DENR-MCTS1 heterodimerization and tRNA recruitment are required for translation reinitiation

Yasar Luqman Ahmed; Sibylle Schleich; Jonathan Bohlen; Nicolas Mandel; Bernd Simon; Irmgard Sinning; Aurelio A Teleman

doi:10.1371/journal.pbio.2005160

DENR-MCTS1 heterodimerization and tRNA recruitment are required for translation reinitiation

PLoS Biol. 2018 Jun 11;16(6):e2005160. doi: 10.1371/journal.pbio.2005160. eCollection 2018 Jun.

Authors

Yasar Luqman Ahmed¹, Sibylle Schleich^{2

3}, Jonathan Bohlen^{2

3}, Nicolas Mandel^{2

3}, Bernd Simon⁴, Irmgard Sinning¹, Aurelio A Teleman^{2

3

5}

Affiliations

¹ Heidelberg University Biochemistry Center (BZH), Heidelberg, Germany.
² German Cancer Research Center (DKFZ), Heidelberg, Germany.
³ Heidelberg University, Heidelberg, Germany.
⁴ European Molecular Biology Laboratory (EMBL), Structural and Computational Biology Unit, Heidelberg, Germany.
⁵ National Center for Tumor Diseases (NCT), Heidelberg, Germany.

Abstract

The succession of molecular events leading to eukaryotic translation reinitiation-whereby ribosomes terminate translation of a short open reading frame (ORF), resume scanning, and then translate a second ORF on the same mRNA-is not well understood. Density-regulated reinitiation and release factor (DENR) and multiple copies in T-cell lymphoma-1 (MCTS1) are implicated in promoting translation reinitiation both in vitro in translation extracts and in vivo. We present here the crystal structure of MCTS1 bound to a fragment of DENR. Based on this structure, we identify and experimentally validate that DENR residues Glu42, Tyr43, and Tyr46 are important for MCTS1 binding and that MCTS1 residue Phe104 is important for tRNA binding. Mutation of these residues reveals that DENR-MCTS1 dimerization and tRNA binding are both necessary for DENR and MCTS1 to promote translation reinitiation in human cells. These findings thereby link individual residues of DENR and MCTS1 to specific molecular functions of the complex. Since DENR-MCTS1 can bind tRNA in the absence of the ribosome, this suggests the DENR-MCTS1 complex could recruit tRNA to the ribosome during reinitiation analogously to the eukaryotic initiation factor 2 (eIF2) complex in cap-dependent translation.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Amino Acid Substitution
Cell Cycle Proteins / chemistry*
Cell Cycle Proteins / genetics
Cell Cycle Proteins / metabolism*
Crystallography, X-Ray
Eukaryotic Initiation Factor-2 / metabolism
Eukaryotic Initiation Factors / chemistry*
Eukaryotic Initiation Factors / genetics
Eukaryotic Initiation Factors / metabolism*
HeLa Cells
Humans
Models, Molecular
Mutagenesis, Site-Directed
Oncogene Proteins / chemistry*
Oncogene Proteins / genetics
Oncogene Proteins / metabolism*
Open Reading Frames
Peptide Chain Initiation, Translational
Protein Interaction Domains and Motifs
Protein Multimerization
RNA, Transfer / genetics
RNA, Transfer / metabolism*
Ribosomes / metabolism

Substances

Cell Cycle Proteins
DENR protein, human
Eukaryotic Initiation Factor-2
Eukaryotic Initiation Factors
MCTS1 protein, human
Oncogene Proteins
RNA, Transfer

Grants and funding

Deutsche Forschungsgemeinschaft (grant number TE 766/7-1). received by AAT. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Deutsche Forschungsgemeinschaft (grant number SFB1036). received by IS and AAT. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Leibniz Programme (grant number SI 586/6-1). received by IS. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. DKFZ NCT3.0 Integrative Project in Cancer Research (grant number NCT3.0_2015.54 DysregPT). received by AAT. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. CellNetworks. AAT and IS are investigators of the Cluster of Excellence: CellNetworks. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.