AID inhibits L1 retrotransposition.

<p>(<b>A</b>) L1 retrotransposition reporter construct. The plasmid contains the human L1_RP element with its proteins, ORF1 and ORF2. The construct also contains a puromycin resistance marker to select for transfected cells (not shown). The intron (in red) in the EGFP is in the same transcriptional orientation as ORF1 and ORF2 (indicated by the arrowhead of the ORF2 exon). When transcribed from the L1 promoter, the GFP gene is spliced, but it cannot be translated, as it is in the inverse orientation and does not encode functional protein. When transcribed from the EGFP promoter (opposite orientation; indicated by the arrowhead of the second EGFP exon), it cannot be spliced and thus not translated. Only when the L1 transcript is spliced, reverse-transcribed and inserted into the genome (a retrotransposition event) is GFP protein expressed. (<b>B</b>) Retrotransposition assay in HEK293 cells. Above, GFP-positive cells as determined by flow cytometry on days 3 and 6 after transfection of the reporter construct (L1) and/or AID. L1 mut is a reporter construct in which the ORF1 contains two missense mutations. Scales on the y-axis differ for day 3 and day 6. Values represent mean ± SD; n = 2 transfection samples. Student's t test; *p≤0.01 and **p≤0.001. Below, Western blots of lysates from cells 3 days after transfection, developed with anti-AID, anti-ORF1, or anti-actin antibody. The position of the molecular mass standard (in kDa) is indicated next to the blots. (<b>C</b>) Retrotransposition assay in WEHI-231 cells. GFP-positive cells as determined by flow cytometry on day 6 after transfection of the reporter constructs L1 and L1 mut. +, AID-positive WEHI-231 subclone; –, AID-negative WEHI-231 subclone. Values represent mean ± SD; n = 2 transfection samples. Student's t test; *p≤0.01.</p

AID binds to L1 mRNA.

<p>(<b>A and B</b>) Agarose gels of products from RT-PCR on RNA-immunoprecipitates (RNA-IP) showing RNA binding for endogenous (A) and exogenous (B) AID from murine B lymphoblasts. Proteins and nucleic acids contained in 1×10⁸ LPS- plus IL-4-stimulated AID-deficient (AID −) and wild-type (AID +) B cells (A) or 7.5×10⁷ LPS- plus IL-4-stimulated AID-deficient B cells, transduced with Flag-GFP (AID −) or Flag-AID (AID +) (B), were cross-linked via treatment with UV (UV +), or not treated (UV−); the cells were then lysed, followed by immunoprecipitation with monoclonal anti-AID (A) or monoclonal anti-Flag (B) antibody. After DNase digest, RT-PCR using oligo(dT) primers, followed by L1-, germ line transcript (GLT)-, Ig κ-light chain (κ chain)-, Ig µ-heavy chain (µ chain)- or GAPDH-specific primers, was performed on the immunoprecipitates (“RT-PCR” panel, “RNA-IP”). To monitor the amount of AID in lysate and immunoprecipitates, we analyzed aliquots of lysates (WB panel, “input”) and immunoprecipitates (WB panel, “IP”), both equivalent to 5×10⁶ cells, and electrophoresed, Western blotted and developed them with an anti-actin, anti-Flag or anti-AID antibody. Lanes 1–4, RNA-IP samples; lanes 5–7, cDNA synthesis and PCR controls, i.e., total RNA of LPS- plus IL-4-stimulated AID-deficient and wild-type B cells with (+ RT) or without (− RT) reverse transcriptase. (<b>C</b>) Left: identity of L1 elements in the immunoprecipitates confirmed by cDNA sequencing. Sequences (nt 151–200) of the 300-bp L1 ORF2 fragments that were amplified from RNA-IP shown in panel A. M13002, L1 reference sequence of BALB/c strain origin; L1.1 and L1.2, sequences obtained from RNA-IP of lane 2 in panel A; L1.3 to L1.5, sequences obtained from lane 4 in panel A. Right: schematic of a full-length L1 element. Arrows indicate the position of the primers used to amplify the 300-bp L1 ORF2 fragment from RNA-IP shown in panel A and B. The numbers represent the nucleotide positions according to the L1Md-A2 sequence [L1 sequence, Genbank:M13002] <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049358#pone.0049358-Loeb1" target="_blank">[68]</a>.</p

AID forms high-molecular-mass complexes in the cytoplasm.

<p>Fractionation according to size by gel filtration of A3G (A) and AID (B and C), followed by Western blot analysis. Numbers above panels, fractions of separation by FPLC; numbers to the left of panels, molecular mass standards (in kDa) of the SDS gel run; numbers below panels, molecular mass standards (in kDa) of the fractions of the FPLC run. (<b>A</b>) Western blots of fractions of FPLC eluates were developed with polyclonal antibody to A3G. Input, untreated lysate of A3G-positive cells; input + RNase, RNase A-treated lysate of A3G-positive cells; A3G minus, lysate of A3G-negative cells; H9, A3G-positive cell line; fractions 3–16, fractionated lysates of LPS- plus IL-4-activated B lymphocytes from human A3 transgenic mice. Upper panel, untreated cell lysate (devoid of nuclei); lower panel, treated with RNase A before fractionation on FPLC. (<b>B and C</b>) Western blots of fractions of FPLC eluates developed with monoclonal antibody to AID. AID minus, lysate of AID-negative HeLa cells; AID plus, lysate of AID-positive HeLa cells; fractions 3–15, fractionated lysates of LPS- plus IL-4-activated B lymphocytes from AID-sufficient mice (B) and of AID-positive HeLa cells (C). Top panel, untreated cell lysate (devoid of nuclei); middle panel, treated with RNase A before fractionation on FPLC; bottom panel, treated with RNase A and RNase inhibitors before fractionation. Concomitant with RNase A treatment, proteins were somewhat digested for unknown reasons.</p

Activated B lymphocytes express L1 mRNA, but not L1 protein.

<p>(<b>A and B</b>) L1, IAP and MLV mRNA expression. After digestion with DNase, mRNA was reverse-transcribed into cDNA using oligo(dT) primers, followed by L1-, IAP- and MLV-specific quantitative real-time PCR. PCR data were quantified according to the comparative <i>C</i>_T method. The amount of target mRNA was normalized to the endogenous control 18S ribosomal RNA. Values represent mean ± SD; y-axis, fold change in mRNA expression. (A) LPS- plus IL-4-stimulated (LPS) and unstimulated B lymphocytes from the spleens of AID-deficient (AID KO; n = 12 mice) and wild-type (WT; n = 12 mice) BALB/c mice. Student's t test, *p≤0.05 and **p≤0.001. (B) Tissues (pancreas, testis) and activated B cells (spleen LPS) from a wild-type BALB/c mouse and mouse cell lines F9 and MOPC104E; F9, mouse embryonal carcinoma cell line expressing high levels of L1 mRNA; spleen LPS, LPS- plus IL-4-stimulated B lymphocytes, as in panel A; n = 2 independent experiments. (<b>C and D</b>) ORF1 protein expression. Cell lysates were electrophoresed, Western blotted and developed with anti-ORF1 or anti-actin antibody. The position of the molecular mass standard (in kDa) is indicated to the left of the blots; the positions of the exogenous (exo) and endogenous (endo) human (hORF1) and mouse (mORF1) ORF1 bands are indicated to the right of the blots; LPS blasts, lysates of LPS- plus IL-4-activated B lymphocytes from BALB/c mice; + AID and – AID, AID-sufficient and AID-deficient, respectively. (C) anti-mORF1 staining; testis, whole testis lysates; IR, cells gamma irradiated 24 h before lysis with 0, 1 or 50 Gy. (D) anti-hORF1 staining; 2102Ep, lysate from a human embryonal carcinoma cell line expressing high levels of ORF1; Phoenix and LPS blasts, lysates of cells transfected (Phoenix) or transduced (LPS Blasts) with a retroviral human ORF1 (+ exo hORF1) or a GFP-only construct (− exo hORF1).</p

AID decreases the steady-state level of L1 protein.

<p>The doxycycline (Dox)-inducible construct encodes AID and GFP, expressed as two proteins; HeLa, untransfected HeLa cells; HeLa-AID, HeLa clone stably transfected with the inducible AID construct; – Dox, uninduced cells; + Dox, induced cells. (<b>A</b>) AID induction in HeLa cells: flow cytometry analysis. GFP expression (x-axis) as an indicator of AID expression. Numbers represent the percentage of GFP-positive cells; empty and black parts of the histogram are – Dox. (<b>B</b>) AID induction in HeLa cells: Western blot analysis. HeLa cell lysates were electrophoresed, Western blotted and developed with monoclonal anti-AID antibody. The position of the molecular mass standard (in kDa) is indicated next to the blot. (<b>C</b>) L1 mRNA levels in HeLa cells. After digestion with DNase, mRNA of HeLa cells was reverse-transcribed into cDNA using oligo(dT) primers, followed by L1-specific quantitative real-time PCR. PCR data were quantified according to the comparative <i>C</i>_T method. The amount of L1 target mRNA was normalized to the endogenous control GAPDH. Values represent mean ± SD; n = 3 independent experiments; y-axis, fold change in L1 mRNA expression (HeLa-AID set to 1.0). (<b>D</b>) ORF1 protein levels in HeLa cells. HeLa cell lysates were electrophoresed, Western blotted and developed with anti-ORF1, anti-AID or anti-actin antibody. 2102Ep, lysate from a human embryonal carcinoma cell line expressing high levels of ORF1. The position of the molecular mass standard (in kDa) is indicated next to the blots. (<b>E</b>) Western blot developed with ORF1 (above) and AID (below). ORF1 protein was co-precipitated with AID in lysates of Flag-AID (+)- and Flag-GFP (−)-transfected Phoenix cells (derived from HEK293 cells). Lysate, input used for IP; IP, immunoprecipitates; anti-Flag, immunoprecipitation with Flag-specific antibody; –, immunoprecipitation control without antibody. The position of the molecular mass standard (in kDa) is indicated next to the blots.</p

AID's binding to agarose is not RNase sensitive.

<p>(A) Agarose binding to AID in the absence (\) and presence of RNase A at various concentrations: low (L) (1 µg/ml), medium (M) (100 µg/ml), or high (H) (1 mg/ml). GFP control (–AID) or AID-transfected (+ AID) human embryonic kidney cell lysates were treated with RNase and incubated with two successive rounds of agarose beads (1st and 2nd Round). The washed 1st and 2nd round beads and the unbound lysate after the second bead incubation (Supernatant) were western blotted and probed with a monoclonal anti-AID antibody. Input, 1st Round, 2nd Round, and Supernatant, all equivalent to 5×10⁵ cells. The positions of the molecular mass standards are indicated next to the blots. (B) Incubation of 1 µg RNA in the absence (\) and presence of RNase A at various concentrations: low (L) (1 µg/ml), medium (M) (100 µg/ml), or high (H) (1 mg/ml). The positions of the size standards and the 18S and 28S rRNA are indicated next to the agarose gel.</p

Agarose binding and immunoprecipitation of AID and GFP.

<p>Lysates of 5×10⁵ Flag-AID-and Flag-GFP-transfected cells were incubated with agarose beads (1st Round). In a second round, the unbound lysate was incubated with 5 µg anti-AID or anti-Flag antibody as indicated, and agarose beads were added (2nd Round + Antibody). The washed 1st and 2nd round beads were western blotted. The positions of the molecular mass standards are indicated next to the blots. (A) Blot developed with a monoclonal anti-AID antibody. (B) Blot developed with a monoclonal anti-Flag antibody.</p

Agarose binding of high molecular mass (HMM) and low molecular mass (LMM) AID complexes.

<p>(A) AID lysate, fractionated by size exclusion chromatography, was incubated with agarose beads (1st Round). The unbound proteins were incubated a second time with agarose beads (2nd Round). The proteins recovered from the washed 1st and 2nd round beads, and the unbound proteins after the second incubation (Supernatant), were western blotted and developed with a monoclonal anti-AID antibody. The positions of the molecular mass standards are indicated next to the blots. Lysate, unfractionated lysate from HeLa (–AID) and HeLa-AID (+ AID) cells; FPLC fractions, size-fractionated HMM and LMM AID from HeLa-AID cells and primary blasts. (B) AID double band from endogenous (lysate from primary blasts), and exogenous (lysate from HeLa-AID cells) AID.</p

Two AID variants with different agarose binding.

<p>Cell lysates were incubated with agarose beads (1st Round). The unbound lysate was incubated a second time with agarose beads (2nd Round). The washed 1st and 2nd round beads and the unbound lysate after the second incubation (Supernatant) were western blotted and developed with a monoclonal anti-AID antibody. The positions of the molecular mass standards are indicated next to the blots. (A) Endogenous AID:–AID, lysate from AID-deficient cells; + AID, lysate from wild-type cells; input equivalent to 2×10⁶ cells; 1st Round, 2nd Round, and Supernatant, equivalent to 1×10⁷ cells. Exogenous AID:–AID, lysate from GFP-transfected cells; + AID, lysate from AID-transfected cells; input, 1st Round, 2nd Round, and Supernatant, all equivalent to 5×10⁵ cells. (B) Incubation with various Sepharose/agarose beads; exogenous AID only. Sepharose is a trade name of agarose with some changes in the charge of the polysaccharides. Protein G1 Sepharose and protein G2 Sepharose are protein G beads from different batches of the same catalog number.</p

Higher magnification of the 5' flanking (upstream of exon 1), and 3' flanking region of

<p><b>Copyright information:</b></p><p>Taken from "-encoded microRNAs in oncogenesis"</p><p>http://www.retrovirology.com/content/5/1/4</p><p>Retrovirology 2008;5():4-4.</p><p>Published online 14 Jan 2008</p><p>PMCID:PMC2257975.</p><p></p> The handle bars in green represent the retroviral insertions; arrows in the line within the bars denote direction of provirus transcription. Proviruses boxed in red are in the same orientation as the gene (from left to right), opposite from the rest. Proviruses are in the same orientation as the gene