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High-Resolution Ribosome Profiling Reveals Gene-Specific Details of UGA Re-Coding in Selenoprotein Biosynthesis

ORCID
0000-0002-1551-9839
Affiliation
Institut für Biochemie und Molekularbiologie, Universitätsklinikum Bonn, Rheinische Friedrich-Wilhelms-Universität Bonn, 53115 Bonn, Germany
Bohleber, Simon;
ORCID
0000-0003-3418-0743
Affiliation
Institut für Biochemie und Molekularbiologie, Universitätsklinikum Bonn, Rheinische Friedrich-Wilhelms-Universität Bonn, 53115 Bonn, Germany
Fradejas-Villar, Noelia;
Affiliation
Institut für Biochemie und Molekularbiologie, Universitätsklinikum Bonn, Rheinische Friedrich-Wilhelms-Universität Bonn, 53115 Bonn, Germany
Zhao, Wenchao;
Affiliation
Institut für Biochemie und Molekularbiologie, Universitätsklinikum Bonn, Rheinische Friedrich-Wilhelms-Universität Bonn, 53115 Bonn, Germany
Reuter, Uschi;
ORCID
0000-0003-1380-4780
Affiliation
Institut für Biochemie und Molekularbiologie, Universitätsklinikum Bonn, Rheinische Friedrich-Wilhelms-Universität Bonn, 53115 Bonn, Germany
Schweizer, Ulrich

Co-translational incorporation of selenocysteine (Sec) into selenoproteins occurs at UGA codons in a process in which translational elongation competes with translational termination. Selenocysteine insertion sequence-binding protein 2 ( SECISBP2 ) greatly enhances Sec incorporation into selenoproteins by interacting with the mRNA, ribosome, and elongation factor Sec (EFSEC). Ribosomal profiling allows to study the process of UGA re-coding in the physiological context of the cell and at the same time for all individual selenoproteins expressed in that cell. Using HAP1 cells expressing a mutant SECISBP2 , we show here that high-resolution ribosomal profiling can be used to assess read-through efficiency at the UGA in all selenoproteins, including those with Sec close to the C-terminus. Analysis of ribosomes with UGA either at the A-site or the P-site revealed, in a transcript-specific manner, that SECISBP2 helps to recruit tRNA Sec and stabilize the mRNA. We propose to assess the effect of any perturbation of UGA read-through by determining the proportion of ribosomes carrying UGA in the P-site, pUGA. An additional, new observation is frameshifting that occurred 3′ of the UGA/Sec codon in SELENOF and SELENOW in SECISBP2 -mutant HAP1 cells, a finding corroborated by reanalysis of neuron-specific Secisbp2 R543Q -mutant brains.

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