Suppressor tRNA Research Articles

Fluorescence Amplification Method for Forward Genetic Discovery of Factors in Human mRNA Degradation

Nonsense-mediated decay (NMD) degrades mRNAs containing a premature termination codon (PTC). PTCs are a frequent cause of human genetic diseases, and the NMD pathway is known to modulate disease severity. Since partial NMD attenuation can potentially enhance nonsense suppression therapies, better definition of human-specific NMD is required. However, the majority of NMD factors were first discovered in model organisms and then subsequently identified by homology in human. Sensitivity and throughput limitations of existing approaches have hindered systematic forward genetic screening for NMD factors in human cells. We developed a method of invivo amplification of NMD reporter fluorescence (Fireworks) that enables CRISPR-based forward genetic screening for NMD pathway defects in human cells. The Fireworks genetic screen identifies multiple known NMD factors and numerous human candidate genes, providing a platform for discovery of additional key factors in human mRNA degradation.

Cellular encoding of Cy dyes for single-molecule imaging.

A general method is described for the site-specific genetic encoding of cyanine dyes as non-canonical amino acids (Cy-ncAAs) into proteins. The approach relies on an improved technique for nonsense suppression with in vitro misacylated orthogonal tRNA. The data show that Cy-ncAAs (based on Cy3 and Cy5) are tolerated by the eukaryotic ribosome in cell-free and whole-cell environments and can be incorporated into soluble and membrane proteins. In the context of the Xenopus laevis oocyte expression system, this technique yields ion channels with encoded Cy-ncAAs that are trafficked to the plasma membrane where they display robust function and distinct fluorescent signals as detected by TIRF microscopy. This is the first demonstration of an encoded cyanine dye as a ncAA in a eukaryotic expression system and opens the door for the analysis of proteins with single-molecule resolution in a cellular environment.

Haploid yeast cells undergo a reversible phenotypic switch associated with chromosome II copy number.

Background SUP35 and SUP45 are essential genes encoding polypeptide chain release factors. However, mutants for these genes may be viable but display pleiotropic phenotypes which include, but are not limited to, nonsense suppressor phenotype due to translation termination defect. [PSI +] prion formation is another Sup35p-associated mechanism leading to nonsense suppression through decreased availability of functional Sup35p. [PSI +] differs from genuine sup35 mutations by the possibility of its elimination and subsequent re-induction. Some suppressor sup35 mutants had also been shown to undergo a reversible phenotypic switch in the opposite direction. This reversible switching had been attributed to a prion termed [ISP +]. However, even though many phenotypic and molecular level features of [ISP +] were revealed, the mechanism behind this phenomenon has not been clearly explained and might be more complex than suggested initially.ResultsHere we took a genomic approach to look into the molecular basis of the difference between the suppressor (Isp−) and non-suppressor (Isp+) phenotypes. We report that the reason for the difference between the Isp+ and the Isp− phenotypes is chromosome II copy number changes and support our finding with showing that these changes are indeed reversible by reproducing the phenotypic switch and tracking karyotypic changes. Finally, we suggest mechanisms that mediate elevation in nonsense suppression efficiency upon amplification of chromosome II and facilitate switching between these states.Conclusions(i) In our experimental system, amplification of chromosome II confers nonsense suppressor phenotype and guanidine hydrochloride resistance at the cost of overall decreased viability in rich medium. (ii) SFP1 might represent a novel regulator of chromosome stability, as SFP1 overexpression elevates frequency of the additional chromosome loss in our system. (iii) Prolonged treatment with guanidine hydrochloride leads to selection of resistant isolates, some of which are disomic for chromosome II.Electronic supplementary materialThe online version of this article (doi:10.1186/s12863-016-0464-4) contains supplementary material, which is available to authorized users.

In Vivo Photo-Cross-Linking to Study T3S Interactions Demonstrated Using the Yersinia pestis T3S System.

Cross-linking of proteins is effective in determining protein-protein interactions. The use of photo-cross-linkers was developed to study protein interactions in several manners. One method involved the incorporation of photo-activatable cross-linking groups into chemically synthesized peptides. A second approach relies on incorporation of photo-activatable cross-linking groups into proteins using tRNAs with chemically bound photo-activatable amino acids with suppressor tRNAs translational systems to incorporate the tags into specific sites. A third system was made possible by the development of photoreactive amino acids that use the normal cellular tRNAs and aminoacyl tRNA synthetases. In this method, the third system is used to demonstrate its utility for the study of T3S system interactions. This method describes how two photo-activatable amino acids, photo-methionine and photo-leucine, that use the normal cellular machinery are incorporated into Yersinia pestis and used to study interactions in the T3S system. To demonstrate the system, the method was used to cross-link the T3S regulatory proteins LcrG and LcrV.

Discovery of Clinically Approved Agents That Promote Suppression of Cystic Fibrosis Transmembrane Conductance Regulator Nonsense Mutations.

Premature termination codons (PTCs) in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause cystic fibrosis (CF). Several agents are known to suppress PTCs but are poorly efficacious or toxic. To determine whether there are clinically available agents that elicit translational readthrough and improve CFTR function sufficient to confer therapeutic benefit to patients with CF with PTCs. Two independent screens, firefly luciferase and CFTR-mediated transepithelial chloride conductance assay, were performed on a library of 1,600 clinically approved compounds using fisher rat thyroid cells stably transfected with stop codons. Select agents were further evaluated using secondary screening assays including short circuit current analysis on primary cells from patients with CF. In addition, the effect of CFTR modulators (ivacaftor) was tested in combination with the most efficacious agents. From the primary screen, 48 agents were selected as potentially active. Following confirmatory tests in the transepithelial chloride conductance assay and prioritizing agents based on favorable pharmacologic properties, eight agents were advanced for secondary screening. Ivacaftor significantly increased short circuit current following forskolin stimulation in cells treated with pyranoradine tetraphosphate, potassium p-aminobenzoate, and escin as compared with vehicle control. Escin, an herbal agent, consistently induced readthrough activity as demonstrated by enhanced CFTR expression and function in vitro. Clinically approved drugs identified as potential readthrough agents, in combination with ivacaftor, may induce nonsense suppression to restore therapeutic levels of CFTR function. One or more agents may be suitable to advance to human testing.

Fitness Trade-Offs Lead to Suppressor Tolerance in Yeast

Genetic variation among individuals within a population provides the raw material for phenotypic diversity upon which natural selection operates. Some given variants can act on multiple standing genomic variations simultaneously and release previously inaccessible phenotypes, leading to increased adaptive potential upon challenging environments. Previously, we identified such a variant related to a tRNA nonsense suppressor in yeast. When introduced into other genetic backgrounds, the suppressor led to an increased population phenotypic variance on various culture conditions, conferring background and environment specific selective advantages. Nonetheless, most isolates are intolerant to the suppressor on rich media due to a severe fitness cost. Here, we found that the tolerance to suppressor is related to a surprising level of fitness outburst, showing a trade-off effect to accommodate the cost of carrying the suppressor. To dissect the genetic basis of such trade-offs, we crossed strains with contrasting tolerance levels on rich media, and analyzed the fitness distribution patterns in the offspring. Combining quantitative tetrad analysis and bulk segregant analysis, we identified two genes, namely MKT1 and RGA1, involved in suppressor tolerance. We showed that alleles from the tolerant parent for both genes conferred a significant gain of fitness, which increased the suppressor tolerance. Our results present a detailed dissection of suppressor tolerance in yeast and provide insights into the molecular basis of trade-offs between fitness and evolutionary potential.

Nonsense Suppression as an Approach to Treat Lysosomal Storage Diseases.

In-frame premature termination codons (PTCs) (also referred to as nonsense mutations) comprise ~10% of all disease-associated gene lesions. PTCs reduce gene expression in two ways. First, PTCs prematurely terminate translation of an mRNA, leading to the production of a truncated polypeptide that often lacks normal function and/or is unstable. Second, PTCs trigger degradation of an mRNA by activating nonsense-mediated mRNA decay (NMD), a cellular pathway that recognizes and degrades mRNAs containing a PTC. Thus, translation termination and NMD are putative therapeutic targets for the development of treatments for genetic diseases caused by PTCs. Over the past decade, significant progress has been made in the identification of compounds with the ability to suppress translation termination of PTCs (also referred to as readthrough). More recently, NMD inhibitors have also been explored as a way to enhance the efficiency of PTC suppression. Due to their relatively low threshold for correction, lysosomal storage diseases are a particularly relevant group of diseases to investigate the feasibility of nonsense suppression as a therapeutic approach. In this review, the current status of PTC suppression and NMD inhibition as potential treatments for lysosomal storage diseases will be discussed.

SFP1-mediated prion-dependent lethality is caused by increased Sup35 aggregation and alleviated by Sis1.

[PSI+ ] is the prion form of the translation termination factor Sup35 (eRF3); [PSI+ ] strains display nonsense suppression. Another prion-like element, [ISP+ ], is linked to antisuppression in a specific background. Transcriptional regulator Sfp1 was shown to be responsible for [ISP+ ] propagation. In this work, we identified SFP1 as a multicopy inducer of [PSI+ ]-dependent lethality. Sfp1 is likely to up-regulate transcription of genes encoding release factors; however, its overproduction increases Sup35, but not Sup45 protein level. Using the synthetic lethality test, we compared the effects of SFP1 and SUP35 over-expression on the viability of [PSI+ ] strains. Together with an observation that Sfp1 overproduction leads to an increased accumulation of Sup35 in [PSI+ ] aggregates, we suggest that excess Sfp1 causes [PSI+ ] toxicity. Even though SUP45 over-expression is known to compensate for the [PSI+ ]-dependent lethality, it fails to do so when the lethality is caused by SFP1 over-expression. We discovered that the increased levels of Hsp40 chaperone Sis1 alleviate prion toxicity caused by either SFP1 or SUP35 over-expression and revert back to normal distribution of Sup35 between monomers and aggregate fractions. Finally, we showed that Sfp1 partially colocalizes with Sup35 aggregates, which may contribute to another mechanism of Sfp1-derived [PSI+ ] prion toxicity.

Atomic mutagenesis in ion channels with engineered stoichiometry.

C-type inactivation of potassium channels fine-tunes the electrical signaling in excitable cells through an internal timing mechanism that is mediated by a hydrogen bond network in the channels' selectively filter. Previously, we used nonsense suppression to highlight the role of the conserved Trp434-Asp447 indole hydrogen bond in Shaker potassium channels with a non-hydrogen bonding homologue of tryptophan, Ind (Pless et al., 2013). Here, molecular dynamics simulations indicate that the Trp434Ind hydrogen bonding partner, Asp447, unexpectedly 'flips out' towards the extracellular environment, allowing water to penetrate the space behind the selectivity filter while simultaneously reducing the local negative electrostatic charge. Additionally, a protein engineering approach is presented whereby split intein sequences are flanked by endoplasmic reticulum retention/retrieval motifs (ERret) are incorporated into the N- or C- termini of Shaker monomers or within sodium channels two-domain fragments. This system enabled stoichiometric control of Shaker monomers and the encoding of multiple amino acids within a channel tetramer.

Transfer RNAs with novel cloverleaf structures.

We report the identification of novel tRNA species with 12-base pair amino-acid acceptor branches composed of longer acceptor stem and shorter T-stem. While canonical tRNAs have a 7/5 configuration of the branch, the novel tRNAs have either 8/4 or 9/3 structure. They were found during the search for selenocysteine tRNAs in terabytes of genome, metagenome and metatranscriptome sequences. Certain bacteria and their phages employ the 8/4 structure for serine and histidine tRNAs, while minor cysteine and selenocysteine tRNA species may have a modified 8/4 structure with one bulge nucleotide. In Acidobacteria, tRNAs with 8/4 and 9/3 structures may function as missense and nonsense suppressor tRNAs and/or regulatory noncoding RNAs. In δ-proteobacteria, an additional cysteine tRNA with an 8/4 structure mimics selenocysteine tRNA and may function as opal suppressor. We examined the potential translation function of suppressor tRNA species in Escherichia coli; tRNAs with 8/4 or 9/3 structures efficiently inserted serine, alanine and cysteine in response to stop and sense codons, depending on the identity element and anticodon sequence of the tRNA. These findings expand our view of how tRNA, and possibly the genetic code, is diversified in nature.

Ataluren stimulates ribosomal selection of near-cognate tRNAs to promote nonsense suppression

A premature termination codon (PTC) in the ORF of an mRNA generally leads to production of a truncated polypeptide, accelerated degradation of the mRNA, and depression of overall mRNA expression. Accordingly, nonsense mutations cause some of the most severe forms of inherited disorders. The small-molecule drug ataluren promotes therapeutic nonsense suppression and has been thought to mediate the insertion of near-cognate tRNAs at PTCs. However, direct evidence for this activity has been lacking. Here, we expressed multiple nonsense mutation reporters in human cells and yeast and identified the amino acids inserted when a PTC occupies the ribosomal A site in control, ataluren-treated, and aminoglycoside-treated cells. We find that ataluren's likely target is the ribosome and that it produces full-length protein by promoting insertion of near-cognate tRNAs at the site of the nonsense codon without apparent effects on transcription, mRNA processing, mRNA stability, or protein stability. The resulting readthrough proteins retain function and contain amino acid replacements similar to those derived from endogenous readthrough, namely Gln, Lys, or Tyr at UAA or UAG PTCs and Trp, Arg, or Cys at UGA PTCs. These insertion biases arise primarily from mRNA:tRNA mispairing at codon positions 1 and 3 and reflect, in part, the preferred use of certain nonstandard base pairs, e.g., U-G. Ataluren's retention of similar specificity of near-cognate tRNA insertion as occurs endogenously has important implications for its general use in therapeutic nonsense suppression.

A simple technique for suppressor detection in Escherichia coli.

To study the viability of agyrA S83 stop mutation found in anEscherichia coli J53 ciprofloxacin-resistant strain (J53 CipR27), a pBR322 derivative was constructed with a TAG mutation in thebla gene knocking out ampicillin resistance. Ampicillin resistance was restored, suggesting that the strain contains tRNA suppressor activity able to suppress the UAG codongyrA and allow viability. The method was applied to 22 unique clinicalE. coli isolates, and all were found to have low-level suppressor activity.

Evolving Orthogonal Suppressor tRNAs To Incorporate Modified Amino Acids.

There have been considerable advancements in the incorporation of noncanonical amino acids (ncAA) into proteins over the last two decades. The most widely used method for site-specific incorporation of noncanonical amino acids, amber stop codon suppression, typically employs an orthogonal translation system (OTS) consisting of a heterologous aminoacyl-tRNA synthetase:tRNA pair that can potentially expand an organism's genetic code. However, the orthogonal machinery sometimes imposes fitness costs on an organism, in part due to mischarging and a lack of specificity. Using compartmentalized partnered replication (CPR) and a newly developed pheS negative selection, we evolved several new orthogonal Methanocaldococcus jannaschii (Mj) tRNA variants tRNAs with increased amber suppression activity, but that also showed up to 3-fold reduction in promiscuous aminoacylation by endogenous aminoacyl-tRNA synthetases (aaRSs). The increased orthogonality of these variants greatly reduced organismal fitness costs associated in part due to tRNA mischarging. Using these methods, we were also able to evolve tRNAs that supported the specific incorporation of 3-halo-tyrosines (3-Cl-Y, 3-Br-Y, and 3-I-Y) in E.coli.

Synthesis of alanyl nucleobase amino acids and their incorporation into proteins

Proteins which bind to nucleic acids and regulate their structure and functions are numerous and exceptionally important. Such proteins employ a variety of strategies for recognition of the relevant structural elements in their nucleic acid substrates, some of which have been shown to involve rather subtle interactions which might have been difficult to design from first principles. In the present study, we have explored the preparation of proteins containing unnatural amino acids having nucleobase side chains. In principle, the introduction of multiple nucleobase amino acids into the nucleic acid binding domain of a protein should enable these modified proteins to interact with their nucleic acid substrates using Watson-Crick and other base pairing interactions. We describe the synthesis of five alanyl nucleobase amino acids protected in a fashion which enabled their attachment to a suppressor tRNA, and their incorporation into each of two proteins with acceptable efficiencies. The nucleobases studied included cytosine, uracil, thymine, adenine and guanine, i.e. the major nucleobase constituents of DNA and RNA. Dihydrofolate reductase was chosen as one model protein to enable direct comparison of the facility of incorporation of the nucleobase amino acids with numerous other unnatural amino acids studied previously. The Klenow fragment of DNA polymerase I was chosen as a representative DNA binding protein whose mode of action has been studied in detail.

Current and emerging treatment strategies for Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is the most common form of muscular dystrophy in childhood. It is caused by mutations of the DMD gene, leading to progressive muscle weakness, loss of independent ambulation by early teens, and premature death due to cardiorespiratory complications. The diagnosis can usually be made after careful review of the history and examination of affected boys presenting with developmental delay, proximal weakness, and elevated serum creatine kinase, plus confirmation by muscle biopsy or genetic testing. Precise characterization of the DMD mutation is important for genetic counseling and individualized treatment. Current standard of care includes the use of corticosteroids to prolong ambulation and to delay the onset of secondary complications. Early use of cardioprotective agents, noninvasive positive pressure ventilation, and other supportive strategies has improved the life expectancy and health-related quality of life for many young adults with DMD. New emerging treatment includes viral-mediated microdystrophin gene replacement, exon skipping to restore the reading frame, and nonsense suppression therapy to allow translation and production of a modified dystrophin protein. Other potential therapeutic targets involve upregulation of compensatory proteins, reduction of the inflammatory cascade, and enhancement of muscle regeneration. So far, data from DMD clinical trials have shown limited success in delaying disease progression; unforeseen obstacles included immune response against the generated mini-dystrophin, inconsistent evidence of dystrophin production in muscle biopsies, and failure to demonstrate a significant improvement in the primary outcome measure, as defined by the 6-minute walk test in some studies. The long-term safety and efficacy of emerging treatments will depend on the selection of appropriate clinical end points and sensitive biomarkers to detect meaningful changes in disease progression. Correction of the underlying mutations using new gene-editing technologies and corticosteroid analogs with better safety profiles offers renewed hope for many individuals with DMD and their families.

Functional rescue of REP1 following treatment with PTC124 and novel derivative PTC-414 in human choroideremia fibroblasts and the nonsense-mediated zebrafish model.

Choroideremia (CHM) is an X-linked chorioretinal dystrophy that is caused by mutations within a single gene, CHM Currently no effective treatment exists for these patients. Since over 30% of patients harbour nonsense mutations in CHM, nonsense suppression therapy using translational readthrough inducing drugs may provide functional rescue of REP1, thus attenuating progressive sight loss. Here, we employed two CHM model systems to systematically test the efficacy and safety of ataluren (PTC124) and its novel analog PTC-414: (1) the chmru848 zebrafish, the only nonsense mutation animal model of CHM harbouring a TAA nonsense mutation, and (2) a primary human fibroblast cell line from a CHM patient harbouring a TAG nonsense mutation. PTC124 or PTC-414 treatment of chmru848 embryos led to a ∼2.0-fold increase in survival, prevented the onset of retinal degeneration with reduced oxidative stress and apoptosis, increased rep1 protein by 23.1% (PTC124) and 17.2% (PTC-414) and restored biochemical function as confirmed through in vitro prenylation assays (98 ± 2% [PTC124] and 68 ± 5% [PTC-414]). In CHMY42X/y fibroblasts, there was a recovery of prenylation activity following treatment with either PTC124 (42 ± 5%) or PTC-414 (36 ± 11%), although an increase in REP1 protein was not detected in these cells, in contrast to the zebrafish model. This comprehensive study on the use of PTC124 and PTC-414 as successful nonsense suppression agents for the treatment of CHM highlights the translational potential of these drugs for inherited retinal disease.

Biofunction-assisted DNA detection through RNase H-enhanced 3′ processing of a premature tRNA probe in a wheat germ extract

We have developed a novel type of biofunction-assisted, signal-turn-on sensor for simply and homogenously detecting DNA. This sensor system is composed of two types of in vitro-transcribed label-free RNAs (a 3′ premature amber suppressor tRNA probe and an amber-mutated mRNA encoding a reporter protein), RNase H, and a wheat germ extract (WGE). A target DNA induces the 3′ end maturation of the tRNA probe, which is enhanced by RNase H and leads to the expression of a full-length reporter protein through amber suppression in WGE, while there is almost no expression without the target due to the inactivity of the premature probe. Therefore, the target can be readily detected with the activity of the translated reporter. The catalytic reuse of the target with the help of RNase H in addition to various bioprocesses in WGE enables this sensor system to exhibit relatively high selectivity and sensitivity.

High frequency of +1 programmed ribosomal frameshifting in Euplotes octocarinatus.

Programmed −1 ribosomal frameshifting (−1 PRF) has been identified as a mechanism to regulate the expression of many viral genes and some cellular genes. The slippery site of −1 PRF has been well characterized, whereas the +1 PRF signal and the mechanism involved in +1 PRF remain poorly understood. Previous study confirmed that +1 PRF is required for the synthesis of protein products in several genes of ciliates from the genus Euplotes. To accurately assess the frequency of genes requiring frameshift in Euplotes, the macronuclear genome and transcriptome of Euplotes octocarinatus were analyzed in this study. A total of 3,700 +1 PRF candidate genes were identified from 32,353 transcripts, and the gene products of these putative +1 PRFs were mainly identified as protein kinases. Furthermore, we reported a putative suppressor tRNA of UAA which may provide new insights into the mechanism of +1 PRF in euplotids. For the first time, our transcriptome-wide survey of +1 PRF in E. octocarinatus provided a dataset which serves as a valuable resource for the future understanding of the mechanism underlying +1 PRF.

A Comparative Analysis of Mitochondrial Genomes in Eustigmatophyte Algae.

Eustigmatophyceae (Ochrophyta, Stramenopiles) is a small algal group with species of the genus Nannochloropsis being its best studied representatives. Nuclear and organellar genomes have been recently sequenced for several Nannochloropsis spp., but phylogenetically wider genomic studies are missing for eustigmatophytes. We sequenced mitochondrial genomes (mitogenomes) of three species representing most major eustigmatophyte lineages, Monodopsis sp. MarTras21, Vischeria sp. CAUP Q 202 and Trachydiscus minutus, and carried out their comparative analysis in the context of available data from Nannochloropsis and other stramenopiles, revealing a number of noticeable findings. First, mitogenomes of most eustigmatophytes are highly collinear and similar in the gene content, but extensive rearrangements and loss of three otherwise ubiquitous genes happened in the Vischeria lineage; this correlates with an accelerated evolution of mitochondrial gene sequences in this lineage. Second, eustigmatophytes appear to be the only ochrophyte group with the Atp1 protein encoded by the mitogenome. Third, eustigmatophyte mitogenomes uniquely share a truncated nad11 gene encoding only the C-terminal part of the Nad11 protein, while the N-terminal part is encoded by a separate gene in the nuclear genome. Fourth, UGA as a termination codon and the cognate release factor mRF2 were lost from mitochondria independently by the Nannochloropsis and T. minutus lineages. Finally, the rps3 gene in the mitogenome of Vischeria sp. is interrupted by the UAG codon, but the genome includes a gene for an unusual tRNA with an extended anticodon loop that we speculate may serve as a suppressor tRNA to properly decode the rps3 gene.

Incorporation of non-canonical amino acids into proteins in yeast

Non-canonical amino acids add extraordinary chemistries to proteins when they gain access to translation. In yeast, they can be incorporated into proteins by replacing a canonical amino acid or in a site-specific manner in response to an amber stop codon. The first approach simply exploits the natural substrate tolerance of the aminoacyl-tRNA synthetases in an auxotrophic host. The latter requires the co-expression of an orthogonal aminoacyl-tRNA synthetase that is specific for the non-canonical amino acid together with an amber suppressor tRNA. This review briefly recaps the residue- and site-specific incorporation techniques for non-canonical amino acids in yeast. It describes the selection system for orthogonal aminoacyl-tRNA synthetase/suppressor tRNA pairs and compares the different expression systems for these pairs. Numerous examples illustrate the application of non-canonical amino acids for protein engineering in yeast. The compilation includes the chemical structures of the amino acid analogs, the orthogonal pairs that were used for their incorporation and the titers of the labeled variant proteins.