A Versatile Strategy to Reduce UGA-Selenocysteine Recoding Efficiency of the Ribosome Using CRISPR-Cas9-Viral-Like-Particles Targeting Selenocysteine-tRNA<sup>[Ser]Sec</sup> Gene
The translation of selenoprotein mRNAs involves a non-canonical ribosomal event in which an in-frame UGA is recoded as a selenocysteine (Sec) codon instead of being read as a stop codon. The recoding machinery is centered around two dedicated RNA components: The selenocysteine insertion sequence (SE...
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doaj-b4f223641178404fa72f0ad99576a24b2020-11-25T01:59:00ZengMDPI AGCells2073-44092019-06-018657410.3390/cells8060574cells8060574A Versatile Strategy to Reduce UGA-Selenocysteine Recoding Efficiency of the Ribosome Using CRISPR-Cas9-Viral-Like-Particles Targeting Selenocysteine-tRNA<sup>[Ser]Sec</sup> GeneCaroline Vindry0Olivia Guillin1Philippe E. Mangeot2Théophile Ohlmann3Laurent Chavatte4Centre International de Recherche en Infectiologie (CIRI), 69007 Lyon, FranceCentre International de Recherche en Infectiologie (CIRI), 69007 Lyon, FranceCentre International de Recherche en Infectiologie (CIRI), 69007 Lyon, FranceCentre International de Recherche en Infectiologie (CIRI), 69007 Lyon, FranceCentre International de Recherche en Infectiologie (CIRI), 69007 Lyon, FranceThe translation of selenoprotein mRNAs involves a non-canonical ribosomal event in which an in-frame UGA is recoded as a selenocysteine (Sec) codon instead of being read as a stop codon. The recoding machinery is centered around two dedicated RNA components: The selenocysteine insertion sequence (SECIS) located in the 3′ UTR of the mRNA and the selenocysteine-tRNA (Sec-tRNA<sup>[Ser]Sec</sup>). This translational UGA-selenocysteine recoding event by the ribosome is a limiting stage of selenoprotein expression. Its efficiency is controlled by the SECIS, the Sec-tRNA<sup>[Ser]Sec</sup> and their interacting protein partners. In the present work, we used a recently developed CRISPR strategy based on murine leukemia virus-like particles (VLPs) loaded with Cas9-sgRNA ribonucleoproteins to inactivate the Sec-tRNA<sup>[Ser]Sec</sup> gene in human cell lines. We showed that these CRISPR-Cas9-VLPs were able to induce efficient genome-editing in Hek293, HepG2, HaCaT, HAP1, HeLa, and LNCaP cell lines and this caused a robust reduction of selenoprotein expression. The alteration of selenoprotein expression was the direct consequence of lower levels of Sec-tRNA<sup>[Ser]Sec</sup> and thus a decrease in translational recoding efficiency of the ribosome. This novel strategy opens many possibilities to study the impact of selenoprotein deficiency in hard-to-transfect cells, since these CRISPR-Cas9-VLPs have a wide tropism.https://www.mdpi.com/2073-4409/8/6/574seleniumselenocysteineselenoproteinSECISSec-tRNA<sup>[Ser]Sec</sup>UGA-recodingCRISPR-Cas9viral-like particlesnanoblades |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Caroline Vindry Olivia Guillin Philippe E. Mangeot Théophile Ohlmann Laurent Chavatte |
spellingShingle |
Caroline Vindry Olivia Guillin Philippe E. Mangeot Théophile Ohlmann Laurent Chavatte A Versatile Strategy to Reduce UGA-Selenocysteine Recoding Efficiency of the Ribosome Using CRISPR-Cas9-Viral-Like-Particles Targeting Selenocysteine-tRNA<sup>[Ser]Sec</sup> Gene Cells selenium selenocysteine selenoprotein SECIS Sec-tRNA<sup>[Ser]Sec</sup> UGA-recoding CRISPR-Cas9 viral-like particles nanoblades |
author_facet |
Caroline Vindry Olivia Guillin Philippe E. Mangeot Théophile Ohlmann Laurent Chavatte |
author_sort |
Caroline Vindry |
title |
A Versatile Strategy to Reduce UGA-Selenocysteine Recoding Efficiency of the Ribosome Using CRISPR-Cas9-Viral-Like-Particles Targeting Selenocysteine-tRNA<sup>[Ser]Sec</sup> Gene |
title_short |
A Versatile Strategy to Reduce UGA-Selenocysteine Recoding Efficiency of the Ribosome Using CRISPR-Cas9-Viral-Like-Particles Targeting Selenocysteine-tRNA<sup>[Ser]Sec</sup> Gene |
title_full |
A Versatile Strategy to Reduce UGA-Selenocysteine Recoding Efficiency of the Ribosome Using CRISPR-Cas9-Viral-Like-Particles Targeting Selenocysteine-tRNA<sup>[Ser]Sec</sup> Gene |
title_fullStr |
A Versatile Strategy to Reduce UGA-Selenocysteine Recoding Efficiency of the Ribosome Using CRISPR-Cas9-Viral-Like-Particles Targeting Selenocysteine-tRNA<sup>[Ser]Sec</sup> Gene |
title_full_unstemmed |
A Versatile Strategy to Reduce UGA-Selenocysteine Recoding Efficiency of the Ribosome Using CRISPR-Cas9-Viral-Like-Particles Targeting Selenocysteine-tRNA<sup>[Ser]Sec</sup> Gene |
title_sort |
versatile strategy to reduce uga-selenocysteine recoding efficiency of the ribosome using crispr-cas9-viral-like-particles targeting selenocysteine-trna<sup>[ser]sec</sup> gene |
publisher |
MDPI AG |
series |
Cells |
issn |
2073-4409 |
publishDate |
2019-06-01 |
description |
The translation of selenoprotein mRNAs involves a non-canonical ribosomal event in which an in-frame UGA is recoded as a selenocysteine (Sec) codon instead of being read as a stop codon. The recoding machinery is centered around two dedicated RNA components: The selenocysteine insertion sequence (SECIS) located in the 3′ UTR of the mRNA and the selenocysteine-tRNA (Sec-tRNA<sup>[Ser]Sec</sup>). This translational UGA-selenocysteine recoding event by the ribosome is a limiting stage of selenoprotein expression. Its efficiency is controlled by the SECIS, the Sec-tRNA<sup>[Ser]Sec</sup> and their interacting protein partners. In the present work, we used a recently developed CRISPR strategy based on murine leukemia virus-like particles (VLPs) loaded with Cas9-sgRNA ribonucleoproteins to inactivate the Sec-tRNA<sup>[Ser]Sec</sup> gene in human cell lines. We showed that these CRISPR-Cas9-VLPs were able to induce efficient genome-editing in Hek293, HepG2, HaCaT, HAP1, HeLa, and LNCaP cell lines and this caused a robust reduction of selenoprotein expression. The alteration of selenoprotein expression was the direct consequence of lower levels of Sec-tRNA<sup>[Ser]Sec</sup> and thus a decrease in translational recoding efficiency of the ribosome. This novel strategy opens many possibilities to study the impact of selenoprotein deficiency in hard-to-transfect cells, since these CRISPR-Cas9-VLPs have a wide tropism. |
topic |
selenium selenocysteine selenoprotein SECIS Sec-tRNA<sup>[Ser]Sec</sup> UGA-recoding CRISPR-Cas9 viral-like particles nanoblades |
url |
https://www.mdpi.com/2073-4409/8/6/574 |
work_keys_str_mv |
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