Stem-loop-induced ribosome queuing in the uORF2/ATF4 overlap fine-tunes stress-induced human ATF4 translational control.

Activating transcription factor 4 (ATF4) is a master transcriptional regulator of the integrated stress response, leading cells toward adaptation or death. ATF4's induction under stress was thought to be due to delayed translation reinitiation, where the reinitiation-permissive upstream open reading frame 1 (uORF1) plays a key role. Accumulating evidence challenging this mechanism as the sole source of ATF4 translation control prompted us to investigate additional regulatory routes. We identified a highly conserved stem-loop in the uORF2/ATF4 overlap, immediately preceded by a near-cognate CUG, which introduces another layer of regulation in the form of ribosome queuing. These elements explain how the inhibitory uORF2 can be translated under stress, confirming prior observations but contradicting the original regulatory model. We also identified two highly conserved, potentially modified adenines performing antagonistic roles. Finally, we demonstrated that the canonical ATF4 translation start site is substantially leaky scanned. Thus, ATF4's translational control is more complex than originally described, underpinning its key role in diverse biological processes.

Stem-loop induced ribosome queuing in the uORF2/ATF4 overlap fine-tunes stress-induced human ATF4 translational control.

ATF4 is a master transcriptional regulator of the integrated stress response leading cells towards adaptation or death. ATF4's induction under stress was thought to be mostly due to delayed translation reinitiation, where the reinitiation-permissive uORF1 plays a key role. Accumulating evidence challenging this mechanism as the sole source of ATF4 translation control prompted us to investigate additional regulatory routes. We identified a highly conserved stem-loop in the uORF2/ATF4 overlap, immediately preceded by a near-cognate CUG, which introduces another layer of regulation in the form of ribosome queuing. These elements explain how the inhibitory uORF2 can be translated under stress, confirming prior observations, but contradicting the original regulatory model. We also identified two highly conserved, potentially modified adenines performing antagonistic roles. Finally, we demonstrate that the canonical ATF4 translation start site is substantially leaky-scanned. Thus, ATF4's translational control is more complex than originally described underpinning its key role in diverse biological processes.

Selective Translation Complex Profiling Reveals Staged Initiation and Co-translational Assembly of Initiation Factor Complexes.

Translational control targeting the initiation phase is central to the regulation of gene expression. Understanding all of its aspects requires substantial technological advancements. Here we modified yeast translation complex profile sequencing (TCP-seq), related to ribosome profiling, and adapted it for mammalian cells. Human TCP-seq, capable of capturing footprints of 40S subunits (40Ss) in addition to 80S ribosomes (80Ss), revealed that mammalian and yeast 40Ss distribute similarly across 5'TRs, indicating considerable evolutionary conservation. We further developed yeast and human selective TCP-seq (Sel-TCP-seq), enabling selection of 40Ss and 80Ss associated with immuno-targeted factors. Sel-TCP-seq demonstrated that eIF2 and eIF3 travel along 5' UTRs with scanning 40Ss to successively dissociate upon AUG recognition; notably, a proportion of eIF3 lingers on during the initial elongation cycles. Highlighting Sel-TCP-seq versatility, we also identified four initiating 48S conformational intermediates, provided novel insights into ATF4 and GCN4 mRNA translational control, and demonstrated co-translational assembly of initiation factor complexes.

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

Protein production must be strictly controlled at its beginning and end to synthesize a polypeptide that faithfully copies genetic information carried in the encoding mRNA. In contrast to viruses and prokaryotes, the majority of mRNAs in eukaryotes contain only one coding sequence, resulting in production of a single protein. There are, however, many exceptional mRNAs that either carry short open reading frames upstream of the main coding sequence (uORFs) or even contain multiple long ORFs. A wide variety of mechanisms have evolved in microbes and higher eukaryotes to prevent recycling of some or all translational components upon termination of the first translated ORF in such mRNAs and thereby enable subsequent translation of the next uORF or downstream coding sequence. These specialized reinitiation mechanisms are often regulated to couple translation of the downstream ORF to various stimuli. Here we review all known instances of both short uORF-mediated and long ORF-mediated reinitiation and present our current understanding of the underlying molecular mechanisms of these intriguing modes of translational control.

Embraced by eIF3: structural and functional insights into the roles of eIF3 across the translation cycle.

Protein synthesis is mediated via numerous molecules including the ribosome, mRNA, tRNAs, as well as translation initiation, elongation and release factors. Some of these factors play several roles throughout the entire process to ensure proper assembly of the preinitiation complex on the right mRNA, accurate selection of the initiation codon, errorless production of the encoded polypeptide and its proper termination. Perhaps, the most intriguing of these multitasking factors is the eukaryotic initiation factor eIF3. Recent evidence strongly suggests that this factor, which coordinates the progress of most of the initiation steps, does not come off the initiation complex upon subunit joining, but instead it remains bound to 80S ribosomes and gradually falls off during the first few elongation cycles to: (1) promote resumption of scanning on the same mRNA molecule for reinitiation downstream-in case of translation of upstream ORFs short enough to preserve eIF3 bound; or (2) come back during termination on long ORFs to fine tune its fidelity or, if signaled, promote programmed stop codon readthrough. Here, we unite recent structural views of the eIF3-40S complex and discus all known eIF3 roles to provide a broad picture of the eIF3's impact on translational control in eukaryotic cells.

Does eIF3 promote reinitiation after translation of short upstream ORFs also in mammalian cells?

Reinitiation after translation of short upstream ORFs (uORFs) represents one of the means of regulation of gene expression on the mRNA-specific level in response to changing environmental conditions. Over the years it has been shown-mainly in budding yeast-that its efficiency depends on cis-acting features occurring in sequences flanking reinitiation-permissive uORFs, the nature of their coding sequences, as well as protein factors acting in trans. We earlier demonstrated that the first two uORFs from the reinitiation-regulated yeast GCN4 mRNA leader carry specific structural elements in their 5' sequences that interact with the translation initiation factor eIF3 to prevent full ribosomal recycling post their translation. Actually, this interaction turned out to be instrumental in stabilizing the mRNA·40S post-termination complex, which is thus capable to eventually resume scanning and reinitiate on the next AUG start site downstream. Recently, we also provided important in vivo evidence strongly supporting the long-standing idea that to stimulate reinitiation, eIF3 has to remain bound to ribosomes elongating these uORFs until their stop codon has been reached. Here we examined the importance of eIF3 and sequences flanking uORF1 of the human functional homolog of yeast GCN4, ATF4, in stimulation of efficient reinitiation. We revealed that the molecular basis of the reinitiation mechanism is conserved between yeasts and humans.

An emergency brake for protein synthesis.

The integrated stress response is able to rapidly shut down the synthesis of proteins in eukaryotic cells.