Investigation of SECISBP2 mutations and potential viral SECIS element recruitment
Investigation of SECISBP2 mutations and potential viral SECIS element recruitment
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DissertationPrüfungsdatum
30.06.2021Datum der Veröffentlichung
15.07.2021Erstgutachter
Schweizer, UlrichZweitgutachter
Schlee, MartinMetadaten
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Inhalt
The 21st amino acid selenocysteine (Sec) is encoded by the UGA codon. In order to recode the canonical stop codon additional factors are needed in mammals including the selenocysteine-insertion-sequence (SECIS) element and SECISBP2. Mutations of SECISBP2 have been identified in patients and this thesis investigates the R540Q and C691R mutations in an in-vitro luciferase assay.Moreover, it has been speculated that
viruses, although lacking a SECIS element, are able to express selenoproteins by using the SECIS element of a human mRNA via an antisense-tethering-interaction (ATI). This possibility was also investigated in an in-vitro luciferase assay.
Murine SECISBP2 was cloned and expressed in E. coli. Both R543Q and C696R mutations were introduced by site-directed mutagenesis. Different murine SECIS elements were cloned into the 3’ UTR of a luciferase reporter that contains a UGA instead of a cysteine codon. All reporters were transcribed in-vitro. In-vitro translation of the luciferase reporters together with SECISBP2 protein was performed and luminescence as a correlate for Sec incorporation was measured. Furthermore, the SECIS element of the same luciferase reporter was replaced by viral sequences predicted to take part in the ATI. Full-length mRNAs of human TXNRD1 and TXNRD3 were transcribed in-vitro, as well as the luciferase reporter. Finally, an in-vitro luciferase assay was performed mixing the reporter and TXNRD mRNAs.
The luciferase assay comparing the mutated SECISBP2 proteins with the wildtype showed that the C696R mutation causes highly reduced Sec-incorporation. No significant difference between the wildtype and the R543Q mutation could be found.
The luciferase assay trying to show an in-trans SECIS element recruitment by viral sequences did not show any luminescence beyond background. The present data show for the first time loss of function of the C691R mutant in an in-vitro assay, thereby showing that RNA binding of SECISBP2 is critical for its function.
Decreased thermal stability of the R540Q mutant protein could be observed which might explain the partial loss of function. A viral in-trans SECIS element recruitment could not be shown. Therefore, the proposed ATI may not take place or it simply could not be shown by the performed approach.
Murine SECISBP2 was cloned and expressed in E. coli. Both R543Q and C696R mutations were introduced by site-directed mutagenesis. Different murine SECIS elements were cloned into the 3’ UTR of a luciferase reporter that contains a UGA instead of a cysteine codon. All reporters were transcribed in-vitro. In-vitro translation of the luciferase reporters together with SECISBP2 protein was performed and luminescence as a correlate for Sec incorporation was measured. Furthermore, the SECIS element of the same luciferase reporter was replaced by viral sequences predicted to take part in the ATI. Full-length mRNAs of human TXNRD1 and TXNRD3 were transcribed in-vitro, as well as the luciferase reporter. Finally, an in-vitro luciferase assay was performed mixing the reporter and TXNRD mRNAs.
The luciferase assay comparing the mutated SECISBP2 proteins with the wildtype showed that the C696R mutation causes highly reduced Sec-incorporation. No significant difference between the wildtype and the R543Q mutation could be found.
The luciferase assay trying to show an in-trans SECIS element recruitment by viral sequences did not show any luminescence beyond background. The present data show for the first time loss of function of the C691R mutant in an in-vitro assay, thereby showing that RNA binding of SECISBP2 is critical for its function.
Decreased thermal stability of the R540Q mutant protein could be observed which might explain the partial loss of function. A viral in-trans SECIS element recruitment could not be shown. Therefore, the proposed ATI may not take place or it simply could not be shown by the performed approach.
Schlagwörter
Selen, Selenoproteine, SECISBP2, SBP2, Mutation, HIV, SECIS, Luciferase-Reporter, antisense-tethering-interaction
Klassifikation (DDC)
500 Naturwissenschaften 570 Biowissenschaften, Biologie 610 Medizin, GesundheitZugehörige Publikation(en)
https://doi.org/10.1074/jbc.RA119.009369Schmidt, Henrik Johannes: Investigation of SECISBP2 mutations and potential viral SECIS element recruitment. - Bonn, 2021. - Dissertation, Rheinische Friedrich-Wilhelms-Universität Bonn.
Online-Ausgabe in bonndoc: https://nbn-resolving.org/urn:nbn:de:hbz:5-63120
Online-Ausgabe in bonndoc: https://nbn-resolving.org/urn:nbn:de:hbz:5-63120
@phdthesis{handle:20.500.11811/9221,
urn: https://nbn-resolving.org/urn:nbn:de:hbz:5-63120,
author = {{Henrik Johannes Schmidt}},
title = {Investigation of SECISBP2 mutations and potential viral SECIS element recruitment},
school = {Rheinische Friedrich-Wilhelms-Universität Bonn},
year = 2021,
month = jul,
note = {The 21st amino acid selenocysteine (Sec) is encoded by the UGA codon. In order to recode the canonical stop codon additional factors are needed in mammals including the selenocysteine-insertion-sequence (SECIS) element and SECISBP2. Mutations of SECISBP2 have been identified in patients and this thesis investigates the R540Q and C691R mutations in an in-vitro luciferase assay.Moreover, it has been speculated that viruses, although lacking a SECIS element, are able to express selenoproteins by using the SECIS element of a human mRNA via an antisense-tethering-interaction (ATI). This possibility was also investigated in an in-vitro luciferase assay.
Murine SECISBP2 was cloned and expressed in E. coli. Both R543Q and C696R mutations were introduced by site-directed mutagenesis. Different murine SECIS elements were cloned into the 3’ UTR of a luciferase reporter that contains a UGA instead of a cysteine codon. All reporters were transcribed in-vitro. In-vitro translation of the luciferase reporters together with SECISBP2 protein was performed and luminescence as a correlate for Sec incorporation was measured. Furthermore, the SECIS element of the same luciferase reporter was replaced by viral sequences predicted to take part in the ATI. Full-length mRNAs of human TXNRD1 and TXNRD3 were transcribed in-vitro, as well as the luciferase reporter. Finally, an in-vitro luciferase assay was performed mixing the reporter and TXNRD mRNAs.
The luciferase assay comparing the mutated SECISBP2 proteins with the wildtype showed that the C696R mutation causes highly reduced Sec-incorporation. No significant difference between the wildtype and the R543Q mutation could be found.
The luciferase assay trying to show an in-trans SECIS element recruitment by viral sequences did not show any luminescence beyond background. The present data show for the first time loss of function of the C691R mutant in an in-vitro assay, thereby showing that RNA binding of SECISBP2 is critical for its function.
Decreased thermal stability of the R540Q mutant protein could be observed which might explain the partial loss of function. A viral in-trans SECIS element recruitment could not be shown. Therefore, the proposed ATI may not take place or it simply could not be shown by the performed approach.},
url = {https://hdl.handle.net/20.500.11811/9221}
}
urn: https://nbn-resolving.org/urn:nbn:de:hbz:5-63120,
author = {{Henrik Johannes Schmidt}},
title = {Investigation of SECISBP2 mutations and potential viral SECIS element recruitment},
school = {Rheinische Friedrich-Wilhelms-Universität Bonn},
year = 2021,
month = jul,
note = {The 21st amino acid selenocysteine (Sec) is encoded by the UGA codon. In order to recode the canonical stop codon additional factors are needed in mammals including the selenocysteine-insertion-sequence (SECIS) element and SECISBP2. Mutations of SECISBP2 have been identified in patients and this thesis investigates the R540Q and C691R mutations in an in-vitro luciferase assay.Moreover, it has been speculated that viruses, although lacking a SECIS element, are able to express selenoproteins by using the SECIS element of a human mRNA via an antisense-tethering-interaction (ATI). This possibility was also investigated in an in-vitro luciferase assay.
Murine SECISBP2 was cloned and expressed in E. coli. Both R543Q and C696R mutations were introduced by site-directed mutagenesis. Different murine SECIS elements were cloned into the 3’ UTR of a luciferase reporter that contains a UGA instead of a cysteine codon. All reporters were transcribed in-vitro. In-vitro translation of the luciferase reporters together with SECISBP2 protein was performed and luminescence as a correlate for Sec incorporation was measured. Furthermore, the SECIS element of the same luciferase reporter was replaced by viral sequences predicted to take part in the ATI. Full-length mRNAs of human TXNRD1 and TXNRD3 were transcribed in-vitro, as well as the luciferase reporter. Finally, an in-vitro luciferase assay was performed mixing the reporter and TXNRD mRNAs.
The luciferase assay comparing the mutated SECISBP2 proteins with the wildtype showed that the C696R mutation causes highly reduced Sec-incorporation. No significant difference between the wildtype and the R543Q mutation could be found.
The luciferase assay trying to show an in-trans SECIS element recruitment by viral sequences did not show any luminescence beyond background. The present data show for the first time loss of function of the C691R mutant in an in-vitro assay, thereby showing that RNA binding of SECISBP2 is critical for its function.
Decreased thermal stability of the R540Q mutant protein could be observed which might explain the partial loss of function. A viral in-trans SECIS element recruitment could not be shown. Therefore, the proposed ATI may not take place or it simply could not be shown by the performed approach.},
url = {https://hdl.handle.net/20.500.11811/9221}
}
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