From the Department of Cellular and Structural Biology and Program in Molecular Biology, University of Colorado School of Medicine, Denver, Colorado 80262
Received for publication, October 13, 2002, and in revised form, December 26, 2002
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ABSTRACT |
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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 KDs 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.
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 (1) and propagate by a unique mechanism,
target-DNA primed reverse transcription (TPRT)1 (2). 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. 3). Only about 3,000 of
the present-day L1 elements are likely to be able to generate progeny
(4). 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 (4-9).
Successful retrotransposition of L1 requires the proteins encoded by
both of its two long open reading frames, ORF1 and ORF2 (5, 10). The
protein product of ORF2 (ORF2p) provides two enzymatic activities that
are required for the TPRT reaction, endonuclease (11) and reverse
transcriptase (10, 12). The functions of the ORF1 protein product
(ORF1p) in L1 retrotransposition are less clear but are likely to be
structural rather than enzymatic (13-17). Both proteins are required
in cis (18).
The ORF1p present in extracts of mouse embryonal carcinoma cells
cofractionates with full-length L1 RNA as a ribonucleoprotein particle
(RNP) (13). Mouse ORF1p, purified following expression in
Escherichia coli, binds both single-stranded RNA and DNA
(17, 19) and functions as a nucleic acid chaperone in vitro
(14). In addition, ORF1p forms multimers via a coiled-coil domain (17, 19). 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 (15). 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 (16).
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 (16) 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 TF
and 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.
DNA Constructs--
The region encoding ORF1 was amplified by
PCR using primers ORF1-start (5'-ATCCGAGCTCGATGGCGAAAGGCAAACG-3')
and ORF1-end (5'-GGGGAATTCGCTGTCTTCTTTTTGGTTTGTTGA-3') with pVK15 (20)
and pTN201 (6) as templates for the A-type and TF-type
elements, respectively. The amplified fragments were digested with
SacI and EcoRI and then cloned into
SacI-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 (21) 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--
Transcripts A-G and the 110/111- and 76/77-nt
versions of MS and MA were generated by run-off in vitro
transcription 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 (22).
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 [ Proteins--
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 (14).
Coimmunoprecipitation--
Protein was incubated with
32P-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 mM
MgCl2, 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 (17). 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 (23) and analyzed by autoradiography.
Nitrocellulose Filter Binding Assay--
Increasing amounts of
ORF1p were incubated with the indicated amounts of in vitro
transcribed 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
(24), 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 concentration versus fraction bound
(nitrocellulose/DE81+nitrocellulose)(y) by fitting the equation y = ([Pf]/KDapp)/(1+[Pf]/KDapp)
where [Pfree]
Control experiments with each RNA determined that the amounts of
32P 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 (17) 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 (14).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP using polynucleotide
kinase. Unincorporated [32P]ATP was removed by gel
filtration as above.
[Ptotal] using
KaleidaGraph software.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Schematic of mouse L1 and location of
transcripts. The canonical mouse L1 structure is shown; the 4-2/3
boxes at the extreme 5'-end represent monomer repeats. These can be
either A or TF type and function to provide a promoter for
transcription (32, 33). The 5'- and 3'-non-coding regions flanking ORF1
and ORF2 are indicated by horizontal lines;
An denotes the poly(A) tail. The two
bars above ORF1 depict TF type and A-type ORF1p,
with the 36-amino acid His tag fusion represented by white
boxes on the N terminus and the 14-amino acid insertion in
TF relative to A2 represented as a box above
TF ORF1p to show their location and extent. The transcripts
used for these studies are mapped below, with the direction of
transcription is indicated by the arrowhead. Transcripts
A-E are A-type, and transcripts F and G are TF-type.
Transcripts A (6323 nt) and B (5339 nt) were described in Kolosha and
Martin (17). Transcripts C-E were derived from digestion of SH8 (17)
with SspI (754 nt), TaqI (342 nt), and
AvaII (199nt), respectively. F and G were transcribed from
pSV1A with either T7 polymerase linearized with SpeI or SP6
polymerase linearized with SacII, generating transcripts of
219 and 250 nt, respectively. MS and
MC/MA indicate the position of the 38-nt T1
fragment, which is also the only L1 sequence present in the 43-nt
transcripts MS and MC that were used for the coimmunoprecipitation
experiments and the 110/111-, 75/76-, and 40-nt sense and antisense
transcripts used in the filter binding assays.
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[in a new window]
Fig. 2.
Coomassie-stained SDS-PAGE of
baculovirus-expressed proteins used for this study.
Lanes contain 3 µg of A-type and TF- type
protein after elution from the nickel-agarose column and prestained
molecular weight marker (M). In addition to amino acid
substitutions, TF differs from A-type ORF1p by the
insertion of 14 amino acids (1724 Da); the N-terminal fusion with the
His-tag adds 4029 Da to both proteins.
Coimmunoprecipitation of RNA after Binding to ORF1p--
Mouse L1
ORF1p was tested for its ability to immunoisolate specific regions of
mouse L1 RNA, as was demonstrated for human L1 ORF1p (16). 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
(17, 25). 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.
3A, lanes 1 and
2). A similar result was obtained using
baculovirus-expressed ORF1p (A-type) and transcript B (Fig. 3A, 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 (16). 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.
3B, lanes 1 and 2), D (not shown), and
E (Fig. 4A) 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. 3B, lanes 3 and
4), 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. 4B). 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.
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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.
5A). Similar results were
obtained using the short transcripts with the 38-nt region, MS and MC
(Fig. 5B). 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.
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High-affinity, Non-sequence-specific ORF1p-RNA Interactions by Filter Binding Assay-- 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 (24). 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 (26, 27).
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 (17, 19), 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 1 M NaCl, the binding of ORF1p to RNA must involve both
electrostatic interactions and other types of molecular contacts
(28).
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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 (KDapp) 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, the KDapp varied between 0.7 and 4.6 nM for 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 versus antisense 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.
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DISCUSSION |
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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 (5). 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 (19). 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 the KDapps for sense/antisense pairs, these differences are within the range (<10-fold) typical of non-sequence-specific nucleic acid binding proteins (29). 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 (17). The apparent affinity of ORF1p for RNA is significantly greater in these experiments, however, as compared with the earlier experiments (17). 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. coli inclusion 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 (19). 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 (14).
Hohjoh and Singer (16) 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 fide species-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 (17, 19, 30). Nevertheless, autonomous retrotransposition of marked elements absolutely requires ORF1p for both mouse and human L1 (5, 10). 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 (31). 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 (16) 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.2 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 (17, 19), 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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants GM40367 (to S. L. M.) and CA46934 (to the University of Colorado Cancer Center for DNA Sequencing and Tissue Culture Cores).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
This work is dedicated to the memory of Vladimir O. Kolosha, who
died on June 16, 2000.
To whom correspondence should be addressed: Dept. of Cellular and
Structural Biology, University of Colorado School of Medicine, 4200 E. Ninth Ave., Box B111, Denver, CO 80262. Tel.: 303-315-6284; Fax:
303-315-4729; E-mail: sandy.martin@uchsc.edu.
Published, JBC Papers in Press, December 27, 2002, DOI 10.1074/jbc.M210487200
2 S.-L. Ooi, G. Cost, and J. D. Boeke, personal communication.
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ABBREVIATIONS |
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The abbreviations used are: TPRT, target DNA-primed reversed transcription; ORF, open reading frame; ORF1p, protein product of ORF1; RNP, ribonucleoprotein particle; nt, nucleotide(s).
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