Alternatively Spliced, Germline Jα11-2-Cα mRNAs Are the Predominant T Cell Receptor α Transcripts in Mouse Kidney

We recently reported the expression of a truncated T cell receptor (TCR) α mRNA in kidney and brain of normal mice. In the kidney, the truncated TCR α transcript was expressed by bone marrow-dependent, non-T large interstitial cells located predominantly in the medulla. Here, we report the molecular characterization of the truncated TCR α transcript from kidney. Using a modified anchored-PCR (A-PCR) technique and directional cloning, 37 cDNA clones extending 5\u27 of the Cα region were generated. cDNA sequencing showed that 29 of the clones (78%) originated in the Jα 11-2 region. Of these clones, 17 started upstream or in the Jα 11-2 exon and contained the entire Jα 11-2 sequence correctly spliced to the first Cα exon. Analysis of the sequence revealed the presence of multiple stop codons in all three reading frames. The other 12 clones originated further upstream of the Jα 11-2 exon and did not include the Jα 11-2 exon, but rather arose from the joining of a cryptic splice donor signal to the usual TCR α C splice acceptor. This alternatively spliced transcript contained an open reading frame extending from the upstream Jα 11-2 region to 82 nucleotides downstream of the beginning of the TCR C α region, and potentially encoded a 36 amino acid polypeptide. The remaining eight clones all contained the Jα TA61 region correctly spliced to Cα with two of these extending upstream of the Jα TA61 exon. The predominance of Jα 11-2-Cα containing clones was confirmed by RNase protection assay using total RNA from kidney and spleen of scid mice. The 3\u27 region of the transcript contained a fully conserved, correctly spliced TCR α C region which was polyadenylated at the 3\u27 end. The truncated TCR a mRNA could be detected in preparations of cytoplasmic RNA, indicating that this transcript follows a normal RNA processing pathway. Our results demonstrate that the truncated TCR α mRNA expressed in normal mouse kidney is a germline J-C transcript resulting from transcription initiated predominantly upstream of the Jα 11-2 region. This germline transcript in the kidney is undergoing alternative splicing leading to the appearance of an open reading frame coding for a short polypeptide. These results suggest that the product of this transcript may be functionally relevant

Alteration of Introns in a Hyaluronan Synthase 1 (HAS1) Minigene Convert Pre-Mrna Splicing to the Aberrant Pattern in Multiple Myeloma (MM): MM Patients Harbor Similar Changes

<div><p>Aberrant pre-mRNA splice variants of hyaluronan synthase 1 (HAS1) have been identified in malignant cells from cancer patients. Bioinformatic analysis suggests that intronic sequence changes can underlie aberrant splicing. Deletions and mutations were introduced into HAS1 minigene constructs to identify regions that can influence aberrant intronic splicing, comparing the splicing pattern in transfectants with that in multiple myeloma (MM) patients. Introduced genetic variations in introns 3 and 4 of HAS1 as shown here can promote aberrant splicing of the type detected in malignant cells from MM patients. HAS1Vd is a novel intronic splice variant first identified here. HAS1Vb, an intronic splice variant previously identified in patients, skips exon 4 and utilizes the same intron 4 alternative 3′splice site as HAS1Vd. For transfected constructs with unaltered introns 3 and 4, HAS1Vd transcripts are readily detectable, frequently to the exclusion of HAS1Vb. In contrast, in MM patients, HAS1Vb is more frequent than HAS1Vd. In the HAS1 minigene, combining deletion in intron 4 with mutations in intron 3 leads to a shift from HAS1Vd expression to HAS1Vb expression. The upregulation of aberrant splicing, exemplified here by the expression of HAS1Vb, is shown here to be influenced by multiple genetic changes in intronic sequences. For HAS1Vb, this includes enhanced exon 4 skipping and increased usage of alternative 3′ splice sites. Thus, the combination of introduced mutations in HAS1 intron3 with introduced deletions in HAS1 intron 4 promoted a shift to an aberrant splicing pattern previously shown to be clinically significant. Most MM patients harbor genetic variations in intron 4, and as shown here, nearly half harbor recurrent mutations in HAS1 intron 3. Our work suggests that aberrant intronic HAS1 splicing in MM patients may rely on intronic HAS1 deletions and mutations that are frequent in MM patients but absent from healthy donors.</p> </div

<i>In vitro</i> splicing analysis of human HAS1 minigene.

<p>Constructs FLc and G345 are shown in (A). Arrows show where PCR primers bind (E3, E5 and E5I4). The length of each intron in G345 is shown in bp. Each construct was transfected into HeLa cells and HAS1 splicing was studied by RT-PCR. Using E3/E5 primer set, products were analyzed by agarose gel electrophoresis (B). For E3/E5I4 primer set, amplicons were analyzed by DNA fragment analysis (C). Splice junctions for each product are also illustrated. Ø, mock transfection; β2m, control.</p

Summary of primer sequences.

<p>Summary of primer sequences.</p

Mutagenesis of G-repeat motifs in del1 promotes HAS1Vb expression.

<p>Selected G-repeat motifs in del1 (striped line) were mutagenized according to sequences shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053469#pone-0053469-g003" target="_blank">Figure 3</a>. Splicing profiles driven by various del1 derivatives were analyzed by RT-PCR using E3/E5 primer set and products were analyzed by agarose gel electrophoresis.</p