Splicing Profile of Human Hyaluronan Synthase 1 (HAS1) Is Altered by Mutagenesis of (A/U)GGG Motifs.

Abstract 2842

Poster Board II-818

In addition to full length (FL) transcripts, clinically significant HAS 1 splice variants (Va, Vb and Vc) have been previously identified in multiple myeloma (MM) and Waldenstrom's macroglobulinemia. Increased HAS1Vb expression correlates with poor survival in a cohort of MM patients. Here, we show that directed mutation of HAS1 intron 3 alters HAS1 splicing and generates a pattern of HAS1 variant expression that mimics patterns detected in MM patients. This suggests that hypermutation of HAS1 and consequent expression of HAS1 splice variants may contribute to oncogenesis in MM. HAS1FL comprises of 5 exons (2089 bp); Va skips exon 4 (133 bp); Vb skips exon 4 and partially retains 59 bp of intron upstream of exon 5 (+59); Vc has all 5 exons and partially retains 26 bp of intron downstream of exon 4. In MM, frequent intronic mutations have been observed in introns 3 and 4, suggesting possible contributions to HAS1 alternative splicing. We have utilized a mammalian expression system to analyse HAS1 splicing by fusing a minigene extending from exon 3 to exon 5 (g345) with the upstream cDNA sequences. HAS1 expression is determined by transfection and RT-PCR using appropriated primer sets. This study focuses on identification of intronic mutations that may affect HAS1 splicing. We target mutations on (A/U)GGG motif because of its high abundance in HAS1 intron 3. The (A/U)GGG repeat was also shown to enhance the splicing of alternative intron in chicken β-tropomyosin (Sirand-Pugnet, P, et al, NAR, 1995, 23, 3501) and intronic G runs could work in a combinatorial way to control the selection of the proper 3' splice site in human thrombopoietin (Marcucci, R, et al, NAR, 2006, 35, 132). A 580 bp long human HAS1 intron 3 is GC-rich and comprises of 28 (A/U)GGG motifs (sequentially identified as G1, G2.., G28). HeLa cells transfected with an unmutated intron 3 construct mainly produce FL with a small amount of HAS1Va, a profile that is similar to CD40L/IL-4 activated normal B cells. Site directed mutagenesis of all 28 (A/U)GGG motifs (G1-28) abolished FL expression, but not HAS1Va, suggesting that these sequence alterations are highly unfavorable for constitutive splicing. It may be due to the loss of essential cis-acting element(s) and/or undesirable conformational changes that prevent spliceosome formation. Mutagenesis of G1-18 is shown to eliminate constitutive expression by increasing the usage of multiple alternative donor sites. Mutagenesis of G19-28 produces more HAS1Va than FL, presumably due to increased exon 4 skipping events. An increased Va/FL ratio could also be achieved by mutagenesis of G25-28 or G27-28, suggesting that this subregion is important for pathway selection. Mutagenesis was also studied in del1 construct, a unique derivative of HAS1 minigene that partially deletes intron 4. Similar to g345, del1 produces FL and HAS1Va as well as promotes expression of novel HAS1Vd, an isoform that includes +59 bp (like Vb) and exon 4. Alteration of HAS1 splicing profile caused by mutagenesis shown in g345 series is also observed in del1 series. Additionally, there is a shift from Vd to Vb expression in all constructs analysed (del1/G1-28, del1/G1-18, del1/G19-28, del1/G25-28 and del1/G27-28), a pattern of aberrant splicing that found in MM patients. Thus, in del1, increased exon 4 skipping events promote both Va and Vb expression. Sequencing of HAS1 intron 3 in a cohort of 50 MM patients indicates that recurrent mutations are found in the G repeat regions and that new repeats are generated by recurrent MM-specific HAS1 mutations. This suggests that mutation of the HAS1 construct mimics HAS1 mutation events that occur in MM patients themselves, and contributes to the clinically significant aberrant HAS1 splicing we have reported in MM (Adamia et al. Blood, 2008, 112, 5111; Blood, 2005, 105, 4836). Overall, critical mutations that could alter HAS1 expression and the ratio of HAS1 variants to FL were identified in intron 3. In intron 4, critical mutations that increase the usage of alternative splice site (+59) remain to be studied. We speculate that cumulative mutations within these two intronic sequences could bring the two events together to promote HAS1Vb splicing. While trans-acting elements are likely to regulate RNA splicing and its pathway, our studies clearly suggest that intronic mutations play an important role in the aberrant splicing of human HAS1, with probable contributions to disease progression in MM.