORF1p antibody but not with the preimmune control (Fig.3 A, lanes 1 and2). A similar result was obtained using baculovirus-expressed ORF1p (A-type) and transcript B (Fig.3 A, lanes 3 and 4); therefore, all further coimmunoprecipitation experiments used ORF1p purified from baculovirus-infected SF9 cells. Inspection of the nucleotide sequence of transcripts A and B revealed that their largest T1 fragment is 38 nt and that it resides 33 nt downstream of the initiation codon for ORF2. This region of the mouse L1 sequence is orthologous to the larger of the two T1 fragments (41 nt) in human L1 RNA that is coimmunoprecipitated by human L1 ORF1p (16Hohjoh H. Singer M.F. EMBO J. 1997; 16: 6034-6043Google Scholar). To confirm that this is the region of the long L1 transcript that is actually bound to ORF1p and coimmunoprecipitated with ORF1 antibody, transcripts C (Fig.3 B, lanes 1 and 2), D (not shown), and E (Fig. 4 A) were tested with the same protocol. Following T1 digestion and immunoprecipitation, the 38-nt fragment was isolated from all of these transcripts. This 38-nt fragment was also efficiently coimmunoprecipitated when the labeled L1 transcript was first treated with T1 nuclease and then incubated with ORF1p and antibody (Fig. 3 B, lanes 3 and4), indicating that the 38-nt region contained all of the sequence or structure needed for ORF1p binding. This prediction was confirmed by coimmunoprecipitation of a 43-nt transcript that contained the 38-nt region from mouse L1 downstream of a minimal T7 promoter (Fig. 4 B). From the results of these coimmunoprecipitation experiments, it appears that the 38-nt T1 fragment from mouse L1 RNA is sufficient to bind ORF1p. However, in addition to isolating the 38- and 43-nt transcripts in Fig.4, we noticed that any larger transcripts present in the RNA population were also coimmunoprecipitated, whereas smaller transcripts were reduced or eliminated, especially those shorter than ∼30 nt. Because the 38-nt sequence is likely to have been included in all of the precipitated RNAs, these data do not address whether the sequences present in the 38-nt T1 fragment are necessary for interaction between RNA and ORF1p.Figure 4Efficient coimmunoprecipation of RNAs that are at least 38 nt. Lanes on these autoradiograms contain the following. A, transcript E digested with T1 nuclease (−), transcript E after binding baculovirus A-type ORF1p followed by T1 digestion and immunoprecipitation with preimmune IgG (P), or ORF1 antibody (I). B, the total in vitro transcription reaction from the MS43 oligonucleotide template (−) or transcripts immunoprecipitated from this transcription reaction by preimmune IgG (P) or ORF1 antibody (I) after incubation with ORF1p. Ab, antibody.View Large Image Figure ViewerDownload (PPT) To test whether the 38-nt region is required for the interaction between ORF1p and RNA that allows coimmunoprecipitation, a 160-nt fragment encompassing this region from mouse L1 was transcribed to generate the sense and the antisense transcripts F and G, respectively. Both of these transcripts are efficiently coimmunoprecipitated (Fig.5 A). Similar results were obtained using the short transcripts with the 38-nt region, MS and MC (Fig. 5 B). Because neither the antisense nor the complementary transcripts of the 38-nt region contain the same sequence as the sense strand, and both were coimmunoprecipitated after binding to ORF1p, the specific sequence present in the sense strand must not be necessary for ORF1p to form a stable interaction with RNA. The availability of highly purified ORF1p makes it possible to quantitatively assess its interactions with RNA. For the remaining studies of the interactions between ORF1p and RNA, we employed a sensitive nitrocellulose filter binding assay (24Wong I. Lohman T.M. Proc. Natl. Acad. Sci, U. S. A. 1993; 90: 5428-5432Google Scholar). Initial assays that measured binding to transcript F revealed no significant differences between A-type and TF-type ORF1p made in either baculovirus or E. coli (data not shown). Therefore, the remainder of these experiments were done using the TF-type ORF1p from the spastic allele of mouse L1 that was expressed and purified from baculovirus, because spa is known to be retrotransposition competent (26Kingsmore S.F. Giros B. Suh D. Bieniarz M. Caron M.G. Seldin M.F. Nat. Genet. 1994; 7: 136-142Google Scholar, 27Mullhard C. Fischer M. Gass P. Simon-Chazottes D. Guenet J.-L. Becker C.-M. Neuron. 1994; 13: 1003-1015Google Scholar). The binding affinity of ORF1p to RNA was unaffected by EDTA concentrations between 0 and 50 mm (data not shown), indicating that magnesium is not required. The nucleic acid binding domain of ORF1p is highly basic (17Kolosha V.O. Martin S.L. Proc. Natl. Acad. Sci, U. S. A. 1997; 94: 10155-10160Google Scholar, 19Martin S.L. Li J. Weisz J.A. J. Mol. Biol. 2000; 304: 11-20Google Scholar), which suggests a role for electrostatic interactions in the binding of ORF1p to RNA. The effect of ionic strength on the affinity of ORF1p for the 110- and 40-nt sense RNA transcripts was determined by varying the concentration of NaCl in the binding reactions. As expected, the apparent affinity of ORF1p for RNA declined as the concentration of NaCl increased. This effect was relatively small at NaCl concentrations below 350 mm but increased significantly at concentrations above 350 mm (Fig. 6). Because neither slope in Fig. 6 extrapolates to zero affinity at 1m NaCl, the binding of ORF1p to RNA must involve both electrostatic interactions and other types of molecular contacts (28Record M.T.J. Anderson C.F. Lohman T.M. Q. Rev. Biophys. 1978; 11: 103-178Google Scholar). The sequence requirements for the interaction between RNA and ORF1p were further examined using the nitrocellulose filter binding assay. In 100 mm NaCl with 100 pm RNA, the apparent binding affinity (K Dapp) of ORF1p for the 110/111-nt and 76/77-nt sense/antisense pairs of RNA varied between 1.3 and 3.6 nm (Table I). This result demonstrates that ORF1p binds RNA with high affinity in a sequence-independent manner. However, because the apparent affinity is so high relative to the concentration of RNA, it is possible that a higher affinity binding site could be masked in these conditions. To address this concern, binding studies were repeated with the 110/111-nt and 76/77-nt sense/antisense pairs, lowering the concentration of RNA to 10 pm and increasing the concentration of NaCl. Using 10 pm RNA and 250 mm NaCl, theK Dapp varied between 0.7 and 4.6 nmfor all four transcripts tested. In these conditions, the apparent affinities for the sense transcripts were 3–7-fold higher than those for the antisense transcripts (Table II). Finally, the 40-nt transcripts were tested using 10 and 40 pm RNA in 410 mm NaCl. As expected from the results of Fig. 6, the affinity of ORF1p for RNA is significantly reduced in this concentration of NaCl. Again little difference was observed between the affinities for sense versusantisense transcript (∼2-fold), and no differences were measured between 10 and 40 pm RNA (Fig.7). Taken together, these results demonstrate high-affinity, non-sequence-specific binding of L1 ORF1p to RNA.Table IKDapp (m) for L1 ORF1p binding to 100 pm RNA in 100 mm NaClLengthSenseAntisense110/1111.3 ± 0.1 × 10−93.6 ± 0.2 × 10−976/773.3 ± 0.4 × 10−92.5 ± 0.3 × 10−9 Open table in a new tab Table IIKDapp (m) for L1 ORF1p binding to 10 pm RNA in 250 mm NaClLengthSenseAntisense110/1116.6 ± 0.4 × 10−104.6 ± 0.6 × 10−976/771.2 ± 0.2 × 10−93.2 ± 0.4 × 10−9 Open table in a new tab In this study, coimmunoprecipitation and nitrocellulose filter binding assays were used to examine the interactions of mouse L1 ORF1p with several in vitro transcribed RNAs. ORF1 proteins from two subtypes of mouse L1 were purified using non-denaturing conditions after expression in baculovirus-infected insect cells. Results obtained from both coimmunoprecipitation and nitrocellulose filter binding assays indicate there are no significant differences between the binding interactions of A-type and TF-type mouse ORF1p with RNA. This is not wholly unexpected, given that the ORF1ps encoded by the A and TF elements used here are 87% identical and 92% similar. A much larger number of A-type than TF-type elements are, however, inactive for transposition (5DeBerardinis R.J. Goodier J.L. Ostertag E.M. Kazazian Jr., H.H. Nat. Genet. 1998; 20: 288-290Google Scholar). Our data indicate that the relative inactivity of A-type elements is not caused by loss of the RNA-binding activity of ORF1p. The sequence of mouse L1 ORF1p lacks any immediately obvious previously identified RNA binding motifs that could elucidate its mode of interaction with RNA. Binding of ORF1p to RNA is unaffected by the presence of EDTA and thus must not require divalent cations. Electrostatic interaction is likely to play a significant role in the interaction between ORF1p and RNA based upon the highly basic nature of the protein. Our experiments have examined the sensitivity of the RNA-protein interaction to increasing concentrations of monovalent cation, and the results demonstrate that binding is dramatically reduced in the presence of high concentrations of NaCl. Consistent with this observation, 500 mm NaCl was used to disrupt L1RNPs from human teratocarcinoma cell extracts in order to isolate the ORF1p (p40) (15,16). We also added 500 mm NaCl to the baculovirus extracts and during binding to the nickel-agarose beads to remove the RNA that would otherwise copurify with the protein (19Martin S.L. Li J. Weisz J.A. J. Mol. Biol. 2000; 304: 11-20Google Scholar). The molecular event(s) responsible for the different slopes of the lines that represent the relationship between binding affinity and salt concentration (Fig. 6) above and below 350 mm NaCl is unknown. High salt concentrations may affect the binding of ORF1p to RNA directly or indirectly, e.g. by changing the conformation of the protein and/or the RNA. The results of this study further demonstrate that mouse ORF1p binds RNA with low nanomolar affinity and little regard for nucleotide sequence in physiological concentrations of monovalent cation. Although reproducible differences were observed between theK Dapps for sense/antisense pairs, these differences are within the range (<10-fold) typical of non-sequence-specific nucleic acid binding proteins (29McGhee J.D. von Hippel P.H. J. Mol. Biol. 1974; 86: 469-489Google Scholar). The results obtained in this study are consistent with our previous findings, which demonstrated sequence-independent binding, not only to RNA but also to single-stranded DNA (17Kolosha V.O. Martin S.L. Proc. Natl. Acad. Sci, U. S. A. 1997; 94: 10155-10160Google Scholar). The apparent affinity of ORF1p for RNA is significantly greater in these experiments, however, as compared with the earlier experiments (17Kolosha V.O. Martin S.L. Proc. Natl. Acad. Sci, U. S. A. 1997; 94: 10155-10160Google Scholar). This may be because a much greater proportion of ORF1p is active following isolation under native conditions from baculovirus-infected insect cells than it was after purification using denaturing conditions from E. coliinclusion bodies. Further support that ORF1p binds non-sequence specifically to RNA with high affinity comes from the observation that ORF1p was bound to RNA after purification from E. coli using non-denaturing conditions in 150 mm NaCl. This is true not only for full-length ORF1p but also for the C-terminal, nucleic acid binding domain expressed separately (19Martin S.L. Li J. Weisz J.A. J. Mol. Biol. 2000; 304: 11-20Google Scholar). Neither of these constructs contained the 38-nt region of L1 that coimmunoprecipitates with ORF1p. Finally, non-sequence-specific single-strand binding is consistent with the nucleic acid chaperone activity of L1 ORF1p (14Martin S.L. Bushman F.D. Mol. Cell. Biol. 2001; 21: 467-475Google Scholar). Hohjoh and Singer (16Hohjoh H. Singer M.F. EMBO J. 1997; 16: 6034-6043Google Scholar) observed stable interactions of human ORF1p with two T1 nuclease fragments contained within human L1 RNA. Binding to other RNAs or DNA was not detected using a coimmunoprecipitation assay. In this study of mouse L1 ORF1p, we observed that a single T1 nuclease fragment interacted with ORF1p by coimmunoprecipitation. Similar to the results obtained with human L1, this region of the mouse L1 transcript was found to be sufficient for binding to ORF1p. In contrast to the findings with human L1, however, this region was not necessary for stable interaction between mouse ORF1p and RNA, because transcripts lacking this sequence also coimmunoprecipitated with ORF1p and were bound in the filter binding assay. The differences in results obtained between human and mouse L1 may be due to either bona fidespecies-specific differences or to unknown experimental differences. The C-terminal sequences of ORF1p from mouse and human L1 are homologous. Sequence similarity breaks down toward their N termini, however, such that the N-terminal one-third or so of these two proteins is probably not related by descent (17Kolosha V.O. Martin S.L. Proc. Natl. Acad. Sci, U. S. A. 1997; 94: 10155-10160Google Scholar, 19Martin S.L. Li J. Weisz J.A. J. Mol. Biol. 2000; 304: 11-20Google Scholar, 30Demers G.W. Matunis M.J. Hardison R.C. J. Mol. Evol. 1989; 29: 3-19Google Scholar). Nevertheless, autonomous retrotransposition of marked elements absolutely requires ORF1p for both mouse and human L1 (5DeBerardinis R.J. Goodier J.L. Ostertag E.M. Kazazian Jr., H.H. Nat. Genet. 1998; 20: 288-290Google Scholar, 10Moran J.V. Holmes S.E. Naas T.P. DeBerardinis R.J. Boeke J.D. Kazazian Jr., H.H. Cell. 1996; 87: 917-927Google Scholar). Thus, it seems highly unlikely that human ORF1p binding to RNA is only sequence-specific, whereas mouse ORF1p binding is only non-sequence-specific. Another explanation of our results is that the specific binding of ORF1p to a region of L1 RNA requires an accessory cofactor that is present in the human cell extract and co-purifies with ORF1p, perhaps because it is bound to it. Such a cofactor would not be expected to be present in the mouse ORF1p that was purified following expression in bacteria or baculovirus-infected insect cells. This explanation is particularly intriguing because there is persuasive evidence for positive selection acting upon the sequences that form the protein-protein interaction domain of ORF1p; there could be selective pressure operating on ORF1p to evade or attract additional proteins that modulate retrotransposition activity (31Boissinot S. Furano A.V. Mol. Biol. Evol. 2001; 18: 2186-2194Google Scholar). Given the location of the 41-nt high affinity site in human L1, which is also conserved in mouse L1 and lies just downstream of the translational initiation codon of ORF2, it is tempting to speculate that regulated binding to this site could affect the translation of ORF2 and hence retrotransposition. Finally, it remains possible that the small but significant differences in the binding affinity of ORF1p for RNA that we do measure may place any given RNA above or below a threshold for coimmunoprecipitation. Such a threshold could differ either directly because of ORF1p or its antibody or indirectly, e.g. if the binding of ORF1p to RNA is modulated by an event like phosphorylation. Modification of ORF1p may have occurred in the complex protein mixture containing human ORF1p that was used for the experiments of Hohjoh and Singer (16Hohjoh H. Singer M.F. EMBO J. 1997; 16: 6034-6043Google Scholar) but not in the relatively highly purified preparations of ORF1p from the heterologous expression systems used here. Additional studies will be required to distinguish among these possibilities, but data indicate that mutations of the 41-nt sequence in human L1 that preserve the open reading frame remain retrotransposition competent in the autonomous retrotransposition assay. 2S.-L. Ooi, G. Cost, and J. D. Boeke, personal communication. This implies that if there is a modulated high-affinity interaction between ORF1 and RNA at the site near the initiation codon of ORF2, it involves a negative regulator that is not present in the cell types used for the autonomous retrotransposition assay. The results of these coimmunoprecipitation and nitrocellulose filter binding assays, taken together with earlier results (17Kolosha V.O. Martin S.L. Proc. Natl. Acad. Sci, U. S. A. 1997; 94: 10155-10160Google Scholar, 19Martin S.L. Li J. Weisz J.A. J. Mol. Biol. 2000; 304: 11-20Google Scholar), firmly establish that ORF1p from mouse L1 is a high affinity, non-sequence-specific RNA binding protein. This feature is likely to be essential for L1 retrotransposition not only in producing the cytoplasmic RNP but also in facilitating the dynamic molecular interactions that are an essential component of the TPRT reaction. We thank M. Churchill, D. Bain, K. Polach, C. Diges, and F. van Breukelen for help and advice, J. Weisz for pJW5, pJW6, and preliminary filter binding assays, J. Li for pSV1A, D. Branciforte for highly purified ORF1p, and members of the laboratory for comments on the manuscript." @default.
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• W2071660897 title "High-affinity, Non-sequence-specific RNA Binding by the Open Reading Frame 1 (ORF1) Protein from Long Interspersed Nuclear Element 1 (LINE-1)" @default.
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• W2071660897 cites W1983856027 @default.
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• W2071660897 cites W1989651677 @default.
• W2071660897 cites W1991333931 @default.
• W2071660897 cites W2001806616 @default.
• W2071660897 cites W2007589425 @default.
• W2071660897 cites W2024364777 @default.
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• W2071660897 cites W2027361898 @default.
• W2071660897 cites W2033178325 @default.
• W2071660897 cites W2034880487 @default.
• W2071660897 cites W2047360265 @default.
• W2071660897 cites W2063830103 @default.
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• W2071660897 cites W2118734051 @default.
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• W2071660897 cites W2147521158 @default.
• W2071660897 cites W2163157311 @default.
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