Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.815516
Title: Biochemical and genetic characterisation of ciliary transition zone transmembrane proteins in cystic kidney disease and ciliopathies
Author: Shoaib, Ekram Abdullah
ISNI:       0000 0004 9358 1341
Awarding Body: University of Leeds
Current Institution: University of Leeds
Date of Award: 2020
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Abstract:
Meckel-Gruber syndrome (MKS) and Joubert syndrome (JBTS) are rare autosomal recessive genetic disorders caused by genetic defects in proteins responsible for primary cilia formation, maintenance or function. Primary cilia regulate essential cellular and developmental processes and have a complex compartmentalized organization. This project focuses on transmembrane proteins (TMEMs) that form the “MKS/JBTS functional module” at the ciliary transition zone compartment. These include TMEM17 TMEM67, TMEM138, TMEM216, TMEM231 and TMEM237 that, when mutated, cause MKS or JBTS. The detailed biochemical and genetic interactions that form this functional module remain unclear. This project describes biochemical and cell biological approaches to understand the functional roles of ciliary TMEMs. Biochemical interactions were defined through a series of co-immunoprecipitation assays of epitope-tagged and endogenous ciliary TMEM proteins. This showed that TMEM17 interacts with TMEM138, TMEM237, TMEM216, TMEM67 and the intraflagellar transport protein IFT88. Tandem affinity purification (TAP) followed by mass spectroscopy confirmed that TMEM17 interacted with TMEM237. In addition, reciprocal biochemical interactions confirmed novel interactions between TMEM237-TMEM216 and TMEM237-TMEM17. TMEM17 also interacted with the pre-mRNA splicing factors (PRPF8 and PRPF6) that do not have an established role in the ciliary apparatus. Genetic interactions were also determined between ciliary TMEMs and other ciliary proteins (RPGRIP1L, CEP290 and IFT88) chosen because they localize to different ciliary compartments and appear to form distinct functional modules. Genetic interactions were inferred from co-localization studies in 3D reconstituted confocal microscopy images following a series of RNAi knockdowns. This highlighted the significance of the TMEM17-TMEM67 genetic interaction in organizing ciliary sub-compartments. Finally, both biochemical and genetic interactions delineated the existence of a novel complex between TMEM237, TMEM17 and TMEM138 with the ciliary protein IFT88. These results provide new insights into how ciliary trafficking and ciliogenesis could be mediated through interactions with TMEM components at the ciliary transition zone.
Supervisor: Johnson, Colin ; Bell, Sandra ; Morrison, Ewan Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.815516  DOI: Not available
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