Rapid 40S scanning and its regulation by mRNA structure during eukaryotic translation initiation.

How the eukaryotic 43S preinitiation complex scans along the 5' untranslated region (5' UTR) of a capped mRNA to locate the correct start codon remains elusive. Here, we directly track yeast 43S-mRNA binding, scanning, and 60S subunit joining by real-time single-molecule fluorescence spectroscopy. 43S engagement with mRNA occurs through a slow, ATP-dependent process driven by multiple initiation factors including the helicase eIF4A. Once engaged, 43S scanning occurs rapidly and directionally at ∼100 nucleotides per second, independent of multiple cycles of ATP hydrolysis by RNA helicases post ribosomal loading. Scanning ribosomes can proceed through RNA secondary structures, but 5' UTR hairpin sequences near start codons drive scanning ribosomes at start codons backward in the 5' direction, requiring rescanning to arrive once more at a start codon. Direct observation of scanning ribosomes provides a mechanistic framework for translational regulation by 5' UTR structures and upstream near-cognate start codons.

Rethinking Unconventional Translation in Neurodegeneration.

Eukaryotic translation is tightly regulated to ensure that protein production occurs at the right time and place. Recent studies on abnormal repeat proteins, especially in age-dependent neurodegenerative diseases caused by nucleotide repeat expansion, have highlighted or identified two forms of unconventional translation initiation: usage of AUG-like sites (near cognates) or repeat-associated non-AUG (RAN) translation. We discuss how repeat proteins may differ due to not just unconventional initiation, but also ribosomal frameshifting and/or imperfect repeat DNA replication, expansion, and repair, and we highlight how research on translation of repeats may uncover insights into the biology of translation and its contribution to disease.

Ribosome queuing enables non-AUG translation to be resistant to multiple protein synthesis inhibitors.

Aberrant translation initiation at non-AUG start codons is associated with multiple cancers and neurodegenerative diseases. Nevertheless, how non-AUG translation may be regulated differently from canonical translation is poorly understood. Here, we used start codon-specific reporters and ribosome profiling to characterize how translation from non-AUG start codons responds to protein synthesis inhibitors in human cells. These analyses surprisingly revealed that translation of multiple non-AUG-encoded reporters and the endogenous GUG-encoded DAP5 (eIF4G2/p97) mRNA is resistant to cycloheximide (CHX), a translation inhibitor that severely slows but does not completely abrogate elongation. Our data suggest that slowly elongating ribosomes can lead to queuing/stacking of scanning preinitiation complexes (PICs), preferentially enhancing recognition of weak non-AUG start codons. Consistent with this model, limiting PIC formation or scanning sensitizes non-AUG translation to CHX. We further found that non-AUG translation is resistant to other inhibitors that target ribosomes within the coding sequence but not those targeting newly initiated ribosomes. Together, these data indicate that ribosome queuing enables mRNAs with poor initiation context-namely, those with non-AUG start codons-to be resistant to pharmacological translation inhibitors at concentrations that robustly inhibit global translation.

Polyamine Control of Translation Elongation Regulates Start Site Selection on Antizyme Inhibitor mRNA via Ribosome Queuing.

Translation initiation is typically restricted to AUG codons, and scanning eukaryotic ribosomes inefficiently recognize near-cognate codons. We show that queuing of scanning ribosomes behind a paused elongating ribosome promotes initiation at upstream weak start sites. Ribosomal profiling reveals polyamine-dependent pausing of elongating ribosomes on a conserved Pro-Pro-Trp (PPW) motif in an inhibitory non-AUG-initiated upstream conserved coding region (uCC) of the antizyme inhibitor 1 (AZIN1) mRNA, encoding a regulator of cellular polyamine synthesis. Mutation of the PPW motif impairs initiation at the uCC's upstream near-cognate AUU start site and derepresses AZIN1 synthesis, whereas substitution of alternate elongation pause sequences restores uCC translation. Impairing ribosome loading reduces uCC translation and paradoxically derepresses AZIN1 synthesis. Finally, we identify the translation factor eIF5A as a sensor and effector for polyamine control of uCC translation. We propose that stalling of elongating ribosomes triggers queuing of scanning ribosomes and promotes initiation by positioning a ribosome near the start codon.

Cysteine tRNA acts as a stop codon readthrough-inducing tRNA in the human HEK293T cell line.

Under certain circumstances, any of the three termination codons can be read through by a near-cognate tRNA; i.e., a tRNA whose two out of three anticodon nucleotides base pair with those of the stop codon. Unless programed to synthetize C-terminally extended protein variants with expanded physiological roles, readthrough represents an undesirable translational error. On the other side of a coin, a significant number of human genetic diseases is associated with the introduction of nonsense mutations (premature termination codons [PTCs]) into coding sequences, where stopping is not desirable. Here, the tRNA's ability to induce readthrough opens up the intriguing possibility of mitigating the deleterious effects of PTCs on human health. In yeast, the UGA and UAR stop codons were described to be read through by four readthrough-inducing rti-tRNAs-tRNATrp and tRNACys, and tRNATyr and tRNAGln, respectively. The readthrough-inducing potential of tRNATrp and tRNATyr was also observed in human cell lines. Here, we investigated the readthrough-inducing potential of human tRNACys in the HEK293T cell line. The tRNACys family consists of two isoacceptors, one with ACA and the other with GCA anticodons. We selected nine representative tRNACys isodecoders (differing in primary sequence and expression level) and tested them using dual luciferase reporter assays. We found that at least two tRNACys can significantly elevate UGA readthrough when overexpressed. This indicates a mechanistically conserved nature of rti-tRNAs between yeast and human, supporting the idea that they could be used in the PTC-associated RNA therapies.

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

Although it was long thought that eukaryotic translation almost always initiates at an AUG start codon, recent advancements in ribosome footprint mapping have revealed that non-AUG start codons are used at an astonishing frequency. These non-AUG initiation events are not simply errors but instead are used to generate or regulate proteins with key cellular functions; for example, during development or stress. Misregulation of non-AUG initiation events contributes to multiple human diseases, including cancer and neurodegeneration, and modulation of non-AUG usage may represent a novel therapeutic strategy. It is thus becoming increasingly clear that start codon selection is regulated by many trans-acting initiation factors as well as sequence/structural elements within messenger RNAs and that non-AUG translation has a profound impact on cellular states.

Structural basis for reduced ribosomal A-site fidelity in response to P-site codon-anticodon mismatches.

Rapid and accurate translation is essential in all organisms to produce properly folded and functional proteins. mRNA codons that define the protein-coding sequences are decoded by tRNAs on the ribosome in the aminoacyl (A) binding site. The mRNA codon and the tRNA anticodon interaction is extensively monitored by the ribosome to ensure accuracy in tRNA selection. While other polymerases that synthesize DNA and RNA can correct for misincorporations, the ribosome is unable to correct mistakes. Instead, when a misincorporation occurs, the mismatched tRNA-mRNA pair moves to the peptidyl (P) site and, from this location, causes a reduction in the fidelity at the A site, triggering post-peptidyl transfer quality control. This reduced fidelity allows for additional incorrect tRNAs to be accepted and for release factor 2 (RF2) to recognize sense codons, leading to hydrolysis of the aberrant peptide. Here, we present crystal structures of the ribosome containing a tRNALys in the P site with a U•U mismatch with the mRNA codon. We find that when the mismatch occurs in the second position of the P-site codon-anticodon interaction, the first nucleotide of the A-site codon flips from the mRNA path to engage highly conserved 16S rRNA nucleotide A1493 in the decoding center. We propose that this mRNA nucleotide mispositioning leads to reduced fidelity at the A site. Further, this state may provide an opportunity for RF2 to initiate premature termination before erroneous nascent chains disrupt the cellular proteome.

Nontraditional translation is the key to UFMylation and beyond.

The Ubiquitin-fold modifier 1 (Ufm1) is a ubiquitin-like protein that can also be conjugated to protein substrates and subsequently alter their fates. Both UFMylation and de-UFMylation are mediated by Ufm1-specific proteases (UFSPs). In humans, it is widely believed that UFSP2 is the only active Ufm1 protease involved in Ufm1 maturation and de-UFMylation, whereas UFSP1 is thought to be inactive. Here, Liang et al. provide strong evidence showing that human UFSP1 is also an active Ufm1 protease. These results solve an age-old mystery in the human Ufm1 conjugation system and could have a greater impact not only on Ufm1 biology but also on the translation of genes employing nontraditional start codons.

Rules are made to be broken: a "simple" model organism reveals the complexity of gene regulation.

Global methods for assaying translation have greatly improved our understanding of the protein-coding capacity of the genome. In particular, it is now possible to perform genome-wide and condition-specific identification of translation initiation sites through modified ribosome profiling methods that selectively capture initiating ribosomes. Here we discuss our recent study applying such an approach to meiotic and mitotic timepoints in the simple eukaryote, budding yeast, as an example of the surprising diversity of protein products-many of which are non-canonical-that can be revealed by such methods. We also highlight several key challenges in studying non-canonical protein isoforms that have precluded their prior systematic discovery. A growing body of work supports expanded use of empirical protein-coding region identification, which can help relieve some of the limitations and biases inherent to traditional genome annotation approaches. Our study also argues for the adoption of less static views of gene identity and a broader framework for considering the translational capacity of the genome.

Deciphering the reading of the genetic code by near-cognate tRNA.

Some codons of the genetic code can be read not only by cognate, but also by near-cognate tRNAs. This flexibility is thought to be conferred mainly by a mismatch between the third base of the codon and the first of the anticodon (the so-called "wobble" position). However, this simplistic explanation underestimates the importance of nucleotide modifications in the decoding process. Using a system in which only near-cognate tRNAs can decode a specific codon, we investigated the role of six modifications of the anticodon, or adjacent nucleotides, of the tRNAs specific for Tyr, Gln, Lys, Trp, Cys, and Arg in Saccharomyces cerevisiae. Modifications almost systematically rendered these tRNAs able to act as near-cognate tRNAs at stop codons, even though they involve noncanonical base pairs, without markedly affecting their ability to decode cognate or near-cognate sense codons. These findings reveal an important effect of modifications to tRNA decoding with implications for understanding the flexibility of the genetic code.

Translation of upstream open reading frames in a model of neuronal differentiation.

BACKGROUND: Upstream open reading frames (uORFs) initiate translation within mRNA 5' leaders, and have the potential to alter main coding sequence (CDS) translation on transcripts in which they reside. Ribosome profiling (RP) studies suggest that translating ribosomes are pervasive within 5' leaders across model systems. However, the significance of this observation remains unclear. To explore a role for uORF usage in a model of neuronal differentiation, we performed RP on undifferentiated and differentiated human neuroblastoma cells. RESULTS: Using a spectral coherence algorithm (SPECtre), we identify 4954 consistently translated uORFs across 31% of all neuroblastoma transcripts. These uORFs predominantly utilize non-AUG initiation codons and exhibit translational efficiencies (TE) comparable to annotated coding regions. On a population basis, the global impact of both AUG and non-AUG initiated uORFs on basal CDS translation were small, even when analysis is limited to conserved and consistently translated uORFs. However, uORFs did alter the translation of a subset of genes, including the Diamond-Blackfan Anemia associated ribosomal gene RPS24. With retinoic acid induced differentiation, we observed an overall positive correlation in translational shifts between uORF/CDS pairs. However, CDSs downstream of uORFs show smaller shifts in TE with differentiation relative to CDSs without a predicted uORF, suggesting that uORF translation buffers cell state dependent fluctuations in CDS translation. CONCLUSION: This work provides insights into the dynamic relationships and potential regulatory functions of uORF/CDS pairs in a model of neuronal differentiation.

A helicase links upstream ORFs and RNA structure.

Upstream open reading frames (uORFs) in 5' UTRs of eukaryotic mRNAs are increasingly recognized as important elements that regulate cellular protein synthesis. Since uORFs can start from non-AUG codons, an enormous number of potential uORF initiation sites exists in 5'UTRs. However, only a subset of these sites is used and it has been unclear how actual start sites are selected. Studies of the DEAD-box helicase Ded1p from S. cerevisiae show that translation of uORFs with non-AUG initiation codons occurs upstream of mRNA structures that emerge with defective Ded1p. The data designate mRNA structure as important determinant for non-AUG initiation sites of uORFs. Ded1p can control this RNA structure and thereby regulate uORF translation.

Increasing the fidelity of noncanonical amino acid incorporation in cell-free protein synthesis.

BACKGROUND: Cell-free protein synthesis provides a robust platform for co-translational incorporation of noncanonical amino acid (ncAA) into proteins to facilitate biological studies and biotechnological applications. Recently, eliminating the activity of release factor 1 has been shown to increase ncAA incorporation in response to amber codons. However, this approach could promote mis-incorporation of canonical amino acids by near cognate suppression. METHODS: We performed a facile protocol to remove near cognate tRNA isoacceptors of the amber codon from total tRNAs, and used the phosphoserine (Sep) incorporation system as validation. By manipulating codon usage of target genes and tRNA species introduced into the cell-free protein synthesis system, we increased the fidelity of Sep incorporation at a specific position. RESULTS: By removing three near cognate tRNA isoacceptors of the amber stop codon [tRNALys, tRNATyr, and tRNAGln(CUG)] from the total tRNA, the near cognate suppression decreased by 5-fold without impairing normal protein synthesis in the cell-free protein synthesis system. Mass spectrometry analyses indicated that the fidelity of ncAA incorporation was improved. CONCLUSIONS: Removal of near cognate tRNA isoacceptors of the amber codon could increase ncAA incorporation fidelity towards the amber stop codon in release factor deficiency systems. GENERAL SIGNIFICANCE: We provide a general strategy to improve fidelity of ncAA incorporation towards stop, quadruplet and sense codons in cell-free protein synthesis systems. This article is part of a Special Issue entitled "Biochemistry of Synthetic Biology - Recent Developments" Guest Editor: Dr. Ilka Heinemann and Dr. Patrick O'Donoghue.

Structural insights into +1 frameshifting promoted by expanded or modification-deficient anticodon stem loops.

Maintenance of the correct reading frame on the ribosome is essential for accurate protein synthesis. Here, we report structures of the 70S ribosome bound to frameshift suppressor tRNA(SufA6) and N1-methylguanosine at position 37 (m(1)G37) modification-deficient anticodon stem loop(Pro), both of which cause the ribosome to decode 4 rather than 3 nucleotides, resulting in a +1 reading frame. Our results reveal that decoding at +1 suppressible codons causes suppressor tRNA(SufA6) to undergo a rearrangement of its 5' stem that destabilizes U32, thereby disrupting the conserved U32-A38 base pair. Unexpectedly, the removal of the m(1)G37 modification of tRNA(Pro) also disrupts U32-A38 pairing in a structurally analogous manner. The lack of U32-A38 pairing provides a structural correlation between the transition from canonical translation and a +1 reading of the mRNA. Our structures clarify the molecular mechanism behind suppressor tRNA-induced +1 frameshifting and advance our understanding of the role played by the ribosome in maintaining the correct translational reading frame.

Translational readthrough potential of natural termination codons in eucaryotes--The impact of RNA sequence.

Termination of protein synthesis is not 100% efficient. A number of natural mechanisms that suppress translation termination exist. One of them is STOP codon readthrough, the process that enables the ribosome to pass through the termination codon in mRNA and continue translation to the next STOP codon in the same reading frame. The efficiency of translational readthrough depends on a variety of factors, including the identity of the termination codon, the surrounding mRNA sequence context, and the presence of stimulating compounds. Understanding the interplay between these factors provides the necessary background for the efficient application of the STOP codon suppression approach in the therapy of diseases caused by the presence of premature termination codons.

Identification of unannotated coding sequences and their physiological functions.

Most protein-coding sequences (CDSs) are predicted sequences based on criteria such as a size sufficient to encode a product of at least 100 amino acids and with translation starting at an AUG initiation codon. However, recent studies based on ribosome profiling and mass spectrometry have shown that several RNAs annotated as long as noncoding RNAs are actually translated to generate polypeptides of fewer than 100 amino acids and that many proteins are translated from near-cognate initiation codons such as CUG and GUG. Furthermore, studies of genetically engineered mouse models have revealed that such polypeptides and proteins contribute to diverse physiological processes. In this review, we describe the latest methods for the identification of unannotated CDSs and provide examples of their physiological functions.

uORF-seqr: A Machine Learning-Based Approach to the Identification of Upstream Open Reading Frames in Yeast.

The identification of upstream open reading frames (uORFs) using ribosome profiling data is complicated by several factors such as the noise inherent to the procedure, the substantial increase in potential translation initiation sites (and false positives) when one includes non-canonical start codons, and the paucity of molecularly validated uORFs. Here we present uORF-seqr, a novel machine learning algorithm that uses ribosome profiling data, in conjunction with RNA-seq data, as well as transcript aware genome annotation files to identify statistically significant AUG and near-cognate codon uORFs.

Overcoming Near-Cognate Suppression in a Release Factor 1-Deficient Host with an Improved Nitro-Tyrosine tRNA Synthetase.

Genetic code expansion (GCE) technologies incorporate non-canonical amino acids (ncAAs) into proteins at amber stop codons. To avoid unwanted truncated protein and improve ncAA-protein yields, genomically recoded strains of Escherichia coli lacking Release Factor 1 (RF1) are becoming increasingly popular expression hosts for GCE applications. In the absence of RF1, however, endogenous near-cognate amber suppressing tRNAs can lead to contaminating protein forms with natural amino acids in place of the ncAA. Here, we show that a second-generation amino-acyl tRNA synthetase (aaRS)/tRNACUA pair for site-specific incorporation of 3-nitro-tyrosine could not outcompete near-cognate suppression in an RF1-deficient expression host and therefore could not produce homogenously nitrated protein. To resolve this, we used Rosetta to target positions in the nitroTyr aaRS active site for improved substrate binding, and then constructed of a small library of variants to subject to standard selection protocols. The top selected variant had an ~2-fold greater efficiency, and remarkably, this relatively small improvement enabled homogeneous incorporation of nitroTyr in an RF1-deficient expression host and thus eliminates truncation issues associated with typical RF1-containing expression hosts. Structural and biochemical data suggest the aaRS efficiency improvement is based on higher affinity substrate binding. Taken together, the modest improvement in aaRS efficiency provides a large practical impact and expands our ability to study the role protein nitration plays in disease development through producing homogenous, truncation-free nitroTyr-containing protein. This work establishes Rosetta-guided design and incremental aaRS improvement as a viable and accessible path to improve GCE systems challenged by truncation and/or near-cognate suppression issues.

Structural insights into mRNA reading frame regulation by tRNA modification and slippery codon-anticodon pairing.

Modifications in the tRNA anticodon loop, adjacent to the three-nucleotide anticodon, influence translation fidelity by stabilizing the tRNA to allow for accurate reading of the mRNA genetic code. One example is the N1-methylguanosine modification at guanine nucleotide 37 (m1G37) located in the anticodon loop andimmediately adjacent to the anticodon nucleotides 34, 35, 36. The absence of m1G37 in tRNAPro causes +1 frameshifting on polynucleotide, slippery codons. Here, we report structures of the bacterial ribosome containing tRNAPro bound to either cognate or slippery codons to determine how the m1G37 modification prevents mRNA frameshifting. The structures reveal that certain codon-anticodon contexts and the lack of m1G37 destabilize interactions of tRNAPro with the P site of the ribosome, causing large conformational changes typically only seen during EF-G-mediated translocation of the mRNA-tRNA pairs. These studies provide molecular insights into how m1G37 stabilizes the interactions of tRNAPro with the ribosome in the context of a slippery mRNA codon.

Translation Initiation Site Profiling Reveals Widespread Synthesis of Non-AUG-Initiated Protein Isoforms in Yeast.

Genomic analyses in budding yeast have helped define the foundational principles of eukaryotic gene expression. However, in the absence of empirical methods for defining coding regions, these analyses have historically excluded specific classes of possible coding regions, such as those initiating at non-AUG start codons. Here, we applied an experimental approach to globally annotate translation initiation sites in yeast and identified 149 genes with alternative N-terminally extended protein isoforms initiating from near-cognate codons upstream of annotated AUG start codons. These isoforms are produced in concert with canonical isoforms and translated with high specificity, resulting from initiation at only a small subset of possible start codons. The non-AUG initiation driving their production is enriched during meiosis and induced by low eIF5A, which is seen in this context. These findings reveal widespread production of non-canonical protein isoforms and unexpected complexity to the rules by which even a simple eukaryotic genome is decoded.