The High Level of Aberrant Splicing of ISCU in Slow-Twitch Muscle May Involve the Splicing Factor SRSF3

Fig 1. Tissue-specific splicing of the human ISCU transgene. A) Schematic representation of the human ISCU transgene. Arrows mark the positions of primers used for semi-qRTPCR. The forward primer, present both in human and mouse, is located in exon 3. The reverse primers, specific to either human or mouse, are located in the 5’ UTR sequence in exon 5. The black thick line represents the last intron of the gene, which includes the pseudoexon represented by the black box. B) Semi-qRTPCR performed on cDNA from the human ISCU transgene and analyzed on a 1.2% agarose gel. Upper panel, human ISCU with incorrect (MUT) and correct (WT) splice variants. Lower panel, mouse endogenous ISCU. C) Quantification of the semi-qRTPCR assays using a 3730X DNA fragment analyzer (Applied Biosystems, Waltham, MA, USA). The graph shows the mean relative proportion ± SD of the splice variants, the wildtype (WT) and the two incorrect splice variants in which either 86 (MUT 86) or 100 bp (MUT 100) of intronic sequence (n = 10–14). D) qRTPCR performed on the mouse tissues (n = 6–10). The graph represents relative proportion incorrectly spliced ISCU (100bp). Tissues used were; muscle (M), heart (H), brain (B)…

Fig 2. Splicing of the human ISCU transgene in mouse muscles. A) Semi- qRTPCR performed on cDNA from the human ISCU transgene and analyzed on a 1.2% agarose gel. Tissues used were soleus muscle (SOL), gastrocnemius muscle (GAS) and quadriceps muscle (QUA) B) Quantification of the semi-qRTPCR assays using a 3730X DNA fragment analyzer. The graph shows the mean relative proportion ± SD of the splice variants, WT and the two incorrect splice variants in which either 86 (MUT 86) or 100 bp (MUT 100) (n = 10–14). C) ISCU qRTPCR performed on mouse muscles (n = 8). The graph represents mean relative proportion ± SD of incorrectly spliced ISCU (100bp). D) SRSF3 qRTPCR using cDNA from mouse muscles (n = 3). The graph represents the mean fold expression ± SD relative to the expression for soleus. GAPDH was used as an internal control. E) Western blot of SRSF3 and ACTIN using nuclear extracts from different mice muscles and protein lysate from control myoblasts (MYO). All semi-qRTPCR and qRTPCR experiments where run in triplicates. (* p < 0.05, ** p < 0.01, *** p < 0.001; Student’s t-test).

Fig 3. SRSF3 binding to normal and mutated ISCU RNA. A) SRSF3 qRTPCR using cDNA from patient (P1, P2) and control (C) myoblasts. The graph presents the mean fold expression ± SD in HML patient samples relative to the control from at least four independent experiments. β-actin was used as an internal control. B) Western blot of SRSF3 and GAPDH in HML patient myoblasts (P1, P2) and a healthy control (C). C) SRSF3 Western blot of the elute fractions (ELUTE) from RNA pull-down assays using patient (P1) and control (C) myoblasts. NE represents input nuclear extract. Nuclear extract was incubated without (-) or with biotinylated RNA oligos; ISCU wildtype oligo (WT), ISCU mutant oligo (MUT), consensus SRSF3 oligo (SRSF3) or scrambled RNA oligo (SCR). GAPDH was used as a negative control for RNA/protein interaction. The pull-down results were reproduced at least once for each oligo. D) Sequence of the ISCU RNA oligos used in the pull-down experiments with proposed SRSF3 binding sites indicated by horizontal square brackets. C/G indicates site of HML mutation. Start of the HML pseudoexon is boxed.

Fig 4. Incorrect splicing of ISCU in myoblasts overexpressing SRSF3. A lentivirus-mediated expression vector for either SRSF3 or mCherry (mChe) was introduced into myoblasts from HML patients (P1, P2) and a healthy control (C). A) SRSF3 qRTPCR using cDNA from uninfected myoblasts (-) as well as myoblasts infected with lentivirus-mediated expression vectors, either SRSF3 (SRSF3) or mCherry (mChe). The graph presents the normalized 2-(ddCt) value ± SD for the SRSF3 expression from four independent experiments. β-actin was used as an internal control. B) Western blot showing levels of SRSF3 in myoblasts overexpressing SRSF3 (SRSF3), mCherry (mC) and non-transduced myoblasts (-). ACTIN was used as a loading control. C) Semi-qRTPCR of human ISCU with incorrect (MUT) and correct (WT) splice variants from uninfected myoblasts (-), myoblasts infected with lentivirus-mediated vectors expressing SRSF3 (SRSF3) or mCherry (mC). D) Quantification of incorrectly spliced ISCU by qRTPCR in uninfected myoblasts (-) as well as myoblasts infected with lentivirus-mediated expression vectors, either SRSF3 (SRSF3) or mCherry (mChe). The graph presents the mean percentage of incorrectly spliced ISCU ± SD from…

Fig 5. Incorrect splicing of ISCU in myoblasts with decreased SRSF3 expression. A lentivirus-mediated expression vector for SRSF3 shRNA (sh3) was introduced into myoblasts from HML patients (P1, P2) and a healthy control (C). A) SRSF3 qRTPCR using cDNA from uninfected myoblasts (-) and myoblasts infected with a shSRSF3 lentivirus-mediated expression vector. The graph present the mean fold change ± SD for the SRSF3 expression from at least three independent experiments. β-actin was used as an internal control. B) Western blot of SRSF3 in non-transduced and transduced myoblasts. ACTIN was used as a loading reference. C) Semi-qRTPCR of human ISCU with incorrect (MUT) and correct (WT) splice variants from uninfected myoblasts, (-) or myoblasts infected with lentivirus-mediated vectors expressing shSRSF3 (sh3). D) Quantification of incorrectly spliced ISCU by qRTPCR in in non-transduced and transduced myoblasts. The graph presents the mean percentage of incorrectly spliced ISCU ± SD from at least three independent experiments (* p < 0.05, ** p < 0.01, *** p < 0.001; Student’s t-test).