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

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

Mutagenesis of G-repeat motifs in HAS1 intron 3 enhances exon 4 skipping.

<p>Selected G-repeat motifs in G345 (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 G345 derivatives were analyzed by RT-PCR using E3/E5 primer set and agarose gel electrophoresis (A). Product in box is not FL as determined by DNA fragment analysis (data not shown). Abnormal HAS1 transcripts driven by G345/G1–18 are summarized in (B). PCR products of G345/G1–18 m transfectants were cloned and spliced junctions were identified by sequencing of subclones. Arrows indicate authentic and cryptic donor sites which located 144 and 279 bp downstream of authentic donor site. The strength of each donor site is determined according to splice site prediction by a neural network (<a href="http://www.fruitfly.org/seq_tools/splice.html" target="_blank">http://www.fruitfly.org/seq_tools/splice.html</a>).</p

<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

Splicing enhancers (ISE) and silencers (ESS) in the G-rich region of HAS1 intron 3.

1<p>Location of each G-motif is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053469#pone-0053469-g003" target="_blank">Figure 3</a>.</p