Identification of transcripts with short stuORFs as targets for DENR•MCTS1-dependent translation in human cells

Abstract

The non-canonical initiation factors DENR and MCTS1 have been linked to cancer and autism. We recently showed in Drosophila that DENR and MCTS1 regulate translation re-initiation on transcripts containing upstream Open Reading Frames (uORFs) with strong Kozak sequences (stuORFs). Due to the medical relevance of DENR and MCTS1, it is worthwhile identifying the transcripts in human cells that depend on DENR and MCTS1 for their translation. We show here that in humans, as in Drosophila, transcripts with short stuORFs require DENR and MCTS1 for their optimal expression. In contrast to Drosophila, however, the dependence on stuORF length in human cells is very strong, so that only transcripts with very short stuORFs coding for 1 amino acid are dependent on DENR and MCTS1. This identifies circa 100 genes as putative DENR and MCTS1 translational targets. These genes are enriched for neuronal genes and G protein-coupled receptors. The identification of DENR and MCTS1 target transcripts will serve as a basis for future studies aimed at understanding the mechanistic involvement of DENR and MCTS1 in cancer and autism.

Non-AUG translation: a new start for protein synthesis in eukaryotes

“…Interestingly, eIF2D was identified as an autoantigen in human hepatocellular carcinoma patients (Wang et al 2002), suggesting a possible functional role for this initiation factor in tumor development. It should also be noted that the MCT-1/ DENR heterodimer (which is homologous to the N-terminal and C-terminal domains of eIF2D, respectively) has also been shown to regulate reinitiation and ribosome recycling (Reinert et al 2006;Schleich et al 2014Schleich et al , 2017Janich et al 2015;Haas et al 2016 Figure 3. Canonical and alternative initiator tRNA i -binding eIFs are differentially regulated and have different tRNA i -binding stringency.…”

Translation acrobatics: how cancer cells exploit alternate modes of translational initiation

“…This process of re‐initiation (Fig iii) has been known to exist for a while, but is only recently starting to be molecularly characterized . The canonical translation initiation factor eIF3h is involved in this process , as well as the non‐canonical initiation factors eIF2D, DENR, and MCTS1 . One key step in this process is likely the de novo recruitment of an initiator tRNA after termination of translation of the uORF, and these non‐canonical factors can recruit tRNA in an eIF2‐independent manner .…”

Please do not recycle! Translation reinitiation in microbes and higher eukaryotes

“…The two subunits of the heterodimeric complex DENR-MCT-1 (MCTS1 in human) were identified as REI-specific trans -acting factors for certain short uORFs in Drosophila and humans (Schleich et al. 2014 , 2017 ). In Drosophila , DENR-MCT-1 was found to regulate a specific group of mRNAs possessing strong Kozak context at the AUG codons of their short uORFs; no additional cis -acting sequences seemed to be necessary (Fig.…”

“…The uORF length requirements were even more strict in human cells, where, somewhat unexpectedly, DENR-MCT-1 supported REI only on reporter mRNAs bearing minimal ‘start-stop uORFs’ with AUGs in strong Kozak context (Schleich et al. 2017 ). Interestingly, mRNA leaders of this type were found to be enriched in neuron-specific genes (Schleich et al.…”