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

Abstract

Hereditary myopathy with lactic acidosis (HML) is an autosomal recessive disease caused by an intronic one-base mutation in the iron-sulfur cluster assembly (ISCU) gene, resulting in aberrant splicing. The incorrectly spliced transcripts contain a 100 or 86 bp intron sequence encoding a non-functional ISCU protein, which leads to defects in several Fe-S containing proteins in the respiratory chain and the TCA cycle. The symptoms in HML are restricted to skeletal muscle, and it has been proposed that this effect is due to higher levels of incorrectly spliced ISCU in skeletal muscle compared with other energy-demanding tissues. In this study, we confirm that skeletal muscle contains the highest levels of incorrect ISCU splice variants compared with heart, brain, liver and kidney using a transgenic mouse model expressing human HML mutated ISCU. We also show that incorrect splicing occurs to a significantly higher extent in the slow-twitch soleus muscle compared with the gastrocnemius and quadriceps. The splicing factor serine/arginine-rich splicing factor 3 (SRSF3) was identified as a potential candidate for the slow fiber specific regulation of ISCU splicing since this factor was expressed at higher levels in the soleus compared to the gastrocnemius and quadriceps. We identified an interaction between SRSF3 and the ISCU transcript, and by overexpressing SRSF3 in human myoblasts we observed increased levels of incorrectly spliced ISCU, while knockdown of SRSF3 resulted in decreased levels. We therefore suggest that SRSF3 may participate in the regulation of the incorrect splicing of mutant ISCU and may, at least partially, explain the muscle-specific symptoms of HML.

PTBP1 acts as a dominant repressor of the aberrant tissue‐specific splicing of ISCU in hereditary myopathy with lactic acidosis

“…It was therefore suggested that PTBP1 can act as a repressor of the incorrect splicing of ISCU (Nordin et al., ). In a recent study, we showed that the levels of incorrectly spliced ISCU vary among a number of mouse tissues, where the highest level of incorrect splicing was observed in the slow fiber muscle soleus (Rawcliffe et al., ). In this study, we examined the same tissues for levels of Ptbp1 RNA and protein to see whether there was a correlation between the levels of ISCU mis‐splicing and the levels of Ptbp1 (Figure a,b).…”

“…ISCU transgenic mice (Rawcliffe et al., ) from a CBA/B6 background were kept in standard cages with free access to water and food (CRM Expanded, SDS). Mice of both genders were sacrificed by cervical dislocation at 9 weeks of age, and the organs of interest were collected and immediately frozen in N 2 (l) followed by storage in −80°C.…”

“…HEK293T Lenti‐X cells (Clontech, Mountain View, CA, USA) were transfected by calcium phosphate precipitates as described previously by Rawcliffe et al. (). In brief, a total of 55 μ g of lentiviral DNA vectors (Addgene), such as psPAX2 (virus envelope), pMD2.G (virus proteins) and DNA of interest, were used in a ratio of 1:0.7:1 in a total volume of 1,500 μ l with 250 mmol/L CaCl 2 .…”

“…The μMACS Streptavidin kit (MACS Miltenyi Biotec, Bergisch Gladbach, Germany) was used to capture RNA‐binding factors as described by Rawcliffe et al., . ISCU oligos representing normal and mutated ISCU were used, where the mutated sequence has a C instead of a G, (5′–[Biotin]AGCUCCAAUCUUU C / G AUUUCAGAAUCUG–3′).…”

“…In contrast, only 30% and 10% mis‐splicing is observed in the heart and liver, respectively (Nordin et al., ). Furthermore, transgenic mice expressing human ISCU , show the highest levels of incorrect splicing in the slow fiber soleus muscle and serine and arginine‐rich splicing factor 3 (SRSF3, OMIM*603364) has been suggested to be involved in this muscle‐type‐specific splicing (Rawcliffe, Osterman, Lindsten, & Holmberg, ).…”