Abstract
BackgroundHereditary 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.MethodsOverexpression 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.ConclusionOur 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.
Highlights
Hereditary myopathy with lactic acidosis (HML, OMIM# 255125) is an autosomal recessive disease characterized by a low tolerance to exercise from an early age (Larsson, Linderholm, M€uller, Ringqvist, & S€orn€as, 1964; Mochel et al, 2008; Olsson, Lind, Thornell, & Holmberg, 2008)
Our results indicate that PTBP1 is a major determinant in the pathology of HML, where the levels of PTBP1 in a tissue controls the levels of mis-splicing of mutant iron-sulfur cluster assembly (ISCU)
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, 2016)
Summary
Hereditary myopathy with lactic acidosis (HML, OMIM# 255125) is an autosomal recessive disease characterized by a low tolerance to exercise from an early age (Larsson, Linderholm, M€uller, Ringqvist, & S€orn€as, 1964; Mochel et al, 2008; Olsson, Lind, Thornell, & Holmberg, 2008). The disease is caused by an intronic G>C mutation (NC_000012.12 (NM_213595):c.418 + 382G>C) in the iron-sulfur cluster assembly gene (ISCU, OMIM *611911) (Kollberg et al, 2009; Mochel et al, 2008; Olsson et al, 2008). A de novo ISCU missense mutation, c.287G>T, that resulted in a dominant form of myopathy, was reported in a 23-year-old Italian male (Legati et al, 2017) In both cases, the mutations resulted in a more severe and progressive phenotype with additional symptoms aside from the myopathy (Kollberg et al, 2009; Legati et al, 2017). Using an ISCU mini-gene system, overexpression of PTBP1 was shown to markedly decrease the mis-splicing (Nordin, Larsson, & Holmberg, 2012). Our results indicate that PTBP1 is a major determinant in the pathology of HML, where the levels of PTBP1 in a tissue controls the levels of mis-splicing of mutant ISCU
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