A Versatile Strategy to Reduce UGA-Selenocysteine Recoding Efficiency of the Ribosome Using CRISPR-Cas9-Viral-Like-Particles Targeting Selenocysteine-tRNA[Ser]Sec Gene.

Caroline Vindry,Olivia Guillin,Théophile Ohlmann,Laurent Chavatte,Philippe E Mangeot

doi:10.3390/cells8060574

Abstract

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[Ser]Sec). 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[Ser]Sec 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[Ser]Sec 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[Ser]Sec 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.

Highlights

Selenocysteine (Sec) is the 21st proteinogenic amino acid and it was the first addition to the genetic code deciphered in the 1960s
We aimed at altering the Sec-tRNA[Ser]Sec gene to decrease overall selenoprotein expression without affecting cell viability
After cutting the genomic DNA (gDNA) at this specific locus, nucleotide insertion or deletion were generated by the non-homologous end-joining (NHEJ) pathway leading to either a less functional tRNA and/or a decreased expression of the tRNA

Summary

Introduction

Selenocysteine (Sec) is the 21st proteinogenic amino acid and it was the first addition to the genetic code deciphered in the 1960s. 25 selenoprotein genes have been identified [8], in which the termination signal is often one of the two other stop codons, namely UAA or UAG. This UGA/Sec recoding process is possible due. The SECIS is necessary and sufficient to drive the efficient recoding of an in-frame UGA codon This feature has been convenient when performing a structure–function analysis of the SECIS element in heterologous gene systems, such as luciferase reporter constructs containing the SECIS in the 30 UTR [9,11,12,13,14,15]. The second key RNA component of Sec insertion machinery is the Sec-tRNA[Ser]Sec (Figure 1A) which associates with a selenocysteine-specific elongation factor (EFSec) [17,18]

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