transcription of genome - encoded information into mrna and translation of mrna into a functional protein are the main layers of gene expression . due to the existential need to adjust gene expression to both intracellular requirements and extracellular stimuli , transcription is a highly controlled process that is extensively regulated at the levels of initiation , elongation , and termination . recent studies in eukaryotes have linked transcription to other levels of mrna regulation , such as alternative splicing ( dujardin et  al . , 2014 ) , polyadenylation ( oktaba et  al . , 2015 ) , localization and translation ( zid and oshea , 2014 ) , and degradation ( dori - bachash et  al . , 2012 ) . although splicing and polyadenylation are thought to be co - transcriptional , and therefore could be directly affected by the rnapii dynamics , the effect on translation and degradation , which have distinct spatial and temporal dynamics , is more complicated to perceive . a recently formulated model explains the imprinting role of transcription by co - transcriptional recruitment of coordinator proteins ( haimovich et  al . , 2013 ) , which accompany the synthesized transcript and are capable of regulating its future fate . translation of mrnas is controlled mainly via initiation ( sonenberg and hinnebusch , 2009 ) and elongation ( richter and coller , 2015 ) . although several recent studies have suggested certain levels of dependency between transcription and translation ( elfakess and dikstein , 2008 , harel - sharvit et  al . , 2010 , tamarkin - ben - harush et  al . , 2014 , zid and oshea , 2014 ) , it is not clear whether these are limited to certain subgroups of mrnas or represent a general link . in general , transcription and translation are still regarded as mutually independent processes , characterized by different timings , cellular locations , functional complexes , and mechanisms of action . n6-methyladenosine ( ma ) is considered to be one of the most abundant rna modifications , detected in thousands of human transcripts ( dominissini et  al . , 2012 , several recent studies have connected ma to the regulation of splicing ( xiao et  al . , 2016 ) , translation ( meyer et  al . , 2015 , wang et  al . , 2015 ) , and degradation ( wang et  al . , 2014 ) . overall , a growing body of evidence suggests that ma plays an important role in multiple levels of mrna regulation . in this study , we tested a hypothesis suggesting a direct flow of information from transcription to translation . combining an unbiased screen for examination of the effect of human promoters on mrna translation and genome - wide analyses , we identified a positive correlation between mrna expression and translation efficiency ( te ) and found that rate of transcription positively affects te . moreover , we observed that transcriptional dynamics are reflected in the relative deposition of ma on mrnas that affects translation . this study establishes a general and robust link between transcription and translation of mrnas and provides a mechanistic insight regarding the way transcription epigenetically imprints mrna molecules . to examine the relationship between transcription and translation , we set out to determine the effect of different human promoters on the translation of a reporter gene ( renilla luciferase [ rluc ] ) . for this purpose , we defined promoters as 0.5- to 2.5-kb - long regions characterized by high h3k4me3 and low h3k4me1 epigenetic marks upstream of transcriptional start sites ( tsss ) , supported by rna sequencing ( rna - seq ) data of mcf7 cells ( loayza - puch et  al . , 2013 ) . to make our screen versatile and diverse , we cloned promoters from genes connected to stress response , autophagy , er metabolism , and metastasis , as well as multiple transcription factors . while cloning the promoter sequences , we avoided regions extending downstream the respective tsss , as these might result in the inclusion of additional 5 utrs into the reporter transcripts , potentially complicating the interpretation of results . because each of the cloned promoters drives the expression of the same reporter gene , we affiliated every promoter with a unique 10 nt barcode ( cloned in the 3 utr of rluc ) in order to follow its expression in a pool of rluc mrnas ( figure  s1a ) . following these guidelines , we cloned 135 human promoters ( table s1 ) in order to create a library named pro - lib , where most of the promoters were associated with two or more different barcodes to provide higher experimental confidence . as expected , cloned promoter regions substantially induced rluc expression ( figure  s1b ) . for normalization of expression , pro - lib included an additional reporter gene , firefly luciferase ( fluc ) , used as an inner control ( figure  s1a ) . last , we employed an flp - frt recombination system and used competent flp - in mcf7 cells to stably integrate a single copy of a pro - lib vector per cell in the identical genetic locus in order to avoid any possible influence of different chromatin neighborhoods on the reporter gene expression . after establishing the library in a stable population , we performed a polysomal profiling experiment by using sucrose gradients , a classical method to separate mrnas according to the amount of bound ribosomes . since we examined only the barcoded rluc mrnas , their relative segregation in the gradient indicates ribosome density and te estimation . we named the whole procedure barcoded polysomal profiling , or bpp ( figure  1a ) . relative enrichment of pro - lib barcodes in the various fractions of the gradient showed that most rluc mrnas are localized in the initial polysomal fractions ( i.e. , 912 ; figure  1b ) . control total rna segregation showed a characteristic pattern of polysomes and edta sensitivity , in line with the known dependence of polysomes on the availability of mg ions ( figures s1c and s1d ) . next , we tested whether bpp can detect changes in te . for this purpose , we employed rluc mrna containing 5-terminal oligopyrimidine tracts ( 5 top ) derived from rpl37a . translation of mrnas possessing 5 top is highly dependent on mtor activity , resulting in a rapid translational arrest after mtor inhibition , as compared to other mrnas ( thoreen et  al . , inhibition of mtorc1 resulted in a global moderate shift of the rluc mrnas to fractions 911 ( e.g. , sptbn1 promoter ) , whereas a 5 top - containing transcript was depleted from the translated fractions of the gradient ( figure  1c ) . interestingly , we also identified several additional rluc transcripts exhibiting similar hypersensitivity toward the inhibition of mtorc1 ( figure  s1e ) . further analysis of the sequences adjacent to their tsss ( figure  s1f ) identified stretches of pyrimidines that could serve as 5 top  signals , providing a probable explanation for their dramatic response . to further test detection capabilities of bpp indeed , we observed that spliced rluc mrna shifts to heavier polysomal fractions and yields more protein ( figure  1d ) , as expected from the known  positive effect of splicing on translation ( nott et  al . , 2004 ) . altogether , our control experiments demonstrate that bpp is capable of examining multiple rluc mrnas in bulk and detecting changes in te of individual transcripts . inspecting the segregation of the barcoded rluc mrnas on sucrose gradients , we identified 12 promoters that caused a shift of the reporter transcript toward higher ribosomal occupancy fractions in at least two independent experiments . intriguingly , four of these promoters contained a tata - box element ( figure  2a ) and four others had ta - rich sequences that could potentially serve as non - canonical tata boxes ( figure  s2a ) , indicating that this promoter element could positively influence translation . to test this possibility , we supplied several tata - less promoters with an artificial tata element ( consisting of the tata sequence followed by the short downstream sequence derived from human actb ) . in all cases , this manipulation resulted in rluc transcripts occupying denser fractions of the gradient ( i.e. , 1113 ; figures 2b and s2b ) , supporting the previous observations . taking the asnsd1 promoter as a model , we observed a positive effect of tata addition on te under various conditions ( figure  s2c ) , thus indicating a robust phenomenon . because artificial introduction of tata may alter the tss , possibly impacting translational capacity ( rojas - duran and gilbert , 2012 ) , we investigated in detail the 5 utrs produced from the tata - containing and tata - less promoter pairs . by northern blotting , we observed that most of the tested promoters resulted in reporter transcripts of a similar length , with a noticeable enhancement of mrna levels in tata - containing promoters ( figure  2c ) . to establish precisely the 5 ends of the transcripts , we performed 5 rapid amplification of cdna ends ( race ) analyses , which showed a very narrow peak 25 nt downstream of the inserted tata element ( figures s3a and s3b ) . to test whether these alterations in the 5 utrs could explain the observed changes in translation , we in  vitro synthesized rluc transcripts bearing the different 5 utrs and examined their relative te ( figure  s3c ) . given that the 5 utr generated after the insertion of tata sequence did not confer higher te , we conclude that the observed positive effect of tata on translation is unlikely to stem from the differences in the 5 utrs of rluc mrnas . so far , we have inferred te from ribosome occupancy measurements reflected by migration within sucrose gradients . next , we tested te changes by measuring separately the levels of the reporter mrna and protein in the promoter pairs . as expected , we observed significantly more mrna produced from tata - containing promoters ( 7- to 9-fold , figure  2d ) , consistent with the levels measured by northern blotting ( figure  2c ) . in contrast , the increase in the level of protein activity was significantly higher ( > 20-fold ) , supporting the connection between the presence of tata in promoter and enhanced protein production . to further test the role of the tata element in translation remarkably , loss of tata reduced the reporter mrna expression by 14-fold and protein activity by 80-fold ( figure  2e ) , suggesting reduced te . indeed , mrna produced by a tata - mutated promoter was enriched in the lighter polysomal fractions ( figure  2f ) , further supporting this indication . we observed similar results upon mutagenesis of tata in other promoters ( figures s2d and s2e ) , suggesting that this effect is not restricted to any particular promoter . next , we tested whether the positive effect of tata on te also applies to the endogenous mammalian gene expression . for this purpose , we chose c - myc , a ubiquitously expressed gene with a single active tata - positive promoter in mcf7 cells ( figure  s2f ) and used the crispr - cas9 system to alter its tata sequence ( figure  2 g ) . this strategy yielded isolated cell clones with disrupted c - myc tata on most of their alleles ( figure  2 g and figure  s2 g ) . examination of c - myc expression in these clones revealed a reduction of 25% in c - myc mrna and 50% in both protein activity and expression ( figures 2h and 2i and s2i and s2j ) . moreover , polysomal profiling of c - myc transcripts showed reduced ribosomal occupancy upon mutation of the tata element ( figures 2j and s2h ) , whereas a control transcript displayed very similar profiles in both clones ( figure  s2k ) , indicating a c - myc - specific effect . thus , we conclude that the presence of the tata element in a promoter enhances the efficiency of mrna translation and note the validity of this observation to multiple promoters and genes . in all the cases we studied , presence of tata in a promoter stimulated both mrna expression levels and te . to examine whether te positively correlates with mrna expression levels , we clustered the pro - lib bpp data to separate the transcripts of our library into two relative groups , one with lower te ( figures s4a and s4b ) and another with higher te ( figures s4c and s4d ) , and compared the expression levels between these two groups . indeed , we found that transcripts with higher te tend to be more abundant ( figure  3a ) . to test whether this coupling is tata dependent , we repeated this analysis while omitting the promoters with artificially added tata elements . notably , this analysis yielded similar albeit less significant results ( figure  3b ) , thus suggesting a positive correlation between the abundance of rluc transcripts and their te , with no dependency on tata . to further test this observation , we employed cells expressing an inducible version of the barcoded reporter gene ( trex - rluc ) , in which the levels of rluc mrna are stimulated 17-fold after induction ( figure  3c ) . importantly , induction of this gene yielded a significantly greater enhancement of protein activity ( 60-fold , figure  3c ) , suggesting a translational boost . indeed , subsequent bpp analysis revealed a strong increase in the ribosome occupancy of the induced reporter mrna ( figure  3d and figure  s5a ) , supporting the notion that increased expression results in higher te . notably , we performed 5 race analysis of both induced and non - induced trex - rluc mrnas and found the scattering of tsss to be very similar ( figure  s3d ) . altogether , we conclude that the observed link between the expression levels of mrnas and their te is not restricted to the tata element and might therefore represent a more general phenomenon . to test whether genome - wide data support a global relationship between mrna level and te , we analyzed pairs of rna - seq and ribo - seq ( ribosomal footprinting sequencing ) datasets from multiple human cell lines . intriguingly , we observed a positive global correlation between mrna expression level and te , which was rather weak but consistent and statistically significant ( figure  3e ) . moreover , this correlation between mrna levels and  te was also apparent in various stress conditions ( figures s5b and s5c ) . these results indicate that there is a positive correlation between expression levels of mrnas and their te in mammalian genomes as well . to examine whether mrna levels could directly regulate te , we transfected different amounts of in  vitro transcribed and purified rluc mrna into mcf7 cells and monitored the activity of the produced rluc protein . we anticipated that if levels of mrna positively stimulated te , transfecting increasing amounts of mrna would result in an exponential increase of protein production . however , we observed a clear linear dependency between the two parameters ( figure  3f ) , suggesting indifference of the protein production rates to mrna abundance . to test this conclusion further , we employed lentiviral - mediated stable integration of a transcriptional unit including a promoter and rluc gene and generated two stable populations with either nearly single or multiple integrations of the same transcriptional unit per cell , resulting in an 9-fold difference in rluc mrna levels ( figure  3 g ) . also here , increase in protein activity was similar to the increase in mrna levels , indicating comparable tes . supporting this conclusion , rluc mrnas derived from both cell populations exhibited similar segregation patterns in sucrose gradients ( figure  3h ) . altogether , these results suggest that while mrna expression levels positively correlate with te in multiple cellular models , mrna abundance does not directly regulate it . next we searched for the causal origin of the observed correlation between mrna levels and te . we considered the rate of transcription to be the most likely candidate since it directly regulates the expression levels of mrnas . to test this possibility , we correlated transcription rates , as estimated by global run - on sequencing ( gro - seq ) ( core et  al . , 2008 ) , and te , as determined by ribo - seq and rna - seq data , in bj and mcf7 cell lines . in both cell types , we observed a significant positive association between the rates of transcription and te of genes that do not possess upstream orfs ( figure  4a and figures s6a and s6b ) . this correlation is notable given that this analysis integrates distinct datasets independently obtained from three genomic techniques in two different cellular systems . to further investigate the relationship between rates of transcription and te , we employed camptothecin ( cpt ) , a chemical compound that inhibits topoisomerase i and , in mild concentrations , slows down the progression of transcribing rna polymerase ii ( rnapii ) ( dujardin et  al . , 2014 ) . indeed , when cpt was applied in parallel to the induction of trex - rluc gene , we observed a reduction of 60% of rluc mrna , but a more prominent reduction of rluc activity of 80% ( figure  4b ) . this difference could be explained by the reduced ribosome occupancy , as reflected by polysomal profiling analyses ( figure  4c and figure  s5d ) . thus , impediment of rnapii progression does not only reduce the transcriptional rate of trex - rluc but also attenuates its te . to further test the role of rnapii dynamics on translation , we assessed te of multiple genes in cpt - treated cells . overall , cpt treatment resulted in te reduction of approximately 700 genes by more than 1.5-fold in two independent experiments ( table s2 ) . given that highly transcribed genes showed higher te ( figure  4a ) , we speculated that cpt treatment would cause a more pronounced repressive effect on the translation of these genes . indeed , intersection with gro - seq data demonstrated that impediment of rnapii caused a significant reduction in te of genes with a relatively high transcription rate ( figure  4d ) . we further examined the polysomal segregation of several mrnas , suggested by the genome - wide experiments , and validated that these mrnas indeed displayed reduced te coupled with reduced expression , as compared to alg8 control mrna ( figure  4e ) . to eliminate the possibility that the transcripts exhibiting translational shifts are truncated due to the cpt treatment , we re - examined these and other mrnas by using primers annealing to 3 utrs only . also in this case , the shift of mrnas was apparent ( figure  s6c ) . taken together , we conclude that transcription rate is an important positive determinant of te . to further examine the mechanism by which transcription affects te , we introduced an internal ribosome entry site ( ires ) sequence into the inducible trex - rluc cassette ( trex - ires - rluc , see schematics in figure  4f ) to bypass the canonical 5 cap - mediated initiation of translation . interestingly , induction of trex - ires - rluc resulted in rluc activity that mirrored the changes in the mrna levels ( i.e. , 12- to 15-fold induction at  both levels , figure  4f ) , which contrasted the previously observed induction of the rluc protein activity ( > 60-fold , figure  3c ) . moreover , bpp analyses revealed similar segregation of both induced and non - induced ires - rluc mrnas ( figure  4 g ) , further suggesting lack of translational induction . furthermore , introduction of ires into constructs containing the artificial tata element ( e.g. , szt2 promoter ) abolished the previously observed enhancement of te ( figure  4h , compare with figure  2d ) and resembled the effect of tata mutagenesis ( figure  s2d ) . thus , we conclude that canonical translation initiation is required for transcription - dependent enhancement of translation . next , we looked for a molecular event that could explain the link  between transcription and translation . since changes in transcription affected translation of multiple genes ( table s2 ) , we reasoned that this molecular event should be robust and widespread . ma is an abundant nuclear dynamic mrna modification identified in thousands of human transcripts ( dominissini et  al . , 2012 , meyer et  al . , 2012 ) , recently implicated in translational regulation ( wang et  al . , 2015 ) . first , we examined whether ma levels could be affected by different rates of transcription . we therefore performed immunoprecipitations of ma - modified rnas ( merip ) from cells with either induced or non - induced trex - rluc and compared the relative recoveries of the different rluc transcripts . strikingly , we observed very significant differences between the two rluc populations ; the non - induced mrna was isolated much more efficiently in all tested mrna regions ( figure  5a ) . importantly , a control mrna ( ahnak ) showed similar recovery in both samples , suggesting a specific enrichment of the non - induced rluc transcripts by merip . we also assessed the methylation levels of rluc mrnas of the two populations expressing different copy numbers of lenti - rluc ( figure  3 g ) and found them to be very similar ( figure  s6d ) , suggesting that the differences observed in merip are unlikely to stem from unequal mrna expression levels . furthermore , we observed differences in the post - merip recovery of rluc mrnas transcribed from either tata - containing or tata - less promoters ( figure  s6e ) , suggesting that promoters with intact tata elements yield mrnas with relatively lower ma content . next , we in  vitro transcribed rluc in  ma - compatible nuclear hela extract ( shimba et  al . , 1995 ) , using conditions that reduce rate of transcription ( e.g. , elevated mgcl2 [ wildeman et  al . , 1984 ] or a template with a mutated tata box ) , and tested the different transcripts for ma content . indeed , both changes resulted in the relatively lower levels of rluc mrna , which were associated with higher deposition of ma ( figure  5b ) . importantly , both the utrs and polya tails of  the different transcripts were similar ( figures s3e and s3f ) . furthermore , treatment with cpt reduced mrna levels in parallel to the relative enhancement of their ma content ( figure  5c ) , suggesting a negative correlation between transcription elongation and ma deposition . additionally , we performed merip analysis of genes that exhibited reduction of both mrna levels and te after cpt treatment ( figure  4e ) . these genes indeed displayed lower levels of mrna coupled with enhanced ma content ( figure  5d ) . to further address the link between the rate of transcription and ma deposition in an additional independent system , we interrogated -amanitin resistant rnapii mutants exhibiting either normal ( n792d ) or slow ( c4/r749h ) rates of elongation ( fong et  al . , 2014 ) . similarly to the previous observations , we found that cells expressing the slow rnapii mutant contained relatively lower levels of mrna , associated with elevated methylation content ( figure  5e ) . thus , various experimental approaches indicate that a suboptimal rate of transcription results in higher ma deposition on mrnas . this conclusion raised the possibility that ma modification of  mrnas occurs co - transcriptionally , and therefore we anticipated a physical interaction between rnapii and the methyltransferase complex ( mtc ) . to test this , we immunoprecipitated rnapii from either normally growing or cpt - treated cells and probed the eluate for mettl3 , a catalytic core of mtc ( wang et  al . , 2016 ) . remarkably , whereas in untreated conditions we observed no visible interaction , upon cpt treatment , mettl3 was efficiently co - precipitated ( figure  5f ) , suggesting co - transcriptional ma modification and enhancement of the interaction between rnapii and mtc upon impediment of rnapii elongation dynamics . we first examined the translation capacity of the in  vitro transcribed rluc mrnas in the conditions of reduced transcription and enhanced ma ( as presented in figure  5b ) . in line with our hypothesis , conditions associated with attenuated transcription resulted in lower efficiency of protein production compared with the normal transcription conditions ( figure  6a ) . next , we examined the global effects of cpt treatment and expression of the slow rnapii mutant on polysomes . figures 6b and 6c show that these two treatments considerably reduced the rna content of the polysomal fractions , suggesting a general reduction of translation and providing further evidence for the positive feedback between transcription and translation . to directly examine the role of ma in translation , we knocked down two genes in the ma pathway ( mettl14 and ythdh1 , figure  s6f ) and examined the effect on the expression of rluc mrna . interestingly , knocking down either factor resulted in a significant boost in the expression of the non - induced rluc , whereas the effect on the induced gene was moderate ( figure  6d ) . importantly , while global polysome segregation analysis indicated only a slight effect of each knockdown on general translation ( figure  s6 g ) , possibly due to compensatory mechanisms , bpp assay verified a considerable increase in the te of the non - induced rluc mrna in these conditions ( figure  6e ) . given that methylation of coding regions ( cdrs ) has been recently suggested to attenuate translation ( choi et  al . , 2016 ) , we re - examined the experiment presented in figure  5a , in order to compare the relative changes in methylation of the different rluc transcript regions . remarkably , the highest gain of methylation upon transcriptional attenuation was observed throughout the cdr of rluc ( up to 11% near the end of the non - induced mrna cds ) , whereas both utrs ( i.e. , 5 and 3 ) displayed the lowest levels ( < 1% , figure  6f ) . this result therefore suggests a plausible explanation for the strong inhibitory effect of transcriptional repression on te that we observed ( figure  3d ) . lastly , we sought to directly examine the effect of ma levels on protein production and produced rluc mrnas in  vitro with different proportions of methylated adenosines . strikingly , increased incorporation of ma led to a progressive attenuation of the te of rluc ( figure  6h ) , suggesting an overall inhibitory effect of ma modification on translation . altogether , these results demonstrate that covalent ma modification of mrnas depends on the dynamics of the transcribing rnapii , and tends to negatively affect te . in this study , we describe a direct and general flow of information from transcription to translation in human cells . we show that genes possessing strong transcriptional activity give rise to mrnas with greater capacity to produce proteins . we propose that this link is mediated , at least in part , by the co - transcriptional n6-methylation of adenosines in mrna  the ma modification . suboptimal transcription results in a relatively higher ma content of transcripts and this , in turn , tends to reduce te ( figure  6h ) . initially , we employed pro - lib , a library consisting of multiple human promoters that drive the transcription of a single reporter gene rluc , and combined it with barcoded polysomal profiling procedure to examine te of the reporter mrnas . first , recombination - mediated single - copy genomic integration resulted in a balanced expression of the reporter constructs . second , all vectors were integrated into the same genetic locus , minimizing the probability of an artificial effect of chromatin neighborhood on transcription . third , establishing a pooled stable reporter population simplified the maintenance and treatments and increased experimental accuracy . lastly , deep sequencing of the barcodes enabled a  quantitative coverage of nearly all reporter transcripts in the polysomal fractions within a single experiment . using pro - lib , we observed that promoters possessing either confirmed or suspected tata - box element ( tata ) yield transcripts characterized by a higher capacity to accommodate ribosomes and produce proteins . this indicated that the presence of this element in a promoter not only boosts transcription , but also enhances mrna translation , suggesting a positive effect of tata - box promoter element on te . indeed , a recent genome - wide bioinformatics analysis has suggested that mammalian genes possessing tata in their promoter tend to be translated more  efficiently ( tamarkin - ben - harush et  al . , 2014 ) , which was attributed to their characteristics ( e.g. , gene length , probability of upstream orf , etc . ) . in this study , we support these observations and conclude , in addition , that a mere presence of intact  tata element in a promoter potentiates mrna translation ( figure 2b ) . the tata element activates transcription ( xiao et  al . , 1995 ) , resulting in elevated levels of mrna . indeed , we have identified a strong correlation between mrna levels and translation in the pro - lib screen ; additionally , we have also shown that this link is not unique to tata , but also holds true for the global mammalian gene expression . we note that the genome - wide correlations between mrna levels and te are rather weak , probably reflecting multiple additional levels of regulation , but statistically significant . interestingly , similar correlations were noticed in both animal cells and yeast ( schwanhusser et  al . , 2011 , weinberg et  al . , 2016 ) , likely indicating an  evolutionarily conserved phenomenon . importantly , while mrna levels could be linked to te via mechanisms that couple mrna stability and translation ( radhakrishnan et  al . , 2016 ) , we found that mrna levels per se do not directly affect translation ( figures 3f3h ) . impediment of transcription , either via cpt treatment or in  the in  vitro assays , demonstrated a role for transcriptional elongation in the determination of te . given that transcription initiation and elongation are related processes ( marbach - bar et  al . , 2013 ) , future studies are necessary in order to precisely  appreciate the role of transcription initiation in the regulation of translation . regarding mrna translation , our observations suggest that it is responsive to transcriptional changes only when the initiation of translation involves 5 cap . when this canonical initiation was bypassed by the introduction of an ires , the translation machinery failed to respond to transcriptional fluctuations ( figures 4f4h ) , indicating that this initial step , which is considered to be rate limiting and highly controlled , might exert additional aspects of translational regulation . here , we suggest that ma , at least partially , mediates the communication between transcription and translation . we present here biochemical evidence for a physical interaction between the mettl3 enzymatic subunit of the mtc and retarded rnapii ( figure  5f ) . these results suggest that ma modification is , at least in part , co - transcriptional and indicate that the affinity between mtc and rnapii depends upon the dynamics of the latter . although it remains unclear how exactly this interaction is induced , we propose that slow progression or frequent pausing of rnapii increases the probability of mtc engagement . the global enrichment of modified nucleotides at the start of mrnas and around stop codons ( meyer et  al . , 2012 ) supports this hypothesis , as these are the locations of probable rnapii pausing ( jonkers and lis , 2015 ) . since a considerable proportion of genes experience slow or paused transcription ( core et  al . , 2008 ) , it would be important to examine , in future studies , whether these genes are preferentially methylated . we note that , together with ma , additional factors might bridge transcription and translation , such as precise sequence of 5 utr ( rojas - duran and gilbert , 2012 , tamarkin - ben - harush et  al . , 2017 ) and recruitment of mrna coordinators ( harel - sharvit et  al . , 2010 ) . interestingly , several recent studies have indeed linked ma to translation , but report mainly a stimulatory role of this modification ( meyer et  al . , 2015 , wang et  al . , 2015 , zhou et  al . , 2015 ) . we anticipate that the precise effect of ma on translation depends on the location of the modified nucleotide within the transcript . remarkably , most of the mentioned studies observed enhanced methylation within utrs of transcripts . in contrast , methylation within cdrs reduced translation ( qi et  al . , 2016 ) , probably due to the attenuated elongation ( hoernes et  al . , 2016 , choi et  al . , 2016 given that most of the methylated residues are located within cdrs , we would anticipate an overall negative effect of ma methylation on translation , which we indeed observed upon random incorporation of modified adenosines ( figure  6 g ) . our study suggests that ma modification of mrnas is co - transcriptional and is used to epigenetically imprint mrnas and control their future activity and fate . this way the dynamics of the transcription process could be recorded in the methylation profile of a given mrna , which , in turn , could further impact multiple steps of mrna biology . reagent or resourcesourceidentifierantibodiesanti - c - myc ( 1:1000)cell signalingcat#d84c12 ; rrid : ab_1903938anti - gapdh ( 1:2500)santa cruzcat#sc-69778 ; rrid : ab_1124759anti - mettl3 ( 1:1000)protein techcat#15073 - 1-ap ; rrid : ab_2142033anti - rnapii ( ctd4h8 ; 1:1000)milliporecat#05 - 623 ; rrid : ab_309852polyclonal goat anti - mouse immunoglobulins / hrpdakocat#p0447 ; rrid : ab_2617137polyclonal goat anti - rabbit immunoglobulins / hrpdakocat#p0448 ; rrid : ab_2617138bacterial and virus strainsdh5biolinecat#bio-85026chemicals , peptides , and recombinant proteinsrna fragmentation reagentambioncat#am8740epimark n6-methyladenosine enrichment kitnebcat#e1610scritical commercial assaysepiquik m6a rna methylation quantification kitepigenetekcat#p-9005 - 963 race system for rapid amplification of cdna endsinvitrogencat#183730195 race system for rapid amplification of cdna ends , v2.0invitrogencat#18374058usb poly(a ) tail - length assay kitaffymetrixcat#76455 1ktdeposited datarna - seq and ribo - seq databj cellsgeo : gse45833pc9 , h1933 cellsgeo : gse96716mcf7 ( nutlin)geo : gse96714mcf7 ( cpt)geo : gse96643gro - seqbj cellsgeo : gse96717mcf7 cellsgeo : gse96718experimental models : cell linesmcf7atccatcc htb-22mcf7/frtthis studyn / amcf7/frt / trthis studyn / ahek293 ( n792d)david bentleyfong et  al . , 2014hek293 ( c4/r749h)david bentleyfong et  al . , 2014oligonucleotidesdna and rna oligonucleotidestable s4n / arecombinant dnalist of cloned promoters and associated barcodestable s1n / alist of used plasmids and detailed restriction sitestable s3n / asoftware and algorithmsbowtielangmead et  al . , 2010http://acgt.cs.tau.ac.il / expander/ further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact , reuven agami ( r.agami@nki.nl ) . human breast adenocarcinoma mcf7 cells ( female ) were purchased from atcc and authenticated by detecting the deficiency of caspase-3 using western blotting . these cells were grown in dmem media ( gibco ) supplemented with 10% bovine serum and penicillin / streptomycin ( gibco ) in 5%co2-buffered incubators at 37c . cells were split twice per week and kept in culture for up to 8  weeks . to create cells compatible with flp recombination system ( thermofisher scientific ) , mcf7 cells were transfected with the pfrt / laczeo and grown in the presence of zeocin ( 400g / ml ) until stable colonies appeared . these cells were named mcf7/frt and used for integration of pro - lib . to integrate pro - lib , these cells were transfected with pro - lib vectors along with pog44 vector ( thermofisher scientific ) in quantities of 1.6g+3g respectively , per 35-mm well . after 48h , the cells were collected , re - plated onto 10-cm dishes , and selected for three weeks in the presence of hygromycin ( 0.1  mg / ml f.c . ) . after appearance of the stable colonies , the cells were collected , frozen , and used for further experiments . to create cells compatible with t - rex inducible system ( invitrogen ) , mcf7/frt cells were transfected with pcdna6/tr vector encoding for the tetracycline - sensitive repressor and separate clones were selected for their resistance to blasticidin s ( 5mg / ml ) . the induction efficiency was examined in separate clones by transient transfection with pcdna4/to - rluc construct , incubation with doxycycline ( sigma , 1  g / ml final conc . ) for 17  hr and measurement of renilla protein activity . one suitable clone was chosen for further experiments and the cells were named mcf7/frt / tr . these cells were stably transfected with pcdna5/frt / to plasmid encoding for barcoded renilla orf ( trex - rluc ) in the presence of pog44 vector ( thermofisher scientific ) and grown for three weeks in the presence of hygromycin ( 0.1  mg / ml f.c . ) . individual colonies were isolated and tested by treatment with doxycycline ( sigma , 1  g / ml final conc . ) . they were maintained in dmem supplemented with 10% fbs , 1% penicillin / streptomycin , 200  g / ml hygromycin , and 6.5  g / ml blasticidin . to stimulate the expression of rpb1 mutants , cells were induced with 2.0  g / ml doxycycline for 16  hr and treated with 2.5  g / ml -amanitin for additional 42  hr . to induce autophagy , mcf7 cells were incubated in ebss ( gibco ) medium for 3  hr . to inhibit mtorc1 , torin1 was added to the medium for 2  hr ( 250  nm ; tocris bioscience , bristol , uk ) . cells were irradiated at 10gy dose and then incubated for additional 18  hr at normal conditions . to arrest cells in mitosis , taxol ( sigma - aldrich , 1  m f.c . for 17  hr ) was added to the growth medium . fugene6 ( roche ) was used for transfection of dna plasmids , dharmafect1 ( dharmacone ) for transfection of sirnas and in - vitro transcribed mrnas , according to the manufacturers instructions . to impede the dynamics of transcribing rna polii , psi - check2 vector with cloned frt sequence and hygromycin - resistance gene served as the basic vector for the construction of pro - lib . to clone human promoters upstream of the renilla orf in pro - lib , putative promoter regions were cloned between bglii and nhei sites of the basic pro - lib vector ( figure  s1a ) after addition of random barcodes and removal of the sv40 promoter . all barcodes were sequenced as well as the proximal parts of the cloned promoter regions to confirm their identity . all other cloning procedures were performed using standard procedures ; dna was extracted from gel using agarose gel dna extraction kit ( roche ) ; all constructs were sequenced . the list of the plasmids used in the study together with their details is available in table s3 . cultured cells were lysed with passive lysis buffer for 15  min at room temperature . renilla ( rluc ) and firefly ( fluc ) enzymatic activities were assayed with the substrates supplied with the kit using centro xs lb960 machine ( berthold technologies ) . myc activity was measured using myc - reporter constructs ( pgl3-m4 and pgl3-m4-mut , ( nagel et  al . mcf7 cells ( 1x10 cells ) were plated in 12-well plates and transfected with 200ng of the reporter constructs using fugene-6 ( promega ) . sucrose gradients for separation of polysomes were usually prepared by gentle sequential addition of 2.2ml of the different sucrose solutions ( e.i . , 47 , 37 , 27 , 17 and 7% in tris - hcl ph  = 7.5 ( f.c . 100 mm ) , supplemented with 2 mm dtt , ribosafe rnase inhibitor ( bioline , 1  l / ml ) and chx ( 100  g / ml ) into a 12  ml tube ( beckman , 9/16   3 1/2 in . ) and left overnight at 4c to achieve continuous gradient prior to the centrifugation . cells growing in subconfluent conditions ( typically 80%85% confluency ) were washed once with ice - cold pbs , collected into 15  ml tubes and incubated with pbs / cycloheximide ( chx , 100  g / ml ) for 5  min on ice . after sedimentation ( 400xg for 3min ) , the cells were lysed in lysis buffer ( 20  mm tris - hcl ( ph 7.5 ) , 10  mm mgcl2 , 100  mm kcl , 1% np40 ) supplemented with edta - free complete protease inhibitors ( roche ) , 2 mm dtt , ribosafe rnase inhibitor ( bioline , 1  l / ml ) and chx ( 100  g / ml ) for 10min on ice . to resolve cell aggregations , the lysates were centrifuged 1300xg for 10  min at 4c and the supernatants were transferred into new tubes . from the cleared lysates , 500  l were loaded on top of each gradient , mounted on sw41ti rotor and centrifuged at 36000rpm for 2  hr at 4c . following the centrifugation , each gradient was split into 15 equal fractions of 760  l , which were subjected to rna isolation using trisure ( bioline ) according to the manufacturer s instructions . rna was precipitated from 750  l of the collected material , using 2  l of glycoblue ( ambion ) and 750  l of isopropanol , washed once with 70% of ethanol and reconstituted in 25  l of water . typically , 7  l of total rna were taken as a template for reverse transcription reactions using tetro cdna synthesis kit ( bioline ) and oligo dt primers . barcodes of the pro - lib transcripts detected by qrt - pcr using specific primers . to construct libraries ready  for deep sequencing , barcodes were pcr amplificed using indexed primers bearing p5 , p7 and illuseq sequences , while each collected fraction was associated with a unique index . the pcr products were cleaned using qiaquick pcr purification kit ( qiagen ) , size selected on e - gel sizeselect 2% gels ( invitrogen ) and sequenced using illumina hi - seq 2000 or 2500 . rna was isolated from cultured cells using tri - sure reagent ( bioline ) according to the manual , using glycoblue ( ambion ) as a carrier . rna was typically reconstituted in 30  l of sterile nuclease - free water ( gibco ) and stored at 20c . to analyze rna on agarose gels , a portion of isolated rna was mixed with equal volume of 2xrna - loading dye ( thermo fisher scientific ) , supplemented with 20ng / ml ethidium bromide , heated at 70c for 5min and loaded on 1.5% agarose gel . reverse transcription was done using tetro cdna synthesis kit ( bioline ) according to the manual . typically , 7  l of total rna was taken for a single rt reaction . real - time pcr experiments were performed using sensifast cybr ( bioline ) reagent and lightcycler 480ii ( roche ) using standard conditions . to synthesize renilla mrna using nuclear extract , we used helascribe nuclear extract in  vitro transcription system ( promega ) according to the manufacturer s protocol . briefly , dna templates including promoter , orf , utrs and polya site were pcr - amplified from the appropriate pgem - t vectors , isolated on agarose gels and cleaned . for a single reaction , 200ng of a clean template were transcribed with 7  l of nuclear extract , in a buffer containing 1.5 mm mgcl2 in a siliconized rnase - free 1.5  ml eppendorf tube ( ambion ) at 30c for 1.5  hr . the reaction was stopped by addition of trisure compound ( bioline ) and rna was isolated , analyzed on agarose gels , quantified using nanodrop , compared using imagej and stored at 20c . transfection was done in 96-wells plates using dharmafect1 agent ( thermo scientific ) , according to the manufacturer s instructions . reconstituted synthetic rna was furthermore cleaned using rneasy kit ( qiagen ) . to synthesize renilla mrna using t7 rna polymerase , we used hiscribe t7 high yield rna synthesis kit ( neb ) according to the manufacturer s instruction . briefly , pcr - produced dna fragments encoding for renilla orf were transcribed in presence of ribo m7 g cap analog ( promega , 8 mm f.c . ) at 37c for 2  hr followed by dnase treatment for 15  min and isolation of rna . to produce transcripts with methylated content , n6-methyladenosine-5-triphosphate ( trilink , 100 mm stock conc . ) was added together with atp ( supplied in the kit ) at the indicated proportions . after recovery , the rnas were polyadenylated using e.coli poly(a ) polymerase ( neb ) and purified . after recovery and analysis on gels , rnas were transfected into living cells as described above or subjected to in - vitro translation . to translate mrnas encoding for rluc protein , rabbit reticulocytes system , all reactions were done in volume of 25  l at 30c for 10  min . to quantify the produced rluc protein , aliquots of 5  l were taken for luciferase measurements using dual - luciferase reporter assay kit ( promega ) . total rna was extracted from growing mcf7 cells as described and enriched for mrna using oligotex kit ( qiagen ) . equal volumes of reconstituted rna were separated on 1.2% agarose gel supplemented with 0.66% ( vol / vol ) formaldehyde and transferred onto amersham hybond - n membrane ( ge healthcare ) . rna probes targeting renilla or firefly mrnas were produced by in vitro transcription ( t7 high yield kit , neb ) of pcr fragments encoding for sequences complementary to these transcripts ( see table s4 for used oligos ) using p()-utp . following cleaning of the probes using microspin g-50 columns ( ge healthcare ) , hybridization was performed in ultrahyb hybridization buffer ( ambion ) rotating at 68c for 4  hr . the membrane was washed twice with 2xssc buffer supplemented with 0.1%sds and twice with 0.2xssc buffer supplemented with 0.1%sds . the signal was detected by fujifilm fla-3000 phosphorimager and analyzed using imagej . to estimate the relative ma content of rna , epiquik m6a rna methylation quantification kit ( epigentek ) , equal volumes of rna solution ( 4 - 8  l ) were processed according to the manufacturer s instructions , aside with the negative , positive , and standards controls . generally , 5race experiments followed the procedure established for 5 race system for rapid amplification of cdna ends , v2.0 ( invitrogen ) . briefly , total rna was extracted with trisure ( bioline ) and subsequently poly - a selected with oligotex direct mrna mini kit ( qiagen ) . for each 5 race assay , 500ng of poly - a selected mrna were used for cdna synthesis with the following primer : cccttctccttgaatg . synthesized cdna were purified with qiaquick pcr purification kit ( qiagen ) . then the cdna were dc - tailed with tdt for 10min at 37c and a first round pcr was performed with a gene specific primer ( gsp2 ) , agtttccgcatgatcttgcttg , and abridged anchor primer ( aap ) . a nested pcr was performed with a nested gene specific primer ( gsp1 ) , gttgatgaaggagtccagcacgtt , and abridged universal amplification primer ( auap ) . for determination of the sequences of  5 race pcr products , the nested pcr products were ligated into pgem - t vector ( promega ) and transfected into competent bacteria . unless otherwise indicated , at least 20 different bacterial colonies from each ligation were subsequently analyzed using sanger sequencing . the protocol was adapted from 3race system for rapid amplification of cdna ends ( invitrogen ) . briefly , total rna was poly - a selected and reverse transcribed with oligo(dt)-containing adaptor primer ( ap ) ggccacgcgtcgactagtacttttttttttttttttt . synthesized cdna was treated with rnase h to remove residual rna templates and pcr - amplified with rluc specific primer ctgagagtgtcgtggacgtga and abridged anchor primer ( aap ) . pcr products from 5 and 3 race preparations were pcr - amplified with illumina sequencing compatible primers ( p5 primer , 5race - p7 primer , and 3race - p7 primer ) . pcr products were then purified with cleanpcr magnetic beads ( cleanna ) , and pooled with equal molar , ran on illumina miseq platform . determination of poly(a ) tail - length was performed with usb poly(a ) tail - length assay kit ( affymetrix ) following the manufactor s protocol . in short , in - vitro transcribed rna was g / i tailed , and reverse - transcribed with specific adaptor primer ( sequence not provided ) . the poly g / i tailed cdna was then amplified with a rluc specific primer ( gcgtgctgaagaacgagcagtaa ) and a provided universal pcr reverse primer . finally , pcr products were resolved on a 2.5% agarose gel with ethidium bromide . to generate viruses with the renilla - expressing plasmid , 3.5  g of pvsv - g , 5  g of pmdl - rre and 2.5  g of prsv - rev was transfected with 10  g of the expression plasmid plenti - puro - rluc . lentivirus - containing supernatant was harvested 48h after the transfection , filtered through a 0.45  m membrane ( milipore steriflip hv / pvdf ) and kept at 80c . subsequently , to infect mcf7 cells , 1x10 cells were plated in 10-cm dish one day prior infection . for low moi infection , cells were infected with 20  l of the virus - containing supernatant ; for high moi infection , cells were infected with 900  l ( 45-fold ) of the supernatant . afterward , low moi - infected mcf7 cells were selected with 1  mg / ml puromycin and high moi - infected mcf7 cells were selected with higher concentrations of puromycin ( up to 25ug / ml ) for 1  week . for the mutagenesis of c - myc tata box sequence , lentivirus containing sgrnas targeting the tata box sequence were generated and subsequently used for infecting mcf7 cells as described above . after puromycin ( 2  g / ml ) selection , cells were plated in a 96-well plate following serial dilutions in order to achieve single cell clones of the cas9-generated mutants . several single cell clones were collected , expanded , and sequenced . to sequence the indel mutations within the c - myc locus , genomic dna was extracted from the grown colonies using dneasy blood and tissue kit ( qiagen ) , pcr amplified using c - myc promoter - specific primers , sub - cloned into pgem - t vector ( promega ) and sequenced . subsequently , cell pellets were lysed , by adding an appropriate amount of ice - cold lysis buffer ( 20 mm tris - hcl at ph 7.5 , 150 mm nacl , 1.8 mm mgcl2 and 0.5% np40 ) supplemented with complete protease inhibitor cocktail ( roche ) and 1 mm dtt . cell lysates were incubated on ice for 20min with occasional vortexing followed by centrifugation at 15000xg for 15min at 4c . proteins were separated on sds - page gels and transferred onto nitrocellulose membrane ( bio - rad , 0.2  m ) , which was blocked in 5% blotting - grade blocker and probed with the indicated antibodies . , blots were reacted with the supersigna west dura extended duration substrate ( thermofisher ) . images were captured with chemidoc xrs+ ( biorad ) and analyzed with image lab software ; quantifications were done using imagej . immunoprecipitation of m6a was adapted from the protocol of epimark n6-methyladenosine enrichment kit ( new england biolabs ) . total rna was isolated as mentioned above and enriched for mrna using oligotex mrna kit ( qiagen ) following manufacturer s instruction . the isolated mrna was fragmented with rna fragmentation reagent ( ambion ) for 5min , 94c and purified through ethanol precipitation . to bind antibody to the beads , protein g beads ( invitrogen ) were pre - incubated with 1  l of anti - maantibody ( neb , cat . e1610s ) in ipp buffer ( 150  mm nacl , 10  mm tris - hcl , ph 7.5 , 0.1% np-40 ) at 4c . subsequently , the beads were washed twice in ipp buffer and incubated with rna for 3h with head - to - tail rotation at 4c ( 10% of the material were kept as input control ) . afterward , beads were washed twice in ipp buffer , twice in low salt buffer ( 50  mm nacl , 10  mm tris - hcl , ph  7.5 , 0.1% np-40 ) , and twice in high salt buffer ( 500  mm nacl , 10  mm tris - hcl , ph 7.5 , 0.1% np-40 ) . for quantification of precipitation , both input samples and ip eluates were examined by qrt - pcr as described above . mcf7 cells growing on 15-cm dishes were treated with cpt ( sigma , 6  m f.c . ) or dmso ( as a control ) for 5  hr . after collection on ice , the cells were incubated in 8ml of pbs supplemented with 1% formaldehyde for 10  min at room temperature in constant rotation . after quenching with glycine ( 0.125 m f.c . ) for 5  min , the cells were spun , washed once with pbs , flash - frozen in liquid nitrogen and kept at 80c . after thawing in 1ml of lysis buffer ( 50  mm hepes - naoh - ph7.5 , 150  mm nacl , 5  mm mgcl2 , 0.1% np40 ) supplemented with edta - free complete protease inhibitors and phosstop ( roche ) , the lysate was divided into three equal 2  ml eppendorf tubes and sonicated at 4c using bioraptor ( diagenode ) for 10 cycles ( each cycle including 30sec of high - power sonication followed by 30sec pause ) . the lysates were centrifuged for 10  min . at 1800xg at 4c to remove unbroken cells . equal volumes of supernatants were added to 20  l of dynabeads - protein g suspension ( life technologies ) pre - coupled with 5  g of anti - rna polymerase ii antibodies ( millipore , clone ctd4h8 ) or normal mouse igg ( santa cruz ) in pbs and 1% bsa for 2  hr at room temperature . the ip reaction was carried out for 2  hr at 4c with constant rotating . after that , the beads were washed once with the regular lysis buffer and three times with stringent lysis buffer ( as detailed above , but with 300  mm nacl ) . the last washing was done with pbs , and the beads were transferred into new 1.5  ml tubes and the bound proteins were eluted and reverse - crosslinked by incubation with 50  l of 1x laemmli sample buffer at 100c for 10min . after collection , the eluates were resolved on 8% sds - page and probed with the indicated antibodies . ribosome profiling ( rp ) was performed as previously described ( loayza - puch et  al . , 2016 ) . briefly , chx ( 100  g / ml ) was added to the medium of growing cells and incubated for 5min at 37c . approximately 30x10 cells were collected in ice - cold pbs and lysed in lysis buffer ( 20  mm tris - hcl ph 7.8 , 10  mm mgcl2 , 100  mm kcl , 1% triton x-100 , 2  mm dtt , 100  g / ml chx , complete protease inhibitors ( roche ) . lysates were passed through a 26 g needle ( bd bioscience ) for further homogenization , and centrifuged at 4c 1300xg for 10  min . i ( ambion ) for 45  min at room temperature in gentle constant rotation . lysates were loaded onto a linear sucrose gradient ( 7%47% , as detailed above ) , and fractionated by ultracentrifugation , using a sw-41ti rotor at 36000  rpm ( 221632.5 g ) during 2  hr . the sucrose gradient was divided into 14 fractions of 830  l . the monosome - enriched sucrose fractions ( 7 to 10 ) were collected and treated with proteinase k ( pcr grade , roche ) , in presence of 1% sds . the so - released ribosome protected fragments ( rpfs ) were purified using trisure reagent ( bioline ) , following the manufacturer s instructions . rpfs between 30 and 33 nucleotides in length were size - selected in 10% acrylamide gel and isolated . the 3  ends of the rpfs were dephosphorylated by treatment with t4 polynucleotide kinase ( new england biolabs ) for 6  hr at 37c in mes - naoh buffer ( 100 mm mes - naoh , ph 5.5 , 10 mm mgcl2 , 10 mm -mercaptoethanol , 300 mm nacl ) . 3 adaptor ( ra3 ) was ligated using t4 rna ligase 1 ( new england biolabs ) for 3.5  hr at 37c , in absence of atp . ligation products were size selected in 10% acrylamide gel , and the 5 ends were phosphorylated by treatment with t4 polynucleotide kinase for 30  min at 37c , in presence of 1 mm atp . 5 adaptor ( ra5 ) was ligated using t4 rna ligase 1 ( new england biolabs ) during 2.5h at 37c , and the ligation products were selected in 10% acrylamide gel . ribosomal rna depletion was performed by biotin - streptavidin affinity purification using biotinylated ribosomal rna probes and streptavidin dynabeads . retro - transcription of the ligation products into cdna was performed using super script iii reverse - transcriptase ( invitrogen ) following the manufacturer s instructions , and the primer rtp . pcr amplification was performed using the forward primer rp1 , and the reverse primer rpi that contained a hexanucleotide index used to multiplex different samples during next generation sequencing ( ngs ) . pcr products were size selected by e - gel electrophoresis ( invitrogen ) , and submitted to ngs using hiseq 2500 system ( illumina ) . the sequence of 3 and 5 adapters , rtp , rp1 and different rpi primers is available in table s5 . total rna was extracted using trizol reagent ( 15596 - 018 , ambion life technologies ) according to the manufactures protocol . briefly , 0.2x volumes of chloroform ( chloroform stab./amylene , biosolve ) was added to the trizol homogenate and the tube(s ) ( falcon , 15ml ) were shaken vigorously . the tube(s ) were incubated for 2 - 3  min at room temperature and centrifuged ( hettich , rotanta 46 rs ) for 1  hr at 4c . approximately 70% of the upper aqueous phase was transferred to a clean 15  ml tube and 0.5x volume of isopropanol ( 33539 , sigma - aldrich , ) was added . the tube(s ) were incubated overnight at 20c and centrifuged for 30  min at 4c . the supernatant was removed and the pellet was washed twice with 80% ethanol ( 32221 - 2.5l , sigma - aldrich ) . the total rna pellet was air - dried for 8  min and dissolved in an appropriate volume of nuclease free water ( ambion ) and quantified using nanodrop uv - vis spectrophotometer . the total rna was further purified using the minelute cleanup kit ( 74204 , qiagen ) according to the manufactures instructions . quality and quantity of the total rna was assessed by the 2100 bioanalyzer using a nano chip ( agilent , santa clara , ca ) . strand - specific libraries were generated using the truseq stranded mrna sample preparation kit ( illumina , rs-122 - 2101/2 ) according to the manufacturer s instructions ( illumina , part # 15031047 rev . e ) . briefly , polyadenylated rna from 1000ng intact total rna was purified using oligo - dt beads . following purification the rna was fragmented , random primed and reverse transcribed using superscript ii reverse transcriptase ( invitrogen ) with the addition of actinomycin d. second strand synthesis was performed using polymerase i and rnaseh with replacement of dttp for dutp . the generated cdna fragments were 3 end adenylated and ligated to illumina paired - end sequencing adapters and subsequently amplified by 12 cycles of pcr . the libraries were analyzed on a 2100 bioanalyzer using a 7500 chip ( agilent , santa clara , ca ) , diluted and pooled equimolar into a 10 nm multiplex sequencing pool . the libraries were sequenced with 65 base single reads on a hiseq2500 using v4 chemistry ( illumina ) . while mentioned , n represents the number of biological repeats of the same experiment . p  values presented in figures 3a , e ; 4a , d ; s5b , c ; s6a , b were calculated using wilcoxon s test . p values mentioned in other figures were calculated using t test ( one - tail distribution , homoscedastic variance ) . in all figures , p  < 0.05 , p  < 0.01 , p  < 0.005 , p  < 0.001 . polysomal fractions and promoter vectors were identified by their unique indexes and barcodes , respectively . expression count matrix was derived by counting the frequency of each barcode in each fraction . to avoid fluctuations due to low coverage , only vectors covered by at least 10,000 counts ( summation over all fractions ) were included in the analyses . for heatmap display , vector counts ( rows ) were standardized to mean  = 0 and sd  = 1 . heatmaps were generated using the expander package ( ulitsky et  al . , 2010 ) . order of promoter vectors in the heatmap was determined by hierarchical clustering of the rows . sequenced reads were aligned to a reference set of human curated protein - coding transcripts ( plus the five human rrna transcripts ) using bowtie ( langmead et  al . , 2009 ) . this reference set of transcripts was based on ensembl s gene annotations ( release 69 ) . for genes with multiple isoforms , the one with longest coding dna sequence ( cds ) region and , in case not unique , the one with longest utrs among the ones with the longest cds , was selected to represent the gene . rna expression levels and ribosome occupancy were estimated by calculating reads per kilobase of mrna per million reads ( rpkm ) per transcript , taking into account either all reads that map to the transcript ( for estimation of rna levels using rna - seq data ) or only those mapping to its cds ( for estimation of ribosome occupancy ) . in estimation of ribosome occupancy in cds , 5 ends of reads were offset 12 nucleotides to the 3 direction to match the p - site location of ribosome ( ingolia et  al . , 2009 ) . translation efficiency ( te ) was estimated by the ( log2 ) ratio between ribosome occupancy and mrna level . only genes with at least 20 reads in rna - seq and ribo - seq samples were included in analyses . sequenced reads were aligned to the human genome ( hg19 ) using bowtie2 , and number of reads mapping to annotated genes ( ensembl v69 ) were counted by htseq ( anders et  al . , 2015 ) . the deep sequencing datasets generated in this study have been deposited in the geo database under accession number geo : gse96643 . planned and designed the experiments , analyzed the results , and wrote the manuscript ; b.s .
summarytranscription and translation are two main pillars of gene expression . due to the different timings , spots of action , and mechanisms of regulation , 
 these processes are mainly regarded as distinct and generally uncoupled , despite serving a common purpose . here , we sought for a possible connection between transcription and translation . employing an unbiased screen of multiple human promoters , we identified a positive effect of tata box on translation and a general coupling between mrna expression and translational efficiency . using a crispr - cas9-mediated approach , genome - wide analyses , and in  vitro experiments , 
 we show that the rate of transcription regulates the efficiency of translation . 
 furthermore , we demonstrate that m6a modification of mrnas is co - transcriptional and depends upon the dynamics of the transcribing rnapii . 
 suboptimal transcription rates lead to elevated m6a content , which may result in reduced translation . 
 this study uncovers a general and widespread link between transcription and translation that is governed by epigenetic modification of mrnas .