Rences ISE (SF1) ESS ESS [28,34] [35,36] 
[35]Location of each G-motif is shown in Figure 3. doi:10.1371/journal.pone.SR3029 web 0053469.tIntronic Changes Alter HAS1 SplicingFigure 5. Mutagenesis of G-repeat motifs in del1 promotes HAS1Vb expression. Selected G-repeat motifs in del1 (striped line) were mutagenized according to sequences shown in Figure 3. 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. doi:10.1371/journal.pone.0053469.gFigure 4. Mutagenesis of G-repeat motifs in HAS1 10236-47-2 biological activity intron 3 enhances exon 4 skipping. Selected G-repeat motifs in G345 (striped line) were mutagenized according to sequences shown in Figure 3. 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?8 are summarized in (B). PCR products of G345/G1?8 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 (http://www.fruitfly.org/seq_tools/ splice.html). doi:10.1371/journal.pone.0053469.gdetected more frequently in patient cells where HAS1Vd is infrequent. For nearly half of MM patients, HAS1Vb is expressed in the MM clone at the time of diagnosis [19,21]. For patients lacking HAS1 splice variants at diagnosis, these transcripts were often detected at later stages of disease [19]. Analysis of a series of directed deletions in HAS1 intron 4 showed that splicing of HAS1Vd could be elevated, but HAS1Vb remained unaffected, despite their use of the same 39 splice site in intron 4. Thus, changes in intron 4 alone were insufficient to promote the splicing pattern observed in patients. Combining deletion in intron 4 with mutations in intron 3 however resulted in skipping of exon 4 and promotion of the splicing pattern that leads to a shift from HAS1Vd expression to HAS1Vb expression, the pattern observed in malignant cells from MM patients. To determine the relevance of these genetic changes in vivo, we sequenced intron 3 from genomic DNA of MM PBMC. Consistent with the influence on HAS1Vb of changes made by site directed mutagenesis, in almost half of MM patients analyzed, we found recurrent mutations in intron 3, some located proximate to G repeats as well as some that increased the GC content and increased or decreased the number of G repeats. Previous work has shown that essentially all MM patients analyzed harbored genetic variations in intron 3 andintron 4 [21]. These observations are consistent with the idea that in MM patients, genetic variations in introns 3 and 4 alter splice site selection resulting in intronic splice variants. Together, these promote use of alternative splice sites to generate intronic splice variants that skip exon 4, operationally resulting in loss of HAS1Vd splicing and enhanced expression of the clinically relevant HAS1Vb variant. Deletion analysis of intron 4 was aimed at identifying an intronic region that is important for aberrant splicing of HAS1. Mutations previously identified in MM and WM are frequent in the two “T” stretches and TTTA repeats of intron 4 [21]. The first T st.Rences ISE (SF1) ESS ESS [28,34] [35,36] [35]Location of each G-motif is shown in Figure 3. doi:10.1371/journal.pone.0053469.tIntronic Changes Alter HAS1 SplicingFigure 5. Mutagenesis of G-repeat motifs in del1 promotes HAS1Vb expression. Selected G-repeat motifs in del1 (striped line) were mutagenized according to sequences shown in Figure 3. 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. doi:10.1371/journal.pone.0053469.gFigure 4. Mutagenesis of G-repeat motifs in HAS1 intron 3 enhances exon 4 skipping. Selected G-repeat motifs in G345 (striped line) were mutagenized according to sequences shown in Figure 3. 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?8 are summarized in (B). PCR products of G345/G1?8 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 (http://www.fruitfly.org/seq_tools/ splice.html). doi:10.1371/journal.pone.0053469.gdetected more frequently in patient cells where HAS1Vd is infrequent. For nearly half of MM patients, HAS1Vb is expressed in the MM clone at the time of diagnosis [19,21]. For patients lacking HAS1 splice variants at diagnosis, these transcripts were often detected at later stages of disease [19]. Analysis of a series of directed deletions in HAS1 intron 4 showed that splicing of HAS1Vd could be elevated, but HAS1Vb remained unaffected, despite their use of the same 39 splice site in intron 4. Thus, changes in intron 4 alone were insufficient to promote the splicing pattern observed in patients. Combining deletion in intron 
4 with mutations in intron 3 however resulted in skipping of exon 4 and promotion of the splicing pattern that leads to a shift from HAS1Vd expression to HAS1Vb expression, the pattern observed in malignant cells from MM patients. To determine the relevance of these genetic changes in vivo, we sequenced intron 3 from genomic DNA of MM PBMC. Consistent with the influence on HAS1Vb of changes made by site directed mutagenesis, in almost half of MM patients analyzed, we found recurrent mutations in intron 3, some located proximate to G repeats as well as some that increased the GC content and increased or decreased the number of G repeats. Previous work has shown that essentially all MM patients analyzed harbored genetic variations in intron 3 andintron 4 [21]. These observations are consistent with the idea that in MM patients, genetic variations in introns 3 and 4 alter splice site selection resulting in intronic splice variants. Together, these promote use of alternative splice sites to generate intronic splice variants that skip exon 4, operationally resulting in loss of HAS1Vd splicing and enhanced expression of the clinically relevant HAS1Vb variant. Deletion analysis of intron 4 was aimed at identifying an intronic region that is important for aberrant splicing of HAS1. Mutations previously identified in MM and WM are frequent in the two “T” stretches and TTTA repeats of intron 4 [21]. The first T st.
