12 research outputs found
The effect of ZAP-S on L1 RNA and L1 protein expression.
<p><i>(A) Schematic of pJM101/L1</i>.<i>3Īneo</i>: Bold black lines indicate the approximate location of probes (5UTR99 and ORF2_5804) used in the northern blot experiments. pJM101/L1.3Īneo is expressed from a pCEP4 vector. A CMV promoter augments L1 expression and an SV40 polyadenylation signal (pA) is located downstream of the native L1 polyadenylation signal. <i>(B) Results of northern blots</i>: Top panel: HeLa cells were co-transfected with pJM101/L1.3Īneo and either the indicated ZAP-S expression plasmids or an empty pCEP4 vector. Northern blot images depict the effect of ZAP-S overexpression on polyadenylated L1 RNA levels. The constructs transfected into HeLa cells are indicated above each lane. UTF indicates untransfected HeLa cells and serves as a negative control. Probes (5UTR99 and ORF2_5804) are indicated in the top left corner of the respective blots. The black arrow indicates the position of the full-length L1 RNA. The blue and yellow arrows indicate shorter L1 RNA species. The experiment was repeated three times with similar results. Actin served as a loading control. RNA size standards (~kb) are shown at the right of the blot image. Bottom panel: Quantification of northern blot bands. The X-axis indicates the cDNA expression construct that was co-transfected with pJM101/L1.3Īneo. The Y-axis indicates relative band intensity normalized to pCEP4 controls (100%). Black bars represent the full-length L1 band. Blue and yellow bars represent the smaller L1 RNA bands, corresponding to the colored arrows, respectively, in the top panel. The results are the average of three independent experiments. Error bars represent standard deviations. <i>(C) Schematic of pJBM2TE1</i>: The construct contains a T7 epitope tag on the carboxyl-terminus of ORF1p and a TAP tag on the carboxyl-terminus of ORF2p. An <i>mneoI</i> retrotransposition indicator cassette is present in the 3ā UTR. pJMB2TE1 is expressed from a pCEP4 backbone, which has been modified to contain a puromycin selectable marker. A CMV promoter augments L1 expression and an SV40 polyadenylation signal (pA) is located downstream of the native L1 polyadenylation signal. <i>(D) ZAP-S decreases the accumulation of the L1-encoded proteins</i>: HeLa cells were co-transfected with pJBM2TE1 and the plasmids indicated above each lane. UTF indicates untransfected HeLa cells and serves as a negative control. Depicted are western blots using whole cell lysates (WCL, top panel) or RNP fractions (RNP, bottom panel). Blue arrows indicate the positions of ORF2p, ORF1p, ZAP-S, and ZAP-S/ā72ā372. The eIF3 protein is used as a loading control. Representative images are shown. The experiments were repeated three times with similar results.</p
The co-localization of ORF1p and ZAP in cytoplasmic foci.
<p><i>(A) Co-localization of transfected ORF1p and ZAP-S in HeLa cells</i>: ORF1p (red) expressed from pJM101/L1.3Īneo co-localizes with ZAP-S-tGFP (green). The experiment was repeated five times with similar results. <i>(B) Co-localization of transfected ORF1p with endogenous ZAP in cytoplasmic foci in HeLa cells</i>: ORF1p-T7 (green) expressed from pAD2TE1 co-localizes with endogenous ZAP (red). The experiment was repeated five times with similar results. <i>(C) Co-localization of transfected ZAP-S-tGFP with endogenous ORF1p in cytoplasmic foci in PA-1 cells</i>: ZAP-S-tGFP (green) co-localizes with endogenous ORF1p (red). PA-1 experiments were repeated twice with similar results. <i>(D-E) The ZAP-S zinc-finger domain is necessary for co-localization with ORF1p in HeLa cells</i>: ORF1p (red) expressed from pJM101/L1.3Īneo co-localizes with ZAP-S/Ī310-645-tGFP (green) (panel D). ORF1p (red) expressed from pJM101/L1.3Īneo forms cytoplasmic foci that do not contain ZAP-S/Ī72-372-tGFP (green) (panel E). The right-most image of each panel represents a merged image. The cell type is indicated at the top left (yellow), the protein name is listed on the bottom left, and the name of the primary antibody used (<i>italicized</i>) is annotated at the bottom right. Nuclei were stained with DAPI (blue) and the scale bar represents 25 Ī¼M.</p
ZAP-S inhibits LINE and Alu retrotransposition.
<p><i>(A) ZAP inhibits L1 retrotransposition</i>: Top panel: Schematics of ZAP constructs. Depicted are the relative positions of the zinc-finger domains (light gray rectangles), cysteine-histidine (CCCH) zinc-fingers (vertical black bars), and PARP-like domain (dark gray rectangles) of the ZAP-L and ZAP-S expression constructs. ZAP-L contains a carboxyl-terminal HA tag (blue rectangle labeled HA). The ZAP-S/1-311 construct contains an additional 31 amino acids at the carboxyl terminus. The ZAP-S/ā72ā372 harbors a deletion that removes the CCCH zinc fingers (See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005121#sec013" target="_blank">Methods</a>). Middle panel: Results of the retrotransposition assays. The X-axis indicates the cDNA co-transfected with pJJ101/L1.3 or pcDNA6/TR. The Y-axis indicates pJJ101/L1.3 retrotransposition activity (black bars), or pcDNA6/TR colony formation activity (white bars). All values have been normalized to the pCEP4 empty vector control (100%). The numbers above the bar graphs indicate the number of independent experiments performed with each cDNA expression construct. Error bars represent standard deviations. Bottom panel: A single well of a representative six-well tissue culture plate, displaying blasticidin-resistant colonies from the pJJ101/L1.3 retrotransposition assay (top, black rectangle) and the pcDNA6/TR control assay (bottom, white rectangle). <i>(B) ZAP inhibits Alu retrotransposition</i>: The X-axis indicates the cDNA co-transfected with pJM101/L1.3Īneo and pAlu<i>neo</i><sup>Tet</sup>. The Y-axis indicates the retrotransposition efficiency. All values are normalized to the pCEP4 empty vector control (100%). Control assays using a plasmid that expresses the neomycin phosphotransferase gene (pcDNA3) were conducted similarly to pcDNA6/TR control assays as outlined in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005121#pgen.1005121.g002" target="_blank">Fig 2B</a>. Representative images of G418-resistant HeLa foci from the Alu retrotransposition assay are shown below the bar graph. The results are the average of three independent experiments. Error bars indicate standard deviations. <i>(C) ZAP inhibits the retrotransposition of mouse and zebrafish LINE elements</i>. The X-axis indicates the cDNA that was co-transfected with human L1 (pJM101/L1.3 (black bars)), mouse L1 (pG<sub>F</sub>21 (dark grey bars)), zebrafish L2 (pZfL2-2 (light grey bars)), or synthetic mouse L1 (pCEPsmL1 (white bars)). The Y-axis indicates the retrotransposition efficiency. Representative images of G418-resistant HeLa cell foci are shown below the bar graph. Control assays using a plasmid that expresses the neomycin phosphotransferase gene (pcDNA3) were conducted similarly to pcDNA6/TR control assays outlined in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005121#pgen.1005121.g002" target="_blank">Fig 2B</a>. All values are normalized to the pCEP4 empty vector control (100%). Error bars indicate standard deviations. <i>(D) The depletion of ZAP enhances L1 retrotransposition</i>: Top panels: Western blots of whole cell lysates derived from mock HeLa cell transfections or HeLa cells transfected with indicated siRNAs. Blue arrows point to the approximate location of ZAP-L and ZAP-S. Bottom panel: The bar graph depicts pLRE-<i>mEGFP1</i> retrotransposition activity following siRNA treatment. The X-axis indicates the siRNA. The Y-axis indicates the pLRE-<i>mEGFP1</i> retrotransposition efficiency normalized to the control siRNA (set to 1). Retrotransposition efficiency values are reported as the mean from four independent experiments. Error bars indicate the standard deviations. Asterisks indicate statistically significant differences from the control siRNA experiments (two-tailed t test/p<0.05).</p
The co-localization of ZAP with L1 RNA and ORF1p in HeLa cells.
<p><i>(A) Co-localization of transfected L1 RNA and ORF1p</i>: ORF1p (red) expressed from pJM101/L1.3 co-localizes with L1 RNA (magenta). <i>(B) Co-localization of transfected L1 RNA and ORF1p with transfected ZAP-S-tGFP in cytoplasmic foci</i>: ORF1p (red) and L1 RNA (magenta) expressed from pJM101/L1.3 co-localize with ZAP-S-tGFP (green). <i>(C-D) The ZAP-S zinc-finger domain is necessary for co-localization with ORF1p</i>: ORF1p (red) and L1 RNA (magenta) expressed from pJM101/L1.3 co-localize with ZAP-S/Ī310-645-tGFP (green) (panel C). ZAP-S/Ī72-372-tGFP (green) diffusely distributes throughout the cytoplasm, while ORF1p (red) expressed from pJM101/L1.3 forms cytoplasmic foci with L1 RNA (magenta) (panel D). The right-most image of each panel represents a merged image. The name of the protein or RNA is indicated at the bottom left, and the name of the primary antibody used (<i>italicized</i>) is annotated at the bottom right of each image. Nuclei were stained with DAPI (blue) and the scale bar represents 25 Ī¼M. Experiments were repeated three times with similar results.</p
The identification of host proteins immunoprecipitated with L1 ORF1p-FLAG.
<p><i>(A) Schematic of L1 constructs</i>: pJM101/L1.3 expresses a human L1 (L1.3) [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005121#pgen.1005121.ref005" target="_blank">5</a>] containing an <i>mneoI</i> retrotransposition indicator cassette within the L1 3' UTR. The pJM101/L1.3FLAG construct is identical to pJM101/L1.3, but contains a single FLAG epitope on the carboxyl-terminus of ORF1p. Both constructs were cloned into a pCEP4 mammalian expression vector. A CMV promoter augments L1 expression and an SV40 polyadenylation signal (pA) is located downstream of the native L1 polyadenylation signal. <i>(B) Results of immunoprecipitation experiments</i>: Whole cell lysates from HeLa cells transfected with either pJM101/L1.3 or pJM101/L1.3FLAG were subjected to immunoprecipitation using an anti-FLAG antibody. The proteins then were separated by SDS-PAGE, visualized by silver staining, and subjected to LC-MS/MS. An ~40 kDa band corresponding to the theoretical molecular weight of ORF1p is visible in the pJM101/L1.3FLAG lane (*). Black bars indicate the approximate molecular weights of the ORF1p-FLAG interacting proteins. Molecular weight standards (kDa) are shown on the left hand side of the gel. <i>(C) Validation of the ORF1p-FLAG immunoprecipitation</i>: Western blot experiments using an antibody specific to amino acids 31ā49 of L1.3 ORF1p verified the enrichment of ORF1p-FLAG in pJM101/L1.3FLAG, but not pJM101/L1.3 immunoprecipitation reactions. Cells transfected with the pCEP4 vector served as a negative control. <i>(D) Validation of putative ORF1p-FLAG interacting proteins</i>: Western blot images of the pJM101/L1.3FLAG and pJM101/L1.3 immunoprecipitation (IP) reactions. The pCEP4 lanes denote whole cell lysates derived from HeLa cells transfected with an empty pCEP4 vector (~ 1.0% input). Primary antibodies used to probe western blots are indicated to the left of the images. Immunoprecipitation reactions were conducted in either the absence (left) or presence (right) of RNaseA (10 Ī¼g/mL). The putative cellular functions of the ORF1p-FLAG interacting proteins are indicated on the right hand side of the blots.</p
The Zinc-Finger Antiviral Protein ZAP Inhibits LINE and Alu Retrotransposition
<div><p>Long INterspersed Element-1 (LINE-1 or L1) is the only active autonomous retrotransposon in the human genome. To investigate the interplay between the L1 retrotransposition machinery and the host cell, we used co-immunoprecipitation in conjunction with liquid chromatography and tandem mass spectrometry to identify cellular proteins that interact with the L1 first open reading frame-encoded protein, ORF1p. We identified 39 ORF1p-interacting candidate proteins including the zinc-finger antiviral protein (ZAP or ZC3HAV1). Here we show that the interaction between ZAP and ORF1p requires RNA and that ZAP overexpression in HeLa cells inhibits the retrotransposition of engineered human L1 and Alu elements, an engineered mouse L1, and an engineered zebrafish LINE-2 element. Consistently, siRNA-mediated depletion of endogenous ZAP in HeLa cells led to a ~2-fold increase in human L1 retrotransposition. Fluorescence microscopy in cultured human cells demonstrated that ZAP co-localizes with L1 RNA, ORF1p, and stress granule associated proteins in cytoplasmic foci. Finally, molecular genetic and biochemical analyses indicate that ZAP reduces the accumulation of full-length L1 RNA and the L1-encoded proteins, yielding mechanistic insight about how ZAP may inhibit L1 retrotransposition. Together, these data suggest that ZAP inhibits the retrotransposition of LINE and Alu elements.</p></div
Spliced integrated retrotransposed element (SpIRE) formation in the human genome
<div><p>Human Long interspersed element-1 (L1) retrotransposons contain an internal RNA polymerase II promoter within their 5ā² untranslated region (UTR) and encode two proteins, (ORF1p and ORF2p) required for their mobilization (i.e., retrotransposition). The evolutionary success of L1 relies on the continuous retrotransposition of full-length L1 mRNAs. Previous studies identified functional splice donor (SD), splice acceptor (SA), and polyadenylation sequences in L1 mRNA and provided evidence that a small number of spliced L1 mRNAs retrotransposed in the human genome. Here, we demonstrate that the retrotransposition of intra-5ā²UTR or 5ā²UTR/ORF1 spliced L1 mRNAs leads to the generation of spliced integrated retrotransposed elements (SpIREs). We identified a new intra-5ā²UTR SpIRE that is ten times more abundant than previously identified SpIREs. Functional analyses demonstrated that both intra-5ā²UTR and 5ā²UTR/ORF1 SpIREs lack <i>Cis</i>-acting transcription factor binding sites and exhibit reduced promoter activity. The 5ā²UTR/ORF1 SpIREs also produce nonfunctional ORF1p variants. Finally, we demonstrate that sequence changes within the L1 5ā²UTR over evolutionary time, which permitted L1 to evade the repressive effects of a host protein, can lead to the generation of new L1 splicing events, which, upon retrotransposition, generates a new SpIRE subfamily. We conclude that splicing inhibits L1 retrotransposition, SpIREs generally represent evolutionary ādead-endsā in the L1 retrotransposition process, mutations within the L1 5ā²UTR alter L1 splicing dynamics, and that retrotransposition of the resultant spliced transcripts can generate interindividual genomic variation.</p></div
A working model for the generation of SpIREs.
<p>(A) Canonical L1 retrotransposition. An L1 is transcribed from a genomic location (red chromosome). Translation of the mRNA (multicolored wavy line) occurs in the cytoplasm and ORF1p (yellow circles) and ORF2p (blue oval) bind back onto their respective mRNA (<i>Cis</i>-preference) to form an RNP. The L1 RNP then enters the nucleus and a de novo L1 insertion occurs at a new genomic location (green chromosome) by TPRT. This insertion, if full length, could act as a source element, giving rise to new insertions (green arrow) at a new genomic location (gray chromosome). (B) Retrotransposition of intra-5ā²UTR spliced L1 isoform. A full-length L1 element is transcribed from its genomic location (red chromosome) and undergoes intra-5ā²UTR splicing. Translation of the mRNA (multicolored wavy line) occurs in the cytoplasm and ORF1p (yellow circles) and ORF2p (blue oval) bind back onto their respective mRNA (<i>Cis</i>-preference) to form an RNP. The L1 RNP then enters the nucleus and L1 mRNAs subject to intra-5ā²UTR splicing can undergo a single round of retrotransposition (green chromosome) by TPRT. However, because the intra-5ā²UTR splicing event deletes sequences required for L1 promoter activity, the resultant insertion is unlikely to undergo subsequent rounds of retrotransposition in future generations (dashed green arrow). (C) Retrotransposition of 5ā²UTR/ORF1 spliced L1 isoform. An L1 is transcribed from its genomic location (red chromosome) and is subject to 5ā²UTR/ORF1 splicing. Translation of the mRNA (multicolored wavy line) occurs in the cytoplasm; however, because translation occurs at downstream AUG codons, ORF1p (yellow circles) is truncated and nonfunctional, the 5ā²UTR/ORF1 spliced L1 mRNA relies on a wild-type source of ORF1p to be supplied from another L1 in <i>trans</i>. In the rare instance that <i>Trans</i>-complementation occurs (dotted arrow), it is highly unlikely that the resultant SpIRE will generate RNAs that can undergo retrotransposition in future generations (dashed thin green arrow). L1, Long interspersed element-1; ORF, open reading frame; RNP, ribonucleoprotein particle; SpIRE, spliced integrated retrotransposed element; TPRT, target-site primed reverse transcription; UTR, untranslated region.</p
L1 mRNA contains potential SD and SA sites.
<p>(A) Schematic of a full-length retrotransposition competent genomic L1. Top: the 5ā² and 3ā² UTRs (gray rectangles), ORF1 (yellow rectangle), and ORF2 (blue rectangle) are indicated in the schematic. The approximate positions of sense transcription initiation and antisense transcription initiation are indicated with black arrows on the top and bottom of the 5ā²UTR, respectively. The approximate positions of the coiled-coil (CC), RNA recognition motif (RRM), and C-terminal domain (CTD) are indicated in black lettering in ORF1. The endonuclease (EN), reverse transcriptase (RT), and cysteine-rich (C) domain are indicated in white lettering in ORF2. The 3ā²UTR ends in an A<sub>N</sub>. The L1 is flanked by target-site duplications (black arrowheads) in genomic DNA (black helical lines). Bottom: a magnified schematic of the 5ā²UTR and 5ā² end of ORF1. The black arrow indicates the relative position of sense transcription initiation. The SD (red) and SA (green) sequences used to generate SpIREs are indicated above the 5ā²UTR (gray rectangle) and ORF1 (yellow rectangle). The position of the SD and SA sequences relative to L1.3 are indicated with superscript numbers. The relative positions of <i>Cis</i>-acting transcription factor binding sequences are indicated in the 5ā²UTR. (BāD) Schematics of the splicing events generating SpIRE<sub>97/622</sub>, SpIRE<sub>97/790</sub>, and SpIRE<sub>97/976</sub>. The SD (red underlined GU nucleotides) and SA (green underlined AG nucleotides) demark the intron boundaries used to generate each class of SpIRE. The left half of the figure depicts the L1 mRNA sequence before splicing and the right half of the figure depicts L1 mRNA after splicing. A<sub>N</sub>, poly(A) tract; C, cysteine-rich; CC, coiled-coil; CTD, C-terminal domain; EN, endonuclease; L1, Long interspersed element-1; ORF, open reading frame; poly(A), polyadenosine; RRM, RNA recognition motif; RT, reverse transcriptase; RUNX3, runt related transcription factor 3; SA, splice acceptor; SD, splice donor; SpIRE, spliced integrated retrotransposed element; SP1, specificity protein 1; SRY, sex determining region Y; UTR, untranslated region; YY1, yin and yang 1.</p
ORF1p expression from intra-5ā²UTR and 5ā²UTR/ORF1 SpIREs.
<p>(A) Schematics of the engineered L1 constructs. The L1 5ā²UTR (gray rectangle), ORF1 (yellow rectangle), and ORF2 (blue rectangle) are indicated in the constructs. Relative positions of the SpIRE<sub>97/622</sub> and SpIRE<sub>97/976</sub> deletions (red triangles) are indicated on the bottom two constructs, respectively. The CMV promoter (white arrowhead) and the <i>mneoI</i> retrotransposition indicator cassette (green rectangle = <i>neo</i> gene sequence; black āvā line = intron interrupting <i>neo</i> coding sequence, SD = splice donor site, SA = splice acceptor site) are indicated at the 5ā² and 3ā² ends of the constructs, respectively. The black lollipop at the 3ā² end on top of the constructs indicates the sense SV40 polyadenylation signal. The black arrow and gray lollipop on the bottom of the constructs are embedded within the <i>mneoI</i> retrotransposition indicator cassette and indicate an SV40 early promoter and herpes simplex virus thymidine kinase polyadenylation signal, respectively, in the antisense orientation. (B) Representative ORF1p western blot from WCLs. Molecular weight standards (kDa) are indicated to the left of the image. The black arrowhead indicates the predicted size of full-length ORF1p (about 40 kDa). Construct names are indicated above the image; pCEP/GFP = negative control. The antibody used in the western blot experiment is indicated to the right of the gel (Ī±-N-ORF1p). The eIF3 protein (110 kDa) served as a loading control. Western blots were performed three times, yielding similar results. (C) Schematic of ORF1 and relative location of antibody binding. Top: The relative positions in ORF1 (yellow rectangle) of the SA sequence at nucleotides 974ā975 (green), the canonical ORF1 initiator methionine (AUG, black, 40 kDa), the two putative initiator methionine codons (AUG, orange, 33 kDa; AUG, blue, 27 kDa), and the N- and C-terminal epitopes recognized by the ORF1p Ab (red and purple stars, respectively) are indicated in the figure. (D) Representative western blots from WCLs: molecular weight standards (kDa) are indicated to the left of the gels. The predicted sizes of full-length ORF1p (black arrowhead) and the N-terminal truncated ORF1p variants (orange and blue arrows, respectively) are highlighted on the gel. Construct names are indicated above the image; pCEP/GFP = negative control. The antibodies used in the western blot experiments are indicated to the left (Ī±-N-ORF1p) and right (Ī±-C-ORF1p) of the gel images, respectively. The eIF3 protein (110 kDa) served as a loading control. The unlabeled band at about 25 kDa in the Ī±-C-ORF1p experiment is an unknown cross-reacting product that was not detected in RNPs or with an antibody to a C-terminal ORF1p T7-<i>gene10</i> epitope tag (S4A Fig and S4B Fig). Western blots were performed three times, yielding similar results. Ī±-C-ORF1p, C-terminal ORF1p antibody; Ī±-elF3, eukaryotic initiation factor 3 antibody; Ī±-N-ORF1p, N-terminal ORF1p antibody; Ab, antibody; AUG, translation initiation codon; CMV, cytomegalovirus; kDa, kilodalton; L1, Long interspersed element-1; ORF, open reading frame; SA, splice acceptor; SD, splice donor; SpIRE, spliced integrated retrotransposed element; UTR, untranslated region; WCL, whole cell lysate.</p