PTBP 1 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 PTBP 1 can act as a repressor of the mis‐splicing of ISCU , where overexpression of PTBP 1 resulted in a decrease of the incorrect splicing. In this study, we wanted to, in more detail, analyze the role of PTBP 1 in the regulation of endogenous ISCU mis‐splicing. Methods Overexpression and knockdown of PTBP 1 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 PTBP 1 in human cell lines and mice tissues were analyzed by qRTPCR and western blot. Results PTBP 1 overexpression in HML patient myoblasts resulted in a substantial decrease of ISCU mis‐splicing while knockdown of PTBP 1 resulted in a drastic increase. The effect could be observed in both patient and control myoblasts. We could also show that PTBP 1 interacts with both the mutant and wild‐type ISCU intron sequence, but with a higher affinity to the mutant sequence. Furthermore, low levels of PTBP 1 among examined mouse tissues correlated with high levels of incorrect splicing of ISCU . Conclusion Our results show that PTBP 1 acts as a dominant repressor of ISCU mis‐splicing. We also show an inverse correlation between the levels of PTBP 1 and ISCU mis‐splicing, suggesting that the high level of mis‐splicing in the skeletal muscle is primarily due to the low levels of PTBP 1.