Shelley R. Starck

Dopaminergic stimulation of local protein synthesis enhances surface expression of GluR1 and synaptic transmission in hippocampal neurons.

The use-dependent modification of synapses is strongly influenced by dopamine, a transmitter that participates in both the physiology and pathophysiology of animal behavior. In the hippocampus, dopaminergic signaling is thought to play a key role in protein synthesis-dependent forms of synaptic plasticity. The molecular mechanisms by which dopamine influences synaptic function, however, are not well understood. Using a GFP-based reporter, as well as a small-molecule reporter of endogenous protein synthesis, we show that dopamine D1/D5 receptor activation stimulates local protein synthesis in the dendrites of hippocampal neurons. We also identify the GluR1 subunit of AMPA receptors as one protein upregulated by dopamine receptor activation, with increased incorporation of surface GluR1 at synaptic sites. The insertion of new GluRs is accompanied by an increase in the frequency of miniature synaptic events. Together, these data suggest a local protein synthesis-dependent activation of previously silent synapses as a result of dopamine receptor stimulation.

Leucine-tRNA Initiates at CUG Start Codons for Protein Synthesis and Presentation by MHC Class I

Noncanonical Pathway The textbook view of translation of messenger RNA to protein is that it is always initiated from open reading frames (ORFs) that begin with an AUG codon (encodes methionine) by an initiator methionine-bound transfer RNA (tRNA). There is evidence, however, that some polypeptides are produced from non–AUG-initiated ORFs. Starck et al. (p. 1719; see the Perspective by Dever) used a variety of biochemical techniques to determine the underlying mechanism for such nontraditional translation initiation. Comparison of translation initiation from AUG-initiated ORFs with those beginning with leucine CUG-initiated ORFs revealed that cells can use an elongator Leu-tRNA to initiate translation at CUG codons. CUG-initiated peptides were presented by major histocompatibility class I molecules and could activate T cells. T cells can use leucyl–transfer RNA (tRNA), instead of methionyl-tRNA, to initiate translation. Effective immune surveillance by cytotoxic T cells requires newly synthesized polypeptides for presentation by major histocompatibility complex (MHC) class I molecules. These polypeptides are produced not only from conventional AUG-initiated, but also from cryptic non–AUG-initiated, reading frames by distinct translational mechanisms. Biochemical analysis of ribosomal initiation complexes at CUG versus AUG initiation codons revealed that cells use an elongator leucine-bound transfer RNA (Leu-tRNA) to initiate translation at cryptic CUG start codons. CUG/Leu-tRNA initiation was independent of the canonical initiator tRNA (AUG/Met-tRNAiMet) pathway but required expression of eukaryotic initiation factor 2A. Thus, a tRNA-based translation initiation mechanism allows non–AUG-initiated protein synthesis and supplies peptides for presentation by MHC class I molecules.

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

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 Protein translation from open reading frames with alternative initiation codons occurs during induction of cellular stress responses. INTRODUCTION 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. RATIONALE 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. RESULTS 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. CONCLUSION 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.

The synthesis of truncated polypeptides for immune surveillance and viral evasion.

Background Cytotoxic T cells detect intracellular pathogens by surveying peptide loaded MHC class I molecules (pMHC I) on the cell surface. Effective immune surveillance also requires infected cells to present pMHC I promptly before viral progeny can escape. Rapid pMHC I presentation apparently occurs because infected cells can synthesize and present peptides from antigenic precursors called defective ribosomal products (DRiPs). The molecular characteristics of DRiPs are not known. Methodology/Principal Findings Here, using a novel method for detecting antigenic precursors and proteolytic intermediates, we tracked the synthesis and processing of Epstein-Barr Virus encoded nuclear antigen 1 (EBNA1). We find that ribosomes initiated translation appropriately, but rapidly produced DRiPs representing ∼120 amino acid truncated EBNA1 polypeptides by premature termination. Moreover, specific sequences in EBNA1 mRNA strongly inhibited the generation of truncated DRiPs and pMHC I presentation. Significance Our results reveal the first characterization of virus DRiPs as truncated translation products. Furthermore, production of EBNA1-derived DRiPs is down-regulated in cells, possibly limiting the antigenicity of EBNA1.

Unnatural RNA display libraries

Combinatorial peptide and protein libraries have now been developed to accommodate unnatural amino acids in a genetically encoded format via in vitro nonsense and sense suppression. General translation features and specific regioselective and stereoselective properties of the ribosome endow these libraries with a broad chemical diversity. Alternatively, amino acid residues can be chemically derivatized post-translationally to add preferred functionality to the encoded peptide. All of these efforts are advancing combinatorial peptide and protein libraries for enhanced ligands against biological targets of interest.

A distinct translation initiation mechanism generates cryptic peptides for immune surveillance.

MHC class I molecules present a comprehensive mixture of peptides on the cell surface for immune surveillance. The peptides represent the intracellular protein milieu produced by translation of endogenous mRNAs. Unexpectedly, the peptides are encoded not only in conventional AUG initiated translational reading frames but also in alternative cryptic reading frames. Here, we analyzed how ribosomes recognize and use cryptic initiation codons in the mRNA. We find that translation initiation complexes assemble at non-AUG codons but differ from canonical AUG initiation in response to specific inhibitors acting within the peptidyl transferase and decoding centers of the ribosome. Thus, cryptic translation at non-AUG start codons can utilize a distinct initiation mechanism which could be differentially regulated to provide peptides for immune surveillance.

Puromycin oligonucleotides reveal steric restrictions for ribosome entry and multiple modes of translation inhibition

Peptidyl transferase inhibitors have generally been studied using simple systems and remain largely unexamined In in vitro translation extracts. Here, we investigate the potency, product distribution, and mechanism of various puromycin-oligonucleotide conjugates (1 to 44 nt with 3-puromycin) In a reticulocyte lysate cell-free translation system. Surprisingly, the potency decreases as the chain length of the oligonucleotide is increased in this series, and only very short puromycin conjugates function efficiently (IC50 < 50 microM). This observation stands in contrast with work on isolated large ribosomal subunits, which Indicates that many of the puromycin-oligonucleotide conjugates we studied should have higher affinity for the peptidyl transferase center than puromycin itself. Two tRNA(Al)-derived minihelices containing puromycin provide an exception to the size trend, and are the only constructs longer than 4 nt with any appreciable potency (IC50 = 40-56 microM). However, the puromycin minihelices inhibit translation by sequestering one or more soluble translation factors, and do not appear to participate in detectable peptide bond formation with the nascent chain. In contrast, puromycin and other short derivatives act in a factor-independent fashion at the peptidyl transferase center and readily become conjugated to the nascent protein chain. However, even for the short derivatives, much of the translation inhibition occurs without peptide bond formation between puromycin and the nascent chain, a revision of the classical model for puromycin function. This peptide bond-independent mode is likely a combination of multiple effects including inhibition of initiation and failure to properly recycle translation complexes that have reacted with puromycin.

Nowhere to hide: unconventional translation yields cryptic peptides for immune surveillance

Effective immune surveillance by CD8+ cytotoxic T cells of intracellular microbes and cancer depends on the antigen presentation pathway. This pathway produces an optimal peptide repertoire for presentation by major histocompatibility (MHC) class I molecules (pMHCs I) on the cell surface. We have known for years that the pMHC I repertoire is a reflection of the intracellular protein pool. However, many studies have revealed that pMHCs I present peptides not only from precursors encoded in open‐reading frames of mRNA transcripts but also cryptic peptides encoded in apparently ‘untranslated’ regions. These sources vastly increase the availability of peptides for presentation and immune evasion. Here, we review studies on the composition of the cryptic pMHC I repertoire, the immunological significance of these pMHC I, and the novel translational mechanisms that generate cryptic peptides from unusual sources.

Presentation of Cryptic Peptides by MHC Class I Is Enhanced by Inflammatory Stimuli

Cytolytic T cells eliminate infected or cancer cells by recognizing peptides presented by MHC class I molecules on the cell surface. The antigenic peptides are derived primarily from newly synthesized proteins including those produced by cryptic translation mechanisms. Previous studies have shown that cryptic translation can be initiated by distinct mechanisms at non-AUG codons in addition to conventional translation initiated at the canonical AUG start codon. In this study, we show that presentation of endogenously translated cryptic peptides is enhanced by TLR signaling pathways involved in pathogen recognition as well as by infection with different viruses. This enhancement of cryptic peptides was caused by proinflammatory cytokines, secreted in response to microbial infection. Furthermore, blocking these cytokines abrogated the enhancement of cryptic peptide presentation in response to infection. Thus, presentation of cryptic peptides is selectively enhanced during inflammation and infection, which could allow the immune system to detect intracellular pathogens that might otherwise escape detection because of inhibition of conventional host translation mechanisms.

Chrysochlamic acid, a new diterpenoid-substituted quinol from Chrysochlamys ulei that inhibits DNA polymerase β

Chrysochlamic acid (1), a new DNA polymerase β inhibitor having an IC50 of 4.3 µM, has been isolated from Chrysochlamys ulei through bioassay-guided fractionation; it is the first example of a prenyl m-methylhydroquinone occurring in a higher terrestrial plant.