cells respond to environmental 
 signals and stresses using a variety 
 of mechanisms that link the external stimuli to changes in cell phenotype 
 by myriad biochemical reactions that ultimately lead to changes in 
 gene expression and protein activity . well - defined pathways of signal 
 transduction affect transcription , mrna stability , protein levels , 
 and protein secondary modification , with the altered protein function 
 and metabolite levels defining a new cell state . among the mechanisms 
 of cell response , the link between translation and environmental changes 
 is the least understood . here , we describe the emerging evidence for 
 a new system by which cells respond and adapt to environmental stresses 
 by reprogramming dozens of modified ribonucleosides in trna , which 
 leads to selective translation of codon - biased mrnas . this system 
 exploits features of both genetics , in the form of a code of codon 
 use in families of genes , and postsynthetic dna and protein modifications , 
 in the idea that editable modifications affect gene expression , with 
 signal transduction pathways linking the environmental stress to controlled 
 changes in gene expression at the level of translational elongation . as a parallel to the system 
 of rna modifications in control of 
 translation , epigenetics is classically defined as heritable changes 
 in gene expression without changing the dna sequence . the prime example 
 involves formation of mc by dna methyltransferases ( dnmt s ) 
 as a well - established regulator of gene expression , with methylation patterns in promoter 
 regions dramatically altered in response to environmental stimuli 
 or in different cancers . although dna methylation 
 patterns are heritable and the patterns previously presumed to be 
 stable in a specific cell type , global reprogramming of mc patterns in the genome has now been observed in response to exposure 
 to drugs and toxicants , which illustrates 
 a dynamic role for epigenetic signals in cellular response and adaptation . methylation of histone tails by protein methyltransferases ( pmt s ) 
 functions in a similar manner to dna methylation as a well - recognized 
 regulator of gene expression . as part 
 of an integrated system with dna methylation , histone methylation 
 is theorized to be part of a complicated histone code that is composed 
 of a variety of other posttranslational modifications and that is 
 altered by environmental signals and disease pathologies to control 
 gene expression . for example , lysine n - methylation ( h3k4 , h3k36 ) in histone h3 and subsequent demethylation 
 are considered dueling signals that regulate transcription . at their simplest , both promoter and histone 
 methylation affect gene expression by regulating how much of a transcript 
 is made , with these signals altered in cancer pathogenesis and reprogrammed 
 after some environmental exposures ( figure 1a ) . however , the complexity of epigenetic dna marks has become more 
 complicated by the emergence of 5-hydroxylmethylcytosine , 5-formylcytosine , 
 and 5-carboxycytosine modifications of dna and by the diversity of histone modifications , including acetylation 
 and phosphorylation . in this context of 
 transcriptional control by dna and histone modification , we introduce 
 the concept of a system of dozens of rna modifications , including 
 rna methylation , that reprogram in response to environmental changes 
 and control gene expression at the level of translation . ( a ) dna and histone methylation 
 marks regulate transcript levels to affect gene expression . dynamic 
 rna methylation signals have recently been demonstrated to regulate 
 how well a transcript is translated to affect gene expression . ( b ) 
 structures of 5-methylcytosine ( mc ) in dna ( x = h ) and 
 rna ( x = oh ) and the trna wobble modification mcmu . ( c ) 
 structures of four of > 120 modified ribonucleosides in prokaryotes 
 and eukaryotes . the rna modification database can be accessed to view 
 more modification structures ( http://mods.rna.albany.edu/ ) . the nucleosome image was prepared 
 by david s. goodsell and the rcsb pdb ( http://www.rcsb.org/pdb/101/motm.do?momid=7 ) and is used with permission . similar to dnmt s and pmt s , rna methytransferases 
 ( rnmt s ) have also been implicated in the pathophysiology of 
 human disease , but a clear understanding of 
 their mechanism of action in human cells has been elusive until recently . 
 for example , the modification of mrna with n - methyladenosine ( ma ) by the methyltransferase 
 mettl3 and removal of ma by the fto demethylase are emerging 
 as determinants of mrna stability and translational efficiency . here , we focus on trna , with recent work with 
 the model eukaryote saccharomyces cerevisiae demonstrating that rna - methylation enzymes specific to trna ( trna ) 
 are vital to cell viability after exposure to agents that generate 
 reactive oxygen species ( ros ) and dna damage . specifically , defects 
 in the mc trna methyltransferase 4 ( trm4 , also called 
 ncl1 ) and mcmu trna methyltransferase 9 ( trm9 ) lead to 
 damage - induced growth and cell cycle phenotypes . this highlights an important connection between trna and stress 
 response : modified ribonucleosides in trna . trna molecules are initially 
 transcribed with u , a , c , and g bases , but the nucleobases and ribose 
 sugars in a trna molecule are subject to chemical modification by 
 a large system of enzymes . there are > 100 chemical trna modifications 
 throughout phylogeny , with 2530 in all trna species 
 in a cell , including s. cerevisiae and 
 humans.s . cerevisiae have an average of 11 modifications spread throughout the 70 
 bases in trna , whereas the average mammalian trna contains 13 modifications 
 ( figure 1b , c ) . in general , trna methyltransferases transfer the methyl group from s - adenosyl methionine ( sam ) to the 2-oh of the ribose 
 sugar , to the heterocyclic or exocyclic nitrogen atoms of the nucleobase , 
 or to nucleophilic sites in modification intermediates . there are 
 18 known trm enzymes in s. cerevisiae , with genomic analyses predicting 36 human trms . in many cases , and for both trm4 and trm9 , there are two 
 or more human homologues for each yeast trna methyltransferases , which 
 suggests diversification or specialization of trm activity to new 
 modifications in humans , modification of different trnas , or functions 
 other than trna modification . regardless of enzyme identify or regulation , 
 modified ribonucleosides can promote trna structural stability and 
 folding , translational fidelity , frame - shift prevention , and translation 
 efficiency , with evidence for roles in trna quality control , cellular 
 stress responses , and cell growth . the modifications are located in conserved positions throughout 
 the four loops and termini of the trna molecule , with a large diversity 
 of chemical structures occurring in the anticodon loop and the wobble 
 position of the anticodon in particular . this is not surprising in light of the role of the wobble ribonucleotide 
 in determining anticodon  codon interactions between trna and 
 mrna during translational elongation . the diversity of both 
 the chemical structures and locations of 
 trna modifications suggests a role for modified ribonucleosides in 
 controlling translation as part of a regulatory system . codon 
 interactions , which gives them great regulatory potential as a result 
 of their ability to control the rate of translational elongation . some wobble base modifications are only found on a subset of trnas 
 for specific amino acids that interact with select codons , which supports 
 the idea that regulation by trna modification can be very specific 
 to a particular codon . if trna modifications are part of a such a 
 regulatory system , then they must satisfy at least two criteria : ( 1 ) 
 that they increase or decrease in response to specific changes in 
 cell state and ( 2 ) that changes in the levels of the modifications 
 alter the codon - reading properties of the associated trna and , in 
 some cases , the selection of redundant codons . these behaviors transcend 
 the chemical structure or location of individual ribonucleoside modifications 
 and require a coordinated system with rules beyond the primary genetic 
 code . only recently have analytical and informatic technologies provided 
 a means to define these transcendent properties of trna modifications . in the field of systems biology , the development 
 of convergent 
 technologies to quantify the thousands of individual components of 
 the transcriptome , proteome , and metabolome has led to the discovery 
 of regulatory networks and interactions that would not have been observed 
 in single - molecule or -pathway analyses . the power of liquid chromatography - coupled 
 mass spectrometry ( lc  ms ) for identifying and quantifying modified 
 ribonucleosides has recently been recognized by several groups . to explore the regulatory potential for trna modifications in cellular 
 stress responses ms platform 
 to measure changes in the relative quantities of all trna modifications 
 in an organism ( figure 2 ) . the platform involves artifact - free rna isolation , purification 
 of individual noncoding rna species by hplc , hydrolysis and hplc resolution of individual ribonucleosides , and 
 mass spectrometric identification and quantification of stress - induced 
 changes in all modified ribonucleosides by quadrupole time - of - flight 
 and tandem quadrupole mass spectrometry , respectively . the data set 
 is subjected to bioinformatic and statistical analysis to define patterns 
 of change and then to define pathways linked to altered ribonucleosides . 
 as shown in figures 2 and 3 , our lc ms method is capable of quantifying 23 of 
 the 25 known ribonucleoside modifications in cytoplasmic trna 
 in s. cerevisiae , with limited detection of two modifications ( ar(p ) and ncmum ) in positive ion mode . of critical importance here is the sensitivity 
 of detection , because low - level modifications are those most likely 
 to be found at wobble positions of specific trna species , as opposed 
 to more abundant modifications found in many trna species . ms 
 analysis reveals that modifications occur roughly at high ( d , mc , mg , m2 g , ma , and y ) , medium ( acc , ta , mu , cm , gm , mg , mg , ia , and am ) , and low levels ( ncmum , mcmsu , ncmu , mcmu , um , mi , i , and mc ) , which generally 
 reflects their presence in all or specific trna species as well as 
 their presence at multiple or single positions in trna . these features 
 make the sensitivity , precision , and accuracy of the analytical method 
 particularly important in first - pass studies of stress - induced changes 
 in trna modifications . for example , trm4 catalyzes the formation of 
 mc in over 34 species of trna , yet trna is the only trna 
 with mc at the anticodon wobble position 34 in addition 
 to position 48 between the variable and tc loops . the observation of stress - dependent changes 
 in mc levels may thus depend on the ability to detect 
 small changes in the total quantity of mc in the trna 
 population . similarly , trm9 catalyzes two modifications , mcmsu and mcmu , at wobble positions in five 
 trna species ( trna , trna , trna , trna , and 
 trna ) such that changes could 
 occur in any or all of the trna species . ultimately , individual trna 
 species must be isolated and analyzed for changes in trna - modification 
 levels in an analysis of the regulatory properties of modified ribonucleosides , 
 and this is accomplished by quantitative localization of modifications 
 using combinations of rnase cleavage and oligonucleotide - based affinity 
 purification along with lc ms analysis . platform for trna modification analytics and computational analysis 
 of codon usage , which allows definition of the link between trna modifications 
 and selective translation of motts in the cell stress response . trna 
 modifications are identified and quantified by hplc - coupled mass spectrometry 
 techniques to identify highly up- and downregulated ribonucleosides . critical modifications are then mapped to wobble positions in specific 
 trna species , the anticodon of which specifies a codon that is subjected 
 to genomic analysis . biased use of this codon in gene families specifies 
 potential motts . in parallel , proteomic analysis of stress - altered 
 protein levels reveals codon - biased translation of motts . ultimately , 
 the stress - altered trna reprogramming is linked to selective translation 
 of codon - biased mrnas , with patterns of gene expression unique to 
 each stress . hierarchical clustering 
 of exposure- and genetic - induced changes 
 in rna modification levels . rna modification data from wild - type cells 
 exposed to different agents and mock - treated cells were identified 
 and quantitated by mass spectrometry . log - based fold - change values 
 were determined relative to untreated , wild - type cells , and these 
 data where hierarchically clustered . as noted earlier , trna modifications fulfill 
 a regulatory function 
 in cell response if they increase or decrease following specific stresses . application of the trna - modification analysis platform to yeast exposed 
 to different chemical stresses revealed that this is indeed the case 
 in yeast . cells were exposed to three equitoxic doses 
 of hydrogen peroxide ( h2o2 ) , methyl methanesulfonate 
 ( mms ) , sodium arsenite ( naaso2 ) , and sodium hypochlorite 
 ( naocl ) , and changes in the levels of 23 modified ribonucleosides 
 in total trna were quantified by lc application 
 of multivariate statistical analysis to the fold - change data ( e.g. , 
 hierarchical clustering ) revealed that specific groups of trna modifications 
 were uniquely up- or downregulated for each agent and for individual 
 doses of each agent , as shown in figure 3 . the highly reproducible changes in trna - modification spectra demonstrate 
 that the exposures promote reprogramming of the system of rna modifications , which has been referred to as the ribonucleome . more recently , we quantitatively compared changes in the 
 complete set of trna modifications in yeast exposed to four different 
 oxidizing agents and five different alkylating agents . multivariate 
 statistical analysis revealed class - specific features that distinguished 
 oxidizing agents from alkylating agents , with 14 modifications forming 
 the basis for a data - driven model that predicted the chemical class 
 of toxicant exposure with greater than 80% sensitivity and specificity 
 ( chan et al . , submitted ) . furthermore , signature changes in trna modification 
 spectra distinguished sn1 from sn2 alkylating 
 agents ( chan et al . , these systems - level changes in trna 
 modifications are analogous to the stress - specific patterns of changes 
 in mrna levels in transcriptional profiling or to proteomic and metabolomic 
 signatures of cell state , which suggests a role for rna modifications 
 in regulating gene expression after ros stress and dna damage . recent 
 evidence for codon biases across the genome has provided a basis for 
 linking trna - modification reprogramming to selective translation of 
 codon - biased transcripts as the regulatory mechanism in question . the second criterion for a translational regulatory role for trna 
 modifications involves their ability to recognize information in mrnas 
 that is separate from the simple amino acid - specifying codon . more 
 specifically , understanding how changes in the levels of specific 
 trna modifications can affect gene expression requires insight into 
 codon  anticodon interactions and the patterns of usage of so - called 
 redundant codons in the genome . the general dogma in thinking about 
 codons is that the rate by which they are translated by the ribosome 
 is tightly linked to the concentration of the decoding trna , with 
 reported correlations between genome - wide codon bias , trna copy number , 
 and gene - expression levels in many model organisms . simply put , current models correlate the most highly translated 
 transcripts with possession of the most frequently used codons , which 
 are specified and decoded by corresponding trna species whose genes 
 have the highest number of copies in the genome and the highest number 
 of trna copies in the pool . although 
 this model holds true for the expression of many genes , it suffers 
 from being a static model : it can not account for stress - induced regulation 
 of translation . there are also many exceptions to the model , as revealed 
 by transcripts showing clustering of low - frequency codons , distinct 
 mrna secondary structure , and internal ribosome binding sites . so there is a need to better understand the information content 
 of biased codon usage in genes and to identify a mechanistic link 
 between codon - usage patterns and specific trna modifications . developing 
 rules , though , is a challenge because of the fact that there are 20 
 standard amino acids , 64 codons , 76 unique trna species , 300 trna 
 genes , and > 23 trna modifications in s. cerevisiae as a model organism . the complexity can be simplified by concentrating 
 on wobble base modifications in specific trnas and then analyzing 
 patterns of codon usage in specific transcripts , but an appreciation 
 of the degeneracy of the genetic code is required to move the model 
 to the next level . there are 64 standard codons possible from the 
 four canonical bases found in mrna , with several ( i.e. , synonymous ) 
 codons translated into the same amino acid . both amino acids have the maximum number 
 of six degenerate codons in whole- ( four ) and split- ( two ) codon boxes 
 ( figure 4a ) . in split - codon boxes , the wobble 
 base trna modifications mc and mcmu can influence 
 codon  anticodon affinity by dramatically enhancing interactions 
 with one codon ( i.e. , ttg for leu and aga for arg ) . transcripts in 
 which one codon from the split box is over - represented therefore have 
 great potential for their translational efficiency to be tied to the 
 levels of specific wobble base modifications . we can extend this idea 
 further by proposing that specific transcripts may have over - representation 
 of many specific codons from split boxes for multiple amino acids , 
 which could lead to translational regulation by multiple trna modifications . we term the mrnas from these codon - biased genes as modification tunable 
 transcripts ( motts ) , and we have identified 425 of them in s. cerevisiae using a recently developed codon - analysis 
 algorithm ( figure 4 ) . these 425 genes contain 
 statistically significant deviations in the usage of 29 codons when 
 compared to all transcripts in the s. cerevisiae genome . several recent studies have validated the 
 use of the term mott by establishing a link between the dynamics of 
 stress - induced tuning of trna wobble modifications and the selective 
 translation of codon - biased mrnas that represent critical stress response 
 genes . ( a ) fold - difference 
 in the average codon frequency of the 425 identified yeast motts when 
 compared to genome averages is noted . those codons overused in the 
 motts are colored yellow ( p < 10 ) , whereas those under - represented are colored purple ( p < 10 ) , with the sum frequency that all degenerate 
 codon are used for each amino acid = 1 . ( b ) run of 25 codons used 
 at the c - terminal end of the yef3 transcript is highly 
 enriched ( n = 24 ) for those over - represented in motts . notably , there are two ( aag)4 codon runs and one ( aag)5 codon run represented in this sequence , with 21 codons specific 
 to trm9 ( aag , gaa , and aga ) and trm4 ( uug ) . the evidence for stress - induced reprogramming 
 of trna modifications 
 and for a link between specific trna modifications and biased codon 
 used in motts suggests that the system of trna modifications composes 
 a mechanism for regulating cellular responses to environmental changes 
 at the level of translation ( figure 5 ) . several 
 recent studies confirm this model , with regulation of codon - biased 
 transcripts demonstrated for mott s linked to mc and mcmu modifications . specifically , trm4-catalyzed 
 modification of c to mc at the wobble position of anticodon 
 of trna has been shown to 
 increase in response h2o2 exposure , with this 
 increase driving increased translation of mrnas ( motts ) derived from 
 the 38 genes in yeast in which 90% or more of the leucines are encoded 
 by uug . among these uug - enriched motts is the fact that rpl22a is one of two alternative 
 proteins for rpl22 , with the gene for its paralogue , rpl22b , lacking 
 significant enrichment of uug . h2o2 exposure 
 did not increase the rate of translation of rpl22b , and only loss 
 of the gene for rpl22a rendered the cells sensitive to killing by 
 oxidative stress . these results provide 
 a direct link between stress - induced increases in a specific wobble 
 trna modification and selective translation of codon - biased mrnas 
 for critical stress response genes . rna modifications and biased codon use 
 form a system that controls 
 cellular stress response at the level of translation . emerging evidence 
 supports a model in which stress induces a reprogramming of trna modifications 
 that leads to selective translation of codon - biased mrnas ( i.e. , motts ) 
 representing critical stress response proteins . similar to trm4 , trm9-specific mcmu and mcmsu modifications have been demonstrated to be 
 regulated 
 in response to dna damage and are required to increase the translation 
 of codon - biased motts . in s. cerevisiae , the mrnas for yeast translation elongation factor 3 ( yef3 ) and ribonucleotide reductases 1 ( rnr1 ) and 3 ( rnr3 ) are over - represented with aga , gaa , and aag codons , 
 and the basal translation of these proteins is dramatically decreased 
 in trm9 cells lacking mcmu and 
 mcmsu . proteins corresponding to transcripts with average 
 codon usage ( i.e. , non - motts ) were found to occur at similar levels 
 in wild - type and trm9 cells . notably , mrna levels for rpl22a , yef3 , rnr1 , and rnr3 were 
 identical in wild - type , trm4 ( for rpl22a ) , 
 and trm9 ( for yef3 , rnr1 , and rnr3 ) cells , which further demonstrates 
 that trna - modification - dependent gene expression operates at the level 
 of translational regulation . computational analysis of the motts rpl22a , yef3 , rnr1 , and rnr3 indicates 
 that each of these transcripts is significantly over - represented in 
 specific groups of codons , with protein analysis technologies clearly 
 demonstrating that their protein levels can be regulated by specific 
 trna modifications . the computed codon signature is an indicator of 
 a mott and is limited at this point from the perspective of developing 
 regulatory rules , as it is a trend for the whole gene . notably , it 
 does not provide any location - specific information detailing where 
 these over - represented codons fall in the gene , and there will most 
 likely be transcript regions that are more severely biased in certain 
 codons and thus represent key regulatory motifs . as an example , the 
 3/c - terminal region of yef3 is shown in figure 4b . in a span of 25 codons , it contains 21 codons 
 whose regulation can be linked to trm4 ( ugu ) and trm9 ( aga , gaa , and 
 aag ) , thus representing a local transcript region that is predicted 
 to be highly dependent on specific trna modifications for translation . furthermore , development and testing of computational rules governing 
 the precise mechanism of translational regulation of motts is needed 
 to identify the most significant regulatory regions where modifications 
 regulate the translation of specific transcripts . motts are 
 a new regulatory term , but they have been described before . 
 there is an important precedent in the form of selenocysteine - containing 
 proteins that also illustrates the concept of motts , rna modifications , 
 and stress response proteins . selenocysteine ( sec ) is commonly called 
 the 21st amino acid , and it is found in cellular detoxification and 
 stress response proteins that include members of the glutathione peroxidase 
 ( gpx ) and thioredoxin reductase ( trxr ) families . importantly , these sec - containing proteins can detoxify h2o2 ( gpx1 and gpx3 ) and lipid peroxidation products ( gpx6 ) 
 and contribute to the regulation of ribonucleotide reductase enzymes 
 ( trxr1 and trxr2 ) . sec lacks its own dedicated codon , and it is incorporated 
 into proteins using a novel mechanism termed stop - codon recoding , 
 which requires a number of key signals . the uga codon ( i.e. , the stop 
 codon ) is normally used to signal the end of translation , but in stop - codon 
 recoding , an internal uga codon is used in conjunction with other 
 factors to signal for the insertion of sec . importantly , the wobble 
 base trna modification mcmu , which is catalyzed by human 
 and mouse alkbh8 , is required for efficient stop - codon recoding . as a side note , it has been proposed that the oxidative demethylation 
 activity of alkbh8 could serve as an off switch by reversing wobble 
 methylation modifications , akin to dna and histone demethylation , 
 but no such activity has been demonstrated . transcripts that encode sec - containing proteins can be thought of 
 as extreme motts because they are over - represented with stop codons 
 and need mcmu for efficient translation . transcripts for 
 sec - containing proteins also fit into the theme of motts because they 
 correspond to important stress response proteins , with specific gpx 
 and trxr activities well established as cellular detoxification enzymes . in conclusion , the connection between rna modifications , biased 
 codon use , and translational regulation of stress response protein 
 highlights a complicated set of mechanisms to regulate gene expression . this parallels other methylation - based signals , as understanding regulation 
 of transcription by mc and histone methylation is also 
 complicated , required new tools at their outset , and can have species - specific 
 rules . it is important to note that the dna , protein , and rna modification 
 activities and modifications specified by dnmt s , pmt s , 
 and rnmt s share a common theme of regulating gene expression 
 by enzyme - catalyzed methylation , with all of them reprogrammable by 
 environmental conditions and during some disease pathologies . there 
 are significant challenges for better defining the roles and mechanisms 
 of motts and rna modifications because codon usage and modification 
 patterns change when studying different organisms and there are numerous , 
 varied , and specialized instrumentation required for the study of 
 rna modifications . the study of motts and rna modifications is therefore 
 required in multiple model systems and settings to define further 
 and to develop general and then species - specific rules . we note that 
 one possible path to make the study of rna modifications simpler and 
 more accessible is to follow the example set by researchers studying 
 dna and histone methylation signals : to develop antibodies for each 
 modification .