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Rawcliffe, Denise F. R.ORCID iD iconorcid.org/0000-0001-6632-5770
Publications (4 of 4) Show all publications
Rawcliffe, D. F. R., Österman, L., Nordin, A. & Holmberg, M. (2018). PTBP1 acts as a dominant repressor of the aberrant tissue-specific splicing of ISCU in hereditary myopathy with lactic acidosis. Molecular Genetics & Genomic Medicine, 6(6), 887-897
Open this publication in new window or tab >>PTBP1 acts as a dominant repressor of the aberrant tissue-specific splicing of ISCU in hereditary myopathy with lactic acidosis
2018 (English)In: Molecular Genetics & Genomic Medicine, ISSN 2324-9269, Vol. 6, no 6, p. 887-897Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
John Wiley & Sons, 2018
Keywords
alternative splicing, hereditary myopathy, ISCU, PTBP1
National Category
Medical Genetics
Identifiers
urn:nbn:se:umu:diva-153102 (URN)10.1002/mgg3.413 (DOI)000454205500003 ()30209894 (PubMedID)2-s2.0-85053400372 (Scopus ID)
Available from: 2018-11-07 Created: 2018-11-07 Last updated: 2019-01-14Bibliographically approved
Rawcliffe, D. F. R. (2018). The regulation of incorrect splicing of ISCU in hereditary myopathy with lactic acidosis. (Doctoral dissertation). Umeå: Umeå Universitet
Open this publication in new window or tab >>The regulation of incorrect splicing of ISCU in hereditary myopathy with lactic acidosis
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Patients suffering from hereditary myopathy with lactic acidosis (HML) can be found in the northern Swedish counties of Ångermanland and Västerbotten. HML is a rare autosomal recessive disease where patients display a low tolerance to exercise at an early age. Exercise can trigger symptoms such as palpitations, tachycardia, muscle cramps and dyspnoea. Extensive exercise or strict diets can result in myoglobinuria and life-threatening levels of lactic acid. The disease is caused by a nonsense G > C mutation (c.418 + 328G < C) in the last intron of the iron-sulphur (FeS) cluster assembly gene (ISCU), resulting in nonsense-mediated decay (NMD) of the transcript due to incorrect splicing. The ISCU protein is involved in the assembly of FeS clusters, which are essential cofactors for a wide range of proteins. Patient muscles display decreased levels of several FeS cluster proteins: mitochondrial aconitase in the tricarboxylic acid (TCA) cycle and Complex I, II (succinate dehydrogenase [SDH]) and III in the electron transport chain (ETC). The incorrect splicing of ISCU occurs to the highest extent in HML patient skeletal muscle, restricting the loss of ISCU protein to muscles, thereby preventing a more severe phenotype.

We found that the incorrect splicing occurs to the highest extent in slow-fibre muscle, which may be caused by the serine/arginine-rich splicing factor (SRSF3) as it is expressed at higher levels in slow-fibre muscle compared to other muscles, and since it is able to activate the incorrect splicing of ISCU. Following muscle, there is a gradual decrease of the incorrect splicing in heart, brain, liver and kidney, which is negatively correlated with the levels of the splicing inhibitor polypyrimidine-tract binding protein 1 (PTBP1). Overexpression of PTBP1 in HML patient myoblasts resulted in a drastic decrease in the incorrect splicing, while a PTBP1 knockdown had the opposite effect. Our results suggest that PTBP1 acts as a dominant inhibitor of the incorrect splicing and is likely the main cause for the tissue-specific splicing of ISCU in HML. We also identified RBM39 and MBNL1 as activators of the incorrect splicing of ISCU, which, together with the low levels of PTBP1, could explain the high levels of incorrect splicing in muscle.

Since almost 95% of all human gene transcripts are alternatively spliced, it is not surprising that a wide range of diseases are caused by mutations that affect splicing. Further knowledge of the function of splicing, such as tissue-specific splicing, can provide vital information for the development of therapies for diseases caused by splicing.

Place, publisher, year, edition, pages
Umeå: Umeå Universitet, 2018. p. 68
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1978
Keywords
HML, ISCU, Splicing, SRSF3, PTBP1, RBM39, MBNL1
National Category
Medical Genetics
Research subject
Medical Genetics
Identifiers
urn:nbn:se:umu:diva-153174 (URN)978-91-7601-954-2 (ISBN)
Public defence
2018-12-04, Betula, Byggnad 6M, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2018-11-13 Created: 2018-11-08 Last updated: 2018-11-09Bibliographically approved
Rawcliffe, D. F. R., Österman, L., Lindsten, H. & Holmberg, M. (2016). The High Level of Aberrant Splicing of ISCU in Slow-Twitch Muscle May Involve the Splicing Factor SRSF3. PLOS ONE, 11(10), Article ID e0165453.
Open this publication in new window or tab >>The High Level of Aberrant Splicing of ISCU in Slow-Twitch Muscle May Involve the Splicing Factor SRSF3
2016 (English)In: PLOS ONE, E-ISSN 1932-6203, Vol. 11, no 10, article id e0165453Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
Public library science, 2016
National Category
Medical Genetics
Identifiers
urn:nbn:se:umu:diva-129745 (URN)10.1371/journal.pone.0165453 (DOI)000389602800075 ()27783661 (PubMedID)2-s2.0-84993965373 (Scopus ID)
Available from: 2017-01-09 Created: 2017-01-09 Last updated: 2023-03-24Bibliographically approved
Rawcliffe, D. F. R., Johansson, M., Österman, L. & Holmberg, M.MBNL1 and RBM39 can activate the incorrect splicing of ISCU and the aberrant transcript is a target for nonsense-mediated decay.
Open this publication in new window or tab >>MBNL1 and RBM39 can activate the incorrect splicing of ISCU and the aberrant transcript is a target for nonsense-mediated decay
(English)Manuscript (preprint) (Other academic)
National Category
Medical Genetics
Identifiers
urn:nbn:se:umu:diva-153148 (URN)
Available from: 2018-11-08 Created: 2018-11-08 Last updated: 2018-11-09
Organisations
Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0001-6632-5770

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