Abstract
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.
Highlights
It is becoming increasingly apparent that splicing defects play a key role in cancer, and that genomic changes in splicing elements [1,2,3], sometimes termed ‘‘splicing spoilers’’ [4,5], can promote aberrant splicing
Transient expression driven by the hyaluronan synthase 1 (HAS1) minigene G345 construct (Figure 1A) yielded mainly full-length transcripts (FL) and varieties of alternatively spliced products similar to those found in ex vivo analysis [19] including a newly identified isoform termed HAS1Vd
In this work we show that mutations and deletions in introns 3 and 4 of HAS1 can alter pre-mRNA splicing events to promote aberrant splicing of the type detected in malignant cells from patients with MM
Summary
It is becoming increasingly apparent that splicing defects play a key role in cancer, and that genomic changes in splicing elements [1,2,3], sometimes termed ‘‘splicing spoilers’’ [4,5], can promote aberrant splicing. It has been estimated that ,50% of mutations underlying genetic diseases cause aberrant splicing [6]. Alterations in a splicing site or splicing control region can have long range implications for splicing events, including altered 3-D architecture of pre-mRNA, activation of cryptic splice sites, exclusion of exons and/or inclusion of all or part of introns. Single mutations can strengthen otherwise weak splice sites and discriminate against otherwise strong splice sites [2,3,4]. Defective mRNA splicing caused by single nucleotide polymorphisms (SNPs) and/or splice site mutations often results in inactivation of tumor suppressor activity (e.g. HRPT2 [7,8], PTEN [9], MLHI [10,11,12], ATR [13]) or generation of dominant negative inhibitors
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