AID binds to L1 mRNA.
2012-11-02T02:45:19Z (GMT) by
<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 1×10<sup>8</sup> LPS- plus IL-4-stimulated AID-deficient (AID −) and wild-type (AID +) B cells (A) or 7.5×10<sup>7</sup> 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 5×10<sup>6</sup> 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"></a>.</p>