Overcoming stalled translation in human mitochondria

Abstract

Protein synthesis is central to life and maintaining a highly accurate and efficient mechanism is essential. What happens when a translating ribosome stalls on a messenger RNA? Many highly intricate processes have been documented in the cytosol of numerous species, but how does organellar protein synthesis resolve this stalling issue? Mammalian mitochondria synthesize just thirteen highly hydrophobic polypeptides. These proteins are all integral components of the machinery that couples oxidative phosphorylation. Consequently, it is essential that stalled mitochondrial ribosomes can be efficiently recycled. To date, there is no evidence to support any particular molecular mechanism to resolve this problem. However, here we discuss the observation that there are four predicted members of the mitochondrial translation release factor family and that only one member, mtRF1a, is necessary to terminate the translation of all thirteen open reading frames in the mitochondrion. Could the other members be involved in the process of recycling stalled mitochondrial ribosomes?

Keywords: ICT1; mitochondria; protein synthesis; release factor; ribosome rescue; ribosome stalling; translation.

FIGURE 1

ICT1 as a mitochondrial translation rescue factor and its possible orthologs. (A) Alignment of human ICT1 (14197) with the Pth4 ortholog from S. pombe (Q9HDZ3) and YaeJ ortholog from E. coli (E2QFB9). Identity is indicated by (*), high levels of similarity by (:) and lower levels by (⋅). The conserved GGQ region is boxed and the YaeJ residues that are required for PTH activity and are highly conserved between bacterial species are indicated by arrows. (B) Release activity of recombinant YaeJ and ICT1. PTH activity was tested on 70S ribosomes primed with either no RNA or UAA triplet in the A-site. (C) Mitochondrial targetted YaeJ-FLAG shows interaction with human mitochondrial ribosomal proteins. FLAG tag mediated immunoprecipitations of ICT1 and mitochondrially targeted YaeJ were performed on lysates of HEK293 cell lines induced for 3 days. The elution fractions (10%) were separated by SDS PAGE and analyzed by silver staining (left panel, FLAG protein indicated by *) or western blot (right panel). Antibodies against MRPL3, MRPL12, ICT1, DAP3, and MRPS18B were used to determine the relative levels of coimmunoprecipitated ribosomal proteins. The presence of FLAG tagged protein in each elution was confirmed by anti-FLAG antibodies. (D) Mitochondrially-targeted YaeJ-FLAG does not co-migrate with the 39S LSU. Lysate (700 μg) of mtYaeJ-FLAG expressing cells was separated on an isokinetic sucrose gradient. Fractions were analyzed by western blot using antibodies against the 39S LSU (MRPL3) and the 28S SSU (DAP3). The distribution of mtYaeJ-FLAG was determined by applying FLAG antibodies. Methods for panels (B–D) were essentially as described in Richter et al. (2010), except a YaeJ-FLAG construct was used to generate a HEK293T overexpression line instead of the ICT1 FLAG.

FIGURE 2

Schematic of a composite ribosome to show relative positions occupied by ICT1 and YaeJ. Truncated mRNA lacking an A-site codon allows ingress of YaeJ such that the C-terminal α-helix aligns within the mRNA entry channel in the small subunit (SSU). This positions the GGQ motif at the peptidyl transferase centre (PTC) allowing cleavage of the ester bond between the P-site tRNA and the truncated polypeptide (as described in Kogure et al., 2014). The aborted product is then released via the polypeptide exit site in the large subunit (LSU). ICT1 by contrast is located at the central protuberance (CP) precluding its interaction with the nascent peptide without a large scale conformational change of the ribosome (as described in Greber et al., 2013).