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

Background Hereditary myopathy with lactic acidosis (HML) is an autosomal recessive disease caused by an intron mutation in the iron‐sulfur cluster assembly (ISCU) gene. The mutation results in aberrant splicing, where part of the intron is retained in the final mRNA transcript, giving rise to a truncated nonfunctional ISCU protein. Using an ISCU mini‐gene system, we have previously shown that PTBP1 can act as a repressor of the mis‐splicing of ISCU, where overexpression of PTBP1 resulted in a decrease of the incorrect splicing. In this study, we wanted to, in more detail, analyze the role of PTBP1 in the regulation of endogenous ISCU mis‐splicing. Methods Overexpression and knockdown of PTBP1 was performed in myoblasts from two HML patients and a healthy control. Quantification of ISCU mis‐splicing was done by qRTPCR. Biotinylated ISCU RNA, representing wildtype and mutant intron sequence, was used in a pull‐down assay with nuclear extracts from myoblasts. Levels of PTBP1 in human cell lines and mice tissues were analyzed by qRTPCR and western blot. Results PTBP1 overexpression in HML patient myoblasts resulted in a substantial decrease of ISCU mis‐splicing while knockdown of PTBP1 resulted in a drastic increase. The effect could be observed in both patient and control myoblasts. We could also show that PTBP1 interacts with both the mutant and wild‐type ISCU intron sequence, but with a higher affinity to the mutant sequence. Furthermore, low levels of PTBP1 among examined mouse tissues correlated with high levels of incorrect splicing of ISCU. Conclusion Our results show that PTBP1 acts as a dominant repressor of ISCU mis‐splicing. We also show an inverse correlation between the levels of PTBP1 and ISCU mis‐splicing, suggesting that the high level of mis‐splicing in the skeletal muscle is primarily due to the low levels of PTBP1. Hereditary myopathy with lactic acidosis (HML) is caused by an intron mutation in the ISCU gene resulting in aberrant splicing. Our results show that PTBP1 acts as a dominant repressor of ISCU mis‐splicing. We also show an inverse correlation between the levels of PTBP1 and ISCU mis‐splicing, suggesting that the high level of mis‐splicing in the skeletal muscle is primarily due to the low levels of PTBP1.