Ribosome rescue and translation termination at non-standard stop codons by ICT1 in mammalian mitochondria

PLoS Genet. 2014 Sep 18;10(9):e1004616. doi: 10.1371/journal.pgen.1004616. eCollection 2014 Sep.

Abstract

Release factors (RFs) govern the termination phase of protein synthesis. Human mitochondria harbor four different members of the class 1 RF family: RF1Lmt/mtRF1a, RF1mt, C12orf65 and ICT1. The homolog of the essential ICT1 factor is widely distributed in bacteria and organelles and has the peculiar feature in human mitochondria to be part of the ribosome as a ribosomal protein of the large subunit. The factor has been suggested to rescue stalled ribosomes in a codon-independent manner. The mechanism of action of this factor was obscure and is addressed here. Using a homologous mitochondria system of purified components, we demonstrate that the integrated ICT1 has no rescue activity. Rather, purified ICT1 binds stoichiometrically to mitochondrial ribosomes in addition to the integrated copy and functions as a general rescue factor, i.e. it releases the polypeptide from the peptidyl tRNA from ribosomes stalled at the end or in the middle of an mRNA or even from non-programmed ribosomes. The data suggest that the unusual termination at a sense codon (AGA/G) of the oxidative-phosphorylation enzymes CO1 and ND6 is also performed by ICT1 challenging a previous model, according to which RF1Lmt/mtRF1a is responsible for the translation termination at non-standard stop codons. We also demonstrate by mutational analyses that the unique insertion sequence present in the N-terminal domain of ICT1 is essential for peptide release rather than for ribosome binding. The function of RF1mt, another member of the class1 RFs in mammalian mitochondria, was also examined and is discussed.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Codon
  • Codon, Terminator*
  • Humans
  • Mitochondria / genetics*
  • Mitochondria / metabolism*
  • Mitochondria, Liver / genetics
  • Mitochondria, Liver / metabolism
  • Models, Biological
  • Molecular Sequence Data
  • Peptide Chain Termination, Translational* / drug effects
  • Protein Binding
  • Protein Biosynthesis
  • Protein Interaction Domains and Motifs
  • Proteins / chemistry
  • Proteins / metabolism*
  • Proteins / pharmacology
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • Ribosomal Proteins
  • Ribosomes / metabolism*
  • Sequence Alignment
  • Swine

Substances

  • Codon
  • Codon, Terminator
  • MRPL58 protein, human
  • Proteins
  • RNA, Messenger
  • Ribosomal Proteins

Grant support

This work was supported by a Grant-in-Aid for Young Scientists (B) and a Grant-in-Aid for Scientific Research (C) from the Japan Society for the Promotion of Science (JSPS), a Grant-in-Aid for Scientific Research on Innovative Areas from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (MEXT), and Grants-in Aid from the Takeda Science Foundation, the Naito Foundation, the Uehara Memorial Foundation, and the Mochida Memorial Foundation for Medical and Pharmaceutical Research to NT. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.