Multiple Codons Research Articles

Predicting synonymous codon usage and optimizing the heterologous gene for expression in E. coli

Of the 20 common amino acids, 18 are encoded by multiple synonymous codons. These synonymous codons are not redundant; in fact, all of codons contribute substantially to protein expression, structure and function. In this study, the codon usage pattern of genes in the E. coli was learned from the sequenced genomes of E. coli. A machine learning based method, Presyncodon was proposed to predict synonymous codon selection in E. coli based on the learned codon usage patterns of the residue in the context of the specific fragment. The predicting results indicate that Presycoden could be used to predict synonymous codon selection of the gene in the E. coli with the high accuracy. Two reporter genes (egfp and mApple) were designed with a combination of low- and high-frequency-usage codons by the method. The fluorescence intensity of eGFP and mApple expressed by the (egfp and mApple) designed by this method was about 2.3- or 1.7- folds greater than that from the genes with only high-frequency-usage codons in E. coli. Therefore, both low- and high-frequency-usage codons make positive contributions to the functional expression of the heterologous proteins. This method could be used to design synthetic genes for heterologous gene expression in biotechnology.

Translation Initiation from Conserved Non-AUG Codons Provides Additional Layers of Regulation and Coding Capacity.

ABSTRACTNeurospora crassa cpc-1 and Saccharomyces cerevisiae GCN4 are homologs specifying transcription activators that drive the transcriptional response to amino acid limitation. The cpc-1 mRNA contains two upstream open reading frames (uORFs) in its >700-nucleotide (nt) 5′ leader, and its expression is controlled at the level of translation in response to amino acid starvation. We used N. crassa cell extracts and obtained data indicating that cpc-1 uORF1 and uORF2 are functionally analogous to GCN4 uORF1 and uORF4, respectively, in controlling translation. We also found that the 5′ region upstream of the main coding sequence of the cpc-1 mRNA extends for more than 700 nucleotides without any in-frame stop codon. For 100 cpc-1 homologs from Pezizomycotina and from selected Basidiomycota, 5′ conserved extensions of the CPC1 reading frame are also observed. Multiple non-AUG near-cognate codons (NCCs) in the CPC1 reading frame upstream of uORF2, some deeply conserved, could potentially initiate translation. At least four NCCs initiated translation in vitro. In vivo data were consistent with initiation at NCCs to produce N-terminally extended N. crassa CPC1 isoforms. The pivotal role played by CPC1, combined with its translational regulation by uORFs and NCC utilization, underscores the emerging significance of noncanonical initiation events in controlling gene expression.

Mitovirus UGA(Trp) codon usage parallels that of host mitochondria

Mitoviruses replicate in mitochondria of their host fungi. They have small RNA genomes that encompass a single ORF encoding the viral RdRp. Since UGA codons encode Trp in fungal mitochondria, the RdRp ORF of a typical mitovirus includes multiple UGA codons. In some mitoviruses, however, the ORF has no such codons, suggesting that these particular viruses may be under selective pressure to exclude them. In this report, new evidence is presented that host fungi whose mitoviruses have no or few UGA codons are distinctive in also having no or few UGA codons in their core mitochondrial genes. Thus, the relative exclusion of such codons in a subset of mitoviruses appears to reflect most fundamentally that UGA(Trp) is a rare mitochondrial codon in their particular hosts. The fact that UGA(Trp) is a rare mitochondrial codon in many fungi appears not to have been widely discussed to date.

An efficient system for incorporation of unnatural amino acids in response to the four-base codon AGGA in Escherichia coli

BackgroundAdding new amino acids to the set of building blocks for protein synthesis expands the scope of protein engineering, and orthogonal pairs of tRNA and aminoacyl-tRNA synthetase have been developed for incorporating unnatural amino acids (UAAs) into proteins. While diverse systems have been developed to incorporate UAAs in response to the amber codon, less research has been focused on four-base codons despites their advantages. In this study, we report an efficient method to incorporate UAA in response to an AGGA codon in Escherichia coli. ResultsThe Methanococcus jannaschii tyrosyl-tRNA synthetase-tRNACUA(MjTyrRS-MjtRNACUA) orthogonal pair has been engineered to incorporate diverse UAAs in response to the amber codon. To apply the engineered MjTyrRS enzymes for UAAs to a four-base codon suppression, we developed an MjTyrRS-MjtRNAUCCU pair system that enabled incorporation of UAAs in response to the AGGA codon in E. coli. Using this system, we demonstrated that several UAAs could be incorporated quantitatively in the AGGA site. In addition, multiple AGGA codons were successfully suppressed in an E. coli strain when the endogenous tRNACCUArg gene was knocked out. ConclusionAn efficient system was developed for the incorporation of UAAs in response to the AGGA four-base codon in E. coli, and the method was successfully demonstrated for several UAAs and for multiple AGGA sites. General significanceThe developed system can expand the repertoire of protein engineering tools based on amino acid analogues in combination with other UAA incorporation methods. 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.

Pseudouridine in the Anticodon of Escherichia coli tRNATyr(QΨA) Is Catalyzed by the Dual Specificity Enzyme RluF

Pseudouridine is found in almost all cellular ribonucleic acids (RNAs). Of the multiple characteristics attributed to pseudouridine, making messenger RNAs (mRNAs) highly translatable and non-immunogenic is one such feature that directly implicates this modification in protein synthesis. We report the existence of pseudouridine in the anticodon of Escherichia coli tyrosine transfer RNAs (tRNAs) at position 35. Pseudouridine was verified by multiple detection methods, which include pseudouridine-specific chemical derivatization and gas phase dissociation of RNA during liquid chromatography tandem mass spectrometry (LC-MS/MS). Analysis of total tRNA isolated from E. coli pseudouridine synthase knock-out mutants identified RluF as the enzyme responsible for this modification. Furthermore, the absence of this modification compromises the translational ability of a luciferase reporter gene coding sequence when it is preceded by multiple tyrosine codons. This effect has implications for the translation of mRNAs that are rich in tyrosine codons in bacterial expression systems.

DNA methylation: conducting the orchestra from exposure to phenotype?

DNA methylation, through 5-methyl- and 5-hydroxymethylcytosine (5mC and 5hmC), is considered to be one of the principal interfaces between the genome and our environment, and it helps explain phenotypic variations in human populations. Initial reports of large differences in methylation level in genomic regulatory regions, coupled with clear gene expression data in both imprinted genes and malignant diseases, provided easily dissected molecular mechanisms for switching genes on or off. However, a more subtle process is becoming evident, where small (<10 %) changes to intermediate methylation levels are associated with complex disease phenotypes. This has resulted in two clear methylation paradigms. The latter “subtle change” paradigm is rapidly becoming the epigenetic hallmark of complex disease phenotypes, although we are currently hampered by a lack of data addressing the true biological significance and meaning of these small differences.Our initial expectation of rapidly identifying mechanisms linking environmental exposure to a disease phenotype led to numerous observational/association studies being performed. Although this expectation remains unmet, there is now a growing body of literature on specific genes, suggesting wide ranging transcriptional and translational consequences of such subtle methylation changes. Data from the glucocorticoid receptor (NR3C1) has shown that a complex interplay between DNA methylation, extensive 5′UTR splicing, and microvariability gives rise to the overall level and relative distribution of total and N-terminal protein isoforms generated. Additionally, the presence of multiple AUG translation initiation codons throughout the complete, processed mRNA enables translation variability, hereby enhancing the translational isoforms and the resulting protein isoform diversity, providing a clear link between small changes in DNA methylation and significant changes in protein isoforms and cellular locations. Methylation changes in the NR3C1 CpG island alters the NR3C1 transcription and eventually protein isoforms in the tissues, resulting in subtle but visible physiological variability.This review addresses the current pathophysiological and clinical associations of such characteristically small DNA methylation changes, the ever-growing roles of DNA methylation and the evidence available, particularly from the glucocorticoid receptor of the cascade of events initiated by such subtle methylation changes, as well as addressing the underlying question as to what represents a genuine biologically significant difference in methylation.

Codon optimality at genome transition

RNA Stability Nucleotide triplets, or codons, designate specific amino acids for protein synthesis. However, that is not their only job. In yeast and bacteria, codons contribute to RNA stability, with “optimal” codons stabilizing RNAs and “suboptimal” codons destabilizing RNAs. This is possible because multiple codons can encode the same amino acid. Bazzini et al. now demonstrate that codon usage in zebrafish, frogs, mice, and flies can affect transcript degradation and polyadenylation at the critical stage in development when transcription switches from the maternal to the zygotic genome. Furthermore, enriching genes with nonoptimal codons can reduce translation efficiency. EMBO J. 10.15252/embj.201694699 (2016).

Abstract 3137: NGS-based detection of KRAS hotspot mutations in plasma cell-free DNA of pancreatic cancer cases

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is characterized by hotspot mutations in the KRAS gene (codons 12, 13 or 61) in 85-90% of cases. Codon 12 KRAS mutations have been detected in pancreatic juice, blood and stool samples from pancreatic cancer cases and represent promising biomarkers for early detection. However, the proportion of tumor-derived KRAS mutations in cell-free DNA fragments (cfDNA) has shown large variations, probably because of the heterogeneity in biosamples and assays tested. Deep sequencing technologies (NGS) allow the identification of low-abundance somatic variants, but have not previously been applied to the detection of KRAS hotspot mutations in cfDNA of PDAC cases. Moreover, variant calling methods have rarely been tested against cancer-free individuals so the proportion of false positives is unknown. We investigated whether deep sequencing of KRAS mutations at codons 12, 13 and 61 in plasma samples could represent a robust assay to distinguish pancreatic cancer from chronic pancreatitis and healthy controls. Methods: We developed an Ion Torrent-based NGS KRAS assay (partial exons 2 and 3, totalling 208bp) to screen cfDNA from plasma samples of 461 PDAC cases, 154 individuals with chronic pancreatitis and 421 healthy controls. cfDNA extraction (&amp;gt;4ng) and sequencing (&amp;gt;1000x coverage on average, and absence of systematic high sequencing error rates on the 208bp) performed well on 431 (93%) PDAC cases, 138 (90%) chronic pancreatitis, and 388 (95%) controls. We fit a robust negative-binomial regression to estimate the distribution of the sequencing errors at each DNA bp position and identified outlying samples, which were considered as KRAS positive when q-value&amp;lt;10-3. We also estimated the detection threshold of our assay using serial dilutions of DNA from SW480 KRAS mutated cell-line (p.G12V) in wild-type DNA. Results: Sequencing of the serial dilutions of KRAS p.G12V mutated DNA indicated a detection threshold at a minor allele frequency of 0.2%. KRAS mutations in cfDNA were detected in 83 (19.3%) PDAC cases (73 on codon 12; 8 on codon 61; 1 on codon 13; and 1 multiple codons, i.e., similar in proportions as reported in tumor tissue from the International Cancer Genomic Consortium); 3 (2.2%) chronic pancreatitis (all on codon 12); and 8 (2.1%) healthy controls (4 on codon 12 and 4 on codon 61). Stage was significantly associated with the proportion of detected mutations in cancer cases (chi-squared p = 0.0005): the proportions of cases with detectable KRAS mutations in plasma were 7.9%, 14.9%, and 31.1% for local, regional, and advanced stages, respectively. Conclusions: The NGS-based KRAS mutation screening is a sensitive approach to detect low allelic fraction in plasma cfDNA, although its utility for early detection is still limited. However, it has the capacity to identify specific KRAS mutations, which could be useful in a panel of other non-invasive biomarkers. Citation Format: Florence Le Calvez-Kelm, Matthieu Foll, Magdalena B. Wozniak, Geoffroy Durand, Priscilia Chopard, Maroulio Pertesi, Tiffany Delhomme, Ivana Holcatova, Lenka Foretova, Vladimir Janout, Eleonora Fabianova, Maxime P. Vallée, Paul Brennan, James D. McKay, Graham Byrnes, Ghislaine Scélo. NGS-based detection of KRAS hotspot mutations in plasma cell-free DNA of pancreatic cancer cases. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3137.

Tryptophan Codon-Dependent Transcription in Chlamydia pneumoniae during Gamma Interferon-Mediated Tryptophan Limitation

In evolving to an obligate intracellular niche, Chlamydia has streamlined its genome by eliminating superfluous genes as it relies on the host cell for a variety of nutritional needs like amino acids. However, Chlamydia can experience amino acid starvation when the human host cell in which the bacteria reside is exposed to interferon gamma (IFN-γ), which leads to a tryptophan (Trp)-limiting environment via induction of the enzyme indoleamine-2,3-dioxygenase (IDO). The stringent response is used to respond to amino acid starvation in most bacteria but is missing from Chlamydia Thus, how Chlamydia, a Trp auxotroph, responds to Trp starvation in the absence of a stringent response is an intriguing question. We previously observed that C. pneumoniae responds to this stress by globally increasing transcription while globally decreasing translation, an unusual response. Here, we sought to understand this and hypothesized that the Trp codon content of a given gene would determine its transcription level. We quantified transcripts from C. pneumoniae genes that were either rich or poor in Trp codons and found that Trp codon-rich transcripts were increased, whereas those that lacked Trp codons were unchanged or even decreased. There were exceptions, and these involved operons or large genes with multiple Trp codons: downstream transcripts were less abundant after Trp codon-rich sequences. These data suggest that ribosome stalling on Trp codons causes a negative polar effect on downstream sequences. Finally, reassessing previous C. pneumoniae microarray data based on codon content, we found that upregulated transcripts were enriched in Trp codons, thus supporting our hypothesis.

Molecular evaluation of PIK3CA gene mutation in breast cancer: determination of frequency, distribution pattern and its association with clinicopathological findings in Indian patients.

Somatic mutations in the PIK3CA gene are common in breast cancer and represent a clinically useful marker for prognosis and therapeutic target. Activating mutations in the PI3K p110 catalytic subunit (PIK3CA) have been identified in 18-40% of breast carcinomas. In this study, we evaluated PIK3CA mutation in 185 Indian breast cancer patients by direct DNA sequencing. PIK3CA mutations were observed in 23.2% (43/185) of breast tumor samples. PIK3CA mutations were more frequent exon 30 (76.8%) than in exon 9 (23.2%). Mutations were mostly clustered within two hotspot region between nucleotides 1624 and 1636 or between 3129 and 3140. Sequencing analysis revealed four different missense mutations at codon 542 and 545 (E542K, E545K, E545A and E545G) in the helical domain and two different amino acid substitutions at codon 1047 (H1047R and H1047L) in the kinase domain. None of the cases harbored concomitant mutations at multiple codons. PIK3CA mutations were more frequent in older patients, smaller size tumors, ductal carcinomas, grade II tumors, lymph node-positive tumors and non-DCIS tumors; however, none of the differences were significant. In addition, PIK3CA mutations were common in ER+, PR+ and HER2+ cases (30%), and a comparatively low frequency were noted in triple-negative tumors (13.6%). In conclusion, to our knowledge, this is the largest study to evaluate the PIK3CA mutation in Indian breast cancer patients. The frequency and distribution pattern of PIK3CA mutations is similar to global reports. Furthermore, identification of molecular markers has unique strengths and can provide insights into the pathogenic process of breast carcinomas.

A novel nuclear genetic code alteration in yeasts and the evolution of codon reassignment in eukaryotes.

The genetic code is the cellular translation table for the conversion of nucleotide sequences into amino acid sequences. Changes to the meaning of sense codons would introduce errors into almost every translated message and are expected to be highly detrimental. However, reassignment of single or multiple codons in mitochondria and nuclear genomes, although extremely rare, demonstrates that the code can evolve. Several models for the mechanism of alteration of nuclear genetic codes have been proposed (including “codon capture,” “genome streamlining,” and “ambiguous intermediate” theories), but with little resolution. Here, we report a novel sense codon reassignment in Pachysolen tannophilus, a yeast related to the Pichiaceae. By generating proteomics data and using tRNA sequence comparisons, we show that Pachysolen translates CUG codons as alanine and not as the more usual leucine. The Pachysolen tRNACAG is an anticodon-mutated tRNAAla containing all major alanine tRNA recognition sites. The polyphyly of the CUG-decoding tRNAs in yeasts is best explained by a tRNA loss driven codon reassignment mechanism. Loss of the CUG-tRNA in the ancient yeast is followed by gradual decrease of respective codons and subsequent codon capture by tRNAs whose anticodon is not part of the aminoacyl-tRNA synthetase recognition region. Our hypothesis applies to all nuclear genetic code alterations and provides several testable predictions. We anticipate more codon reassignments to be uncovered in existing and upcoming genome projects.

Alternative Translation Initiation of a Haloarchaeal Serine Protease Transcript Containing Two In-Frame Start Codons.

Recent studies have shown that haloarchaea employ leaderless and Shine-Dalgarno (SD)-less mechanisms for translation initiation of leaderless transcripts with a 5' untranslated region (5' UTR) of <10 nucleotides (nt) and leadered transcripts with a 5' UTR of ≥10 nt, respectively. However, whether the two mechanisms can operate on the same naturally occurring haloarchaeal transcript carrying multiple potential start codons is unknown. In this study, the transcript of the sptA gene (encoding an extracellular serine protease of Natrinema sp. strain J7-2) was experimentally determined and found to contain two potential in-frame AUG codons (AUG(1) and AUG(2)) located 5 and 29 nt, respectively, downstream of the transcription start site. Mutational analysis revealed that both AUGs can function as the translation start codon for production of active SptA, although AUG(1) is more efficient than AUG(2) for translation initiation. Insertion of a stable stem-loop structure between the two AUGs completely abolished initiation at AUG(1) but did not affect initiation at AUG(2), indicating that AUG(2)-initiated translation does not involve ribosome scanning from the 5' end of the transcript. Furthermore, the efficiency of AUG(2)-initiated translation was not influenced by an upstream SD-like sequence. In addition, both AUG(1) and AUG(2) contribute to transcript stability, probably by recruiting ribosomes to protect the transcript against degradation. These data suggest that depending on which of two in-frame start codons is used, the sptA transcript can act as either a leaderless or a leadered transcript for SptA production in haloarchaea. In eukaryotes and bacteria, alternative translation start sites contribute to proteome complexity and can be used as a functional mechanism to increase translation efficiency. However, little is known about alternative translation initiation in archaea. Our results demonstrate that leaderless and SD-less mechanisms can be used for translation initiation of the sptA transcript from two in-frame start codons, raising the possibility that in haloarchaea, alternative translation initiation on one transcript functions to increase translation efficiency and/or contribute to proteome complexity.

Forced Ambiguity of the Leucine Codons for Multiple-Site-Specific Incorporation of a Noncanonical Amino Acid.

Multiple-site-specific incorporation of a noncanonical amino acid into a recombinant protein would be a very useful technique to generate multiple chemical handles for bioconjugation and multivalent binding sites for the enhanced interaction. Previously combination of a mutant yeast phenylalanyl-tRNA synthetase variant and the yeast phenylalanyl-tRNA containing the AAA anticodon was used to incorporate a noncanonical amino acid into multiple UUU phenylalanine (Phe) codons in a site-specific manner. However, due to the less selective codon recognition of the AAA anticodon, there was significant misincorporation of a noncanonical amino acid into unwanted UUC Phe codons. To enhance codon selectivity, we explored degenerate leucine (Leu) codons instead of Phe degenerate codons. Combined use of the mutant yeast phenylalanyl-tRNA containing the CAA anticodon and the yPheRS_naph variant allowed incorporation of a phenylalanine analog, 2-naphthylalanine, into murine dihydrofolate reductase in response to multiple UUG Leu codons, but not to other Leu codon sites. Despite the moderate UUG codon occupancy by 2-naphthylalaine, these results successfully demonstrated that the concept of forced ambiguity of the genetic code can be achieved for the Leu codons, available for multiple-site-specific incorporation.

Expanding the amino acid repertoire of ribosomal polypeptide synthesis via the artificial division of codon boxes.

In ribosomal polypeptide synthesis the library of amino acid building blocks is limited by the manner in which codons are used. Of the proteinogenic amino acids, 18 are coded for by multiple codons and therefore many of the 61 sense codons can be considered redundant. Here we report a method to reduce the redundancy of codons by artificially dividing codon boxes to create vacant codons that can then be reassigned to non-proteinogenic amino acids and thereby expand the library of genetically encoded amino acids. To achieve this, we reconstituted a cell-free translation system with 32 in vitro transcripts of transfer RNASNN (tRNASNN) (S = G or C), assigning the initiator and 20 elongator amino acids. Reassignment of three redundant codons was achieved by replacing redundant tRNASNNs with tRNASNNs pre-charged with non-proteinogenic amino acids. As a demonstration, we expressed a 32-mer linear peptide that consists of 20 proteinogenic and three non-proteinogenic amino acids, and a 14-mer macrocyclic peptide that contains more than four non-proteinogenic amino acids.

GC-Content of Synonymous Codons Profoundly Influences Amino Acid Usage.

Amino acids typically are encoded by multiple synonymous codons that are not used with the same frequency. Codon usage bias has drawn considerable attention, and several explanations have been offered, including variation in GC-content between species. Focusing on a simple parameter—combined GC proportion of all the synonymous codons for a particular amino acid, termed GCsyn—we try to deepen our understanding of the relationship between GC-content and amino acid/codon usage in more details. We analyzed 65 widely distributed representative species and found a close association between GCsyn, GC-content, and amino acids usage. The overall usages of the four amino acids with the greatest GCsyn and the five amino acids with the lowest GCsyn both vary with the regional GC-content, whereas the usage of the remaining 11 amino acids with intermediate GCsyn is less variable. More interesting, we discovered that codon usage frequencies are nearly constant in regions with similar GC-content. We further quantified the effects of regional GC-content variation (low to high) on amino acid usage and found that GC-content determines the usage variation of amino acids, especially those with extremely high GCsyn, which accounts for 76.7% of the changed GC-content for those regions. Our results suggest that GCsyn correlates with GC-content and has impact on codon/amino acid usage. These findings suggest a novel approach to understanding the role of codon and amino acid usage in shaping genomic architecture and evolutionary patterns of organisms.

MTB-DR-RIF 9G membrane: a platform for multiplex SNP detection of multidrug-resistant TB

The MTB-DR-RIF 9G membrane can detect by detecting multiple mutations in multiple codons. The MTB-DR-RIF 9G membrane possesses clinical applicability in point-of-care settings for the following reasons: (i) 100% similar results with that of the sequencing analysis for clinical samples, (ii) discrimination of the multiple mutations in multiple codons, (iii) a specific/non-specific hybridization ratio higher than 350:1, and (iv) the sensitivity was found to be 1-10 copies/test for detection and discrimination of the wild and mutant TB strains. Graphical abstract Schematic illustration of the effect of controller DNA on the hybridization of the immobilized probes (corresponding to the wild TB strain) with the PCR product of (a) wild TB strain and (b) mutant TB strain.

Abstract P2-03-06: FoundationOne profiling of TSC1 and TSC2-mutated advanced breast cancers

Abstract Background: The TSC1/TSC2 complex inhibits the mTOR pathway. Loss of function of this complex leads to hyperperactivation of the mTOR pathway which promotes growth and survival. Few studies have examined the role of TSC1 and TSC2 mutations in breast cancer and as a clinically relevant target for molecularly targeted therapy, such as everolimus, a rapamycin analog. Methods: Hybridization capture of 3769 exons of 236 cancer-related genes and 47 introns from 19 genes that are frequently rearranged in cancer were fully sequenced to high, uniform coverage from a commercial CLIA-certified laboratory (Foundation Medicine). Results: 30 of 2,208 breast cancer pt samples (1.3%) harbored either TSC1 (13/30, 43%) or TSC2 (17/30, 56%) genomic alterations (GAs). The samples with TSC1/2 consisted of breast carcinoma n.o.s. (17/30) and invasive ductal carcinoma (12/30); one invasive lobular carcinoma (ILC) harbored a TSC2 alteration. Sequencing was performed on metastatic (18/30, 60%) and primary (12/30, 40%) tumors. All TSC1 alterations resulted in truncation of the protein product; TSC2 GA consisted of truncations (14/17) and point mutations (3/17). TSC1/2 GAs frequently co-occurred with GAs in TP53 (19/30; 63%) and amplifications of MYC (9/30; 30%) and FGFR1 (7/30; 23%). Of the TSC1/2-mutant cases where hormone receptor and HER2/neu status was known (23/30), 65% were ER-/PR-/HER2- (TNBC), 26% were ER+/HER2- and 9% were ER-/HER2+. Patient case: We describe a postmenopausal node positive ILC (ER+/PR+/HER2-) pt who received adjuvant chemotherapy followed by 9 yrs adjuvant anastrazole then with disease recurrence. On FoundationOne testing a TSC2 A1141T missense mutation, along with GAs in CDH1, ETV6, and CSF1R, were found in her metastatic disease in the soft tissues of her left orbit associated with substantial swelling resulting in loss of vision. While TSC2 A1141T has not been functionally characterized, in vitro and in vivo data suggest sensitivity to mTOR inhibitors. Her disease stabilized but did not decrease in size with vinorelbine + capecitabine. She was treated with letrozole + everolimus and had a near clinical complete response with restoration of vision for 5 + months. Collection of clinical data on additional pts is currently ongoing. Conclusion: 1.3% of breast cancer cases harbored GAs in either TSC1 orTSC2. The majority of TSC1/2 GAs cause inactivation of these negative regulatory proteins leading to activation of the mTOR signaling pathway. TSC1/2 GAs were enriched for TNBC (65%) versus non-TNBC (35%) (p= 0.077; Fisher’s exact test). Truncation of TSC1/2 occurred at multiple codons, underscoring the importance of examining the entire coding region of tumor suppressor genes for these inactivating events. Comprehensive genomic profiling has the potential to identify the broad spectrum of these inactivating events, a subset of which may be clinically relevant. Citation Format: Norma A Palma, Juliann Chmielecki, Garrett Frampton, Siraj Ali, Maren Levin, Jeffrey S Ross, Deborah Morosini, Gary Palmer, Vincent A Miller, Phil Stephens, Joyce O'Shaughnessy. FoundationOne profiling of TSC1 and TSC2-mutated advanced breast cancers [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P2-03-06.

Determinants of the CmoB carboxymethyl transferase utilized for selective tRNA wobble modification

Enzyme-mediated modifications at the wobble position of tRNAs are essential for the translation of the genetic code. We report the genetic, biochemical and structural characterization of CmoB, the enzyme that recognizes the unique metabolite carboxy-S-adenosine-L-methionine (Cx-SAM) and catalyzes a carboxymethyl transfer reaction resulting in formation of 5-oxyacetyluridine at the wobble position of tRNAs. CmoB is distinctive in that it is the only known member of the SAM-dependent methyltransferase (SDMT) superfamily that utilizes a naturally occurring SAM analog as the alkyl donor to fulfill a biologically meaningful function. Biochemical and genetic studies define the in vitro and in vivo selectivity for Cx-SAM as alkyl donor over the vastly more abundant SAM. Complementary high-resolution structures of the apo- and Cx-SAM bound CmoB reveal the determinants responsible for this remarkable discrimination. Together, these studies provide mechanistic insight into the enzymatic and non-enzymatic feature of this alkyl transfer reaction which affords the broadened specificity required for tRNAs to recognize multiple synonymous codons.

Identification of Determinants that Govern Processive and Efficient Selenocysteine (Sec) Incorporation

Selenoproteins include essential proteins and are unique as they incorporate selenocysteine (Sec), the 21st amino acid, in response to an stop codon (UGA). Thus selenoprotein expression is a challenge as it requires stop codon redefinition. To date, five essential factors for Sec incorporation have been identified. This includes the mRNA with an inframe UGA codon, a stem loop structure referred to as SECIS element located in the 3′UTR, a dedicated elongation factor (eEFSec), a SECIS binding protein (SBP2) and a unique Sec tRNA Sec‐tRNASec. However the mechanistic details of Sec incorporation still remains unclear. Among the known selenoproteins, Selenoprotein P (SEPP1) is unique mainly as it contains multiple UGA codons and possesses two SECIS elements in the 3′UTR. Thus, full length SEPP1 synthesis requires processive Sec incorporation which in vivo appears to be efficient. The focus of this research is to identify core determinants that govern processive and efficient Sec incorporation. Using 75Se labeling in vitro we demonstrate that selenium supplementation stimulates processive Sec incorporation by 4‐fold thus linking active Sec‐tRNASec synthesis with processive Sec incorporation. We also establish that while the SEPP1 mRNA is inherently processive for Sec incorporation, there exists an interplay between the coding region and the 3′UTR that governs the degree of processivity. Furthermore cellular transfection studies indicate RNA stability as a crucial determinant of efficiency in mammalian SEPP1 synthesis. Thus translation regulation primarily governs processive and efficient Sec incorporation via channeling of Sec incorporation components and also by recruiting novel cis elements.

Post-transcriptional modifications to tRNA--a response to the genetic code degeneracy.

We used to think of the genetic code degeneracy as an obligatory process resulting from assigning 61 sense codons to the 20 amino acids in protein. In this degeneracy, 18 of the 20 amino acids are endowed with multiple codons, and the conventional wisdom was that each of the 18 was translated equally from each codon. However, due to the degeneracy mostly manifest at the wobble position for each amino acid, we were led to believe that the degeneracy is simply to provide a deck of codons for safety control, in case one or two codons are damaged by nucleotide substitutions to the wobble position. However, work over a long path has now shown that the degeneracy has a much deeper meaning, with the ability to confer codon-by-codon translational regulation. At its core, translation of a codon requires pairing interaction with the anticodon of its cognate tRNA. Due to the degeneracy, multiple codons are paired with one anticodon in different efficiencies and stabilities. This is a form of translational regulation executed at the level of individual codons. Nature has developed this translational regulation by two means: one by employing biased codon usage and the other by modifying the chemical structure of the tRNA anticodon or adjacent nucleotides via post-transcriptional modifications. While biased codon usage is genetically encoded, post-transcriptional modifications are epigenetic and have more flexibility to modulate the fidelity and efficiency of codon-anticodon pairing interactions. Indeed, among codons for the same amino acid, some require tRNA modifications that are stress-induced, conferring a capacity to regulate cellular adaptation to stress at individual codons. Thus, in response to the genetic code degeneracy, tRNA has evolved to carry post-transcriptional modifications to fine-tune translation one codon at a time. Enzymes catalyzing tRNA modifications are often linked to human diseases, ranging from metabolic defects, mitochondrial dysfunctions, and neurological disorders, to cancer. The greatest diversity of tRNAmodifications is at positions 34 (the wobble position) and 37 (on the 3′ side of the anticodon), two positions critical for translational accuracy and reading-frame maintenance. Three modifications at these positions have strong effects on codon-anticodon pairing interactions. First, U34 is modified to 5-methoxycarbonyl-methyl-2-thiouridine (mcmsU34) in tRNA (anticodon 5′-UUU-3′), tRNA, tRNA, and tRNA. In the case of tRNA for example, while it can read both Lys codons (5′-AAA and 5′-AAG), the codon-anticodon pairing in either case is weak, consisting of A-U and G-U base pairs. The addition of mcms to U34 can stabilize the A-U and G-U pairs at the wobble position by providing an extended chemical group to enhance the stacking interaction within each pair. The importance of this mcms modification is emphasized by its response to cellular stress. Specifically, mcmsU34 is synthesized in two sequential steps, first via the addition of mcm to U34 by the ELP complex and second via the addition of s to the mcmU34 intermediate by the URM1 pathway. While the ELP complex is formally associated with the RNA pol II transcription complex in yeast, it catalyzes themcm addition to U34 upon cellular oxidative stress. Similarly, the URM1 complex is activated by cellular stress in yeast, such as oxidative stress and temperature up-shift stress. Recent work shows that mcms addition to U34 facilitates translation of codonbiased mRNAs required for stress response. For example, upon stress with the alkylating agent methyl methane sulfonate, the methyl transferase Trm9 associated with the ELP complex is activated to help to translate DNA damage response genes each with a pattern of codon usage dependent on mcmsU34-tRNA for translation. Similarly, a human Trm9-like protein in cancer cells helps to translate the LIN9 protein, which has a codon bias requiring mcmsU34tRNA for translation. LIN9 interacts with the tumor suppressor protein Rb to suppress cancer progression, thus implicating the Trm9-like protein and the mcms modification of U34-tRNA with a role in inhibition of tumor growth.