Research Article

Translation from the 5′ untranslated region shapes the integrated stress response

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Science  29 Jan 2016:
Vol. 351, Issue 6272, aad3867
DOI: 10.1126/science.aad3867

How cells keep going in the face of adversity

When cells experience stresses that affect their ability to process newly synthesized proteins, they turn down their rates of translation to help them survive the stress. They also turn on the translation of proteins that will help them cope with the misfolded proteins generated during stress. How do they turn down translation in general, but maintain or increase translation of specific proteins? Starck et al. developed an approach that allowed them to look at the translation of specific messenger RNAs that were not down-regulated by stress. They identified a motif that helped keep chaperone protein synthesis going.

Science, this issue p. 10.1126/science.aad3867

Structured Abstract


Protein synthesis is controlled by a plethora of developmental and environmental conditions. One intracellular signaling network, the integrated stress response (ISR), activates one of four kinases in response to a variety of distinct stress stimuli: the endoplasmic reticulum (ER)–resident kinase (PERK), the interferon-induced double-stranded RNA–dependent eIF2α kinase (PKR), the general control nonderepressible 2 (GCN2), or the heme-regulated inhibitor kinase (HRI). These four kinases recognize a central target and phosphorylate a single residue, Ser51, on the α subunit of the eukaryotic initiation factor 2 (eIF2α), which is a component of the trimeric initiation factor eIF2 that catalyzes translation initiation at AUG start codons. Phosphorylation of eIF2α down-regulates eIF2-dependent protein synthesis, which is important in development and immunity but also is implicated in neurodegeneration, cancer, and autoimmunity. However, protein synthesis does not cease on all mRNAs during the ISR. Rather, eIF2α phosphorylation is required for expression of select mRNAs, such as ATF4 and CHOP, that harbor small upstream open reading frames (uORFs) in their 5′ untranslated regions (5′ UTRs). Still other mRNAs sustain translation despite ISR activation. We developed tracing translation by T cells (3T) as an exquisitely sensitive technique to probe the translational dynamics of uORFs directly during the ISR. With 3T, we measured the peptide products of uORFs present in the 5′ UTR of the essential ER-resident chaperone, binding immunoglobulin protein (BiP), also known as heat shock protein family A member 5 (HSPA5), and characterized their requirement for BiP expression during the ISR.


We repurposed the sensitivity and specificity of T cells to interrogate the translational capacity of RNA outside of annotated protein coding sequences (CDSs). 3T relies on insertion of a tracer peptide coding sequence into a candidate DNA sequence. The resulting mRNAs harboring the nested tracer peptide coding sequence are translated to produce tracer peptides. These translation products are processed and loaded onto major histocompatibility complex class I (MHC I) molecules in the ER and transit to the cell surface, where they can be detected by specific T cell hybridomas that are activated and quantified using a colorimetric reagent. 3T provides an approach to interrogate the thousands of predicted uORFs in mammalian genomes, characterize the importance of uORF biology for regulation, and generate fundamental insights into uORF mutation-based diseases.


3T proved to be a sensitive and robust indicator of uORF expression. We measured uORF expression in the 5′ UTR of mRNAs at multiple distinct regions, while simultaneously detecting expression of the CDS. We directly measured uORF peptide expression from ATF4 mRNA and showed that its translation persisted during the ISR. We applied 3T to study BiP expression, an ER chaperone stably synthesized during the ISR. We showed that the BiP 5′ UTR harbors uORFs that are exclusively initiated by UUG and CUG start codons. BiP uORF expression bypassed a requirement for eIF2 and was dependent on the alternative initiation factor eIF2A. Both translation of the UUG-initiated uORF and eIF2A were necessary for BiP expression during the ISR. Unexpectedly, the products of uORF translation are predicted to generate MHC I peptides active in adaptive immunity. We propose that this phenomenon presents an extracellular signature during the ISR.


Our findings introduce the notion that cells harbor a distinct translation initiation pathway to respond to a variety of environmental conditions and cellular dysfunction. We showed that cells utilize a distinct, eIF2A-mediated initiation pathway, which includes uORF translation, to sustain expression of particular proteins during the ISR. 3T offers a valuable method to characterize the thousands of predicted translation events in 5′ UTRs and other noncoding RNAs and, expanded to a genome-wide scale, can complement ribosome profiling and mass spectrometry in uORF and short ORF discovery. Our observations underscore the importance of translation outside of annotated CDSs and challenge the very definition of the U in 5′ UTR.

3T reveals the translational landscape of the genome outside of annotated coding sequences.

Tracer peptide coding sequences are inserted into regions outside the annotated CDS, such as uORFs. When translated, they generate peptides that are transported into the ER, loaded onto MHC I, and transit to the cell surface. T cell hybridomas that recognize the specific tracer peptide–MHC I complex become activated, which is detected using a colorimetric substrate.


Translated regions distinct from annotated coding sequences have emerged as essential elements of the proteome. This includes upstream open reading frames (uORFs) present in mRNAs controlled by the integrated stress response (ISR) that show “privileged” translation despite inhibited eukaryotic initiation factor 2–guanosine triphosphate–initiator methionyl transfer RNA (eIF2·GTP·Met-tRNAiMet). We developed tracing translation by T cells to directly measure the translation products of uORFs during the ISR. We identified signature translation events from uORFs in the 5′ untranslated region of binding immunoglobulin protein (BiP) mRNA (also called heat shock 70-kilodalton protein 5 mRNA) that were not initiated at the start codon AUG. BiP expression during the ISR required both the alternative initiation factor eIF2A and non–AUG-initiated uORFs. We propose that persistent uORF translation, for a variety of chaperones, shelters select mRNAs from the ISR, while simultaneously generating peptides that could serve as major histocompatibility complex class I ligands, marking cells for recognition by the adaptive immune system.

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