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A cell culture Model of MRPS2-related cutis laxa

Requena, Martin (2017) A cell culture Model of MRPS2-related cutis laxa. Master's Thesis, University of Pittsburgh. (Unpublished)

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Cutis laxa (CL) is a disorder with multiple etiologies characterized by defective or deficient production of elastin. Previous research has suggested that a significant portion of cases of CL are due to some type of mitochondrial dysfunction. Recent clinical cases implicate mitochondrial ribosomal protein S2 (MRPS2) as a possible cause of autosomal recessive CL. Despite this, in vitro tissue culture knockout of MRPS2 has not been seriously investigated as a model for studying the pathology of mitoribosomal mutation-caused wrinkly skin at a cellular and tissue level. In this study, we used CRISPR/Cas9 to lower the activity of MRPS2 in rat lung fibroblasts. We generated one heterozygous mutant with a mutation in one allele of exon 4, and two biallelic mutants, each with unique mutations in both alleles of exon 3. The biallelic mutations resulted in aberrant mRNA products, including skipping of exon 3 in both biallelic mutants and insertion/deletion mutations unique to each mutant. Analysis of secreted protein and lysate by immunoblotting revealed two MRPS2 fragments, around 8 and 33 kilodaltons in mass, respectively. Immunostaining of biallelic mutant cell lines revealed altered organization and reduced abundance of elastic fibers along with alterations in nuclear and cellular morphology and cell growth rates.
Public Health Significance: The study of the importance of mitoribosomal assembly for fibroblast health has consequences for both the study of cutis laxa and the more general study of the molecular pathogenesis of skin wrinkling.


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Item Type: University of Pittsburgh ETD
Status: Unpublished
CreatorsEmailPitt UsernameORCID
Requena, Martinmdr53@pitt.edumdr530000-0001-7997-5932
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Thesis AdvisorUrban, Zsolturbanz@pitt.eduurbanz0000-0003-3890-1843
Committee MemberPadiath, Quasarqpadiath@pitt.eduqpadiath0000-0002-2468-6364
Committee MemberGoetzman, EricEric.Goetzman@chp.eduesg50000-0001-9476-309X
Date: 29 June 2017
Date Type: Publication
Defense Date: 27 April 2017
Approval Date: 29 June 2017
Submission Date: 3 April 2017
Access Restriction: 1 year -- Restrict access to University of Pittsburgh for a period of 1 year.
Number of Pages: 49
Institution: University of Pittsburgh
Schools and Programs: School of Public Health > Human Genetics
Degree: MS - Master of Science
Thesis Type: Master's Thesis
Refereed: Yes
Uncontrolled Keywords: MRPS2 Cutis Laxa Elastin Fibrillin CRISPR CRISPR/cas9 RFL6 Fibroblast Extracellular Matrix Microfibrils Tropoelastin Elastogenesis Wrinkly Skin Mitochondria Mitochondrion Mitoribosome Mitochondrial Ribosome
Date Deposited: 29 Jun 2017 23:31
Last Modified: 01 May 2018 05:15


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