Plasmodiumfalciparum has been a tremendous resource for the understanding of themolecular physiology of this parasite. However, the translational dynamics that linksteady-state mRNA with protein levels are not well understood. In this study, webridge this disconnect by measuring genome-wide translation using ribosome profiling,through five stages of the P. falciparum blood phase developmentalcycle. Our findings show that transcription and translation are tightly coupled, withovert translational control occurring for less than 10% of the transcriptome.Translationally regulated genes are predominantly associated with merozoite egressfunctions. We systematically define mRNA 5′ leader sequences, and 3′UTRs, as well as antisense transcripts, along with ribosome occupancy for each, andestablish that accumulation of ribosomes on 5′ leaders is a common transcriptfeature. This work represents the highest resolution and broadest portrait of geneexpression and translation to date for this medically important parasite.The characterization of the transcriptome and proteome of DOI:http://dx.doi.org/10.7554/eLife.04106.001 The genome of an organism includes all of the genes or information necessary tobuild, maintain, and replicate that organism. However, cells with the samegenome—such as a skin cell and a liver cell from the same person—canlook and behave very differently depending on which of the genes in their genomesthey express, and to what extent.For a gene to be expressed, its DNA is ‘transcribed’ to make an RNAmolecule, which is then ‘translated’ to make a protein. Efforts tomeasure the transcription and translation processes in diseased cells, or in themicroorganisms that cause infections, may lead to new treatments and preventativemedicines. Such work is currently ongoing in the global effort to treat and preventmalaria.Plasmodium. Mosquitoes carry the parasites in their salivaryglands, and when a mosquito bites a human, these parasites are injected into thebloodstream with the mosquito's saliva. Plasmodium parasites thentravel to and infect the liver, before bursting out of this tissue into thebloodstream. Here, the parasites infect red blood cells and undergo rounds ofreplication during which the symptoms of the disease are manifested. It is alsoduring this bloodstream phase that parasites can develop into forms capable ofinfecting another mosquito and continuing the transmission cycle.Malaria is both preventable and curable, yet over 600,000 people are estimated to diefrom this disease each year. The disease is caused by a single-celled parasite calledPlasmodium falciparumparasite—which causes the most serious cases of malaria in humans—havebeen cataloged to better understand the biology of this parasite. However, theprocesses that control how, and when, an RNA transcript is translated into a proteinare not well understood.The genes, RNA molecules, and proteins of the Plasmodium development within the blood. Thetranscription and translation of genes in this parasite were found to be tightlylinked processes; the expression of only a few genes was controlled more by thetranslation process than by transcription. These translationally regulated genes werefound mainly to be those that encode proteins involved in the parasite's exit fromthe red blood cells and spread throughout the bloodstream.Now Caro et al. have uncovered which RNA molecules are being translated, and by howmuch, during Plasmodium and preventing its implementation could prove aneffective strategy to curb the spread of malaria.Caro et al. discovered that genetic regulation of the malaria parasite resembles apreset genetic program, rather than a system that responds to changes and externalsignals. As such, these findings suggest that targeting such a genetic program withinDOI:http://dx.doi.org/10.7554/eLife.04106.002 P.falciparum is characterized by a continuous cascade wherein the expressionof the majority of genes is maximally induced once per cycle and their timing correlateswell with the timing for the respective protein's biological function  binds within the coding region ofits own cognate mRNA to repress translation  coding region, a unique variant of the surfaceantigen PfEMP1 that mediates adhesion to placenta in pregnant women  of function . The appfunction . This isr stages . Similarr stages . Gene-spnslation  and antinslation . In Plastor CITH , 2010. Utor CITH . In P. fnt women . In thisP. falciparum genome was fully sequenced , mapped uniquely to the genome  to the corresponding 11, 21, 31, 45, and 2 hr postmerozoite invasion time points of the microarray data set, despite the use ofdifferent methodologies (microarray vs RNA-seq) and the use of different P.falciparum strains . Because of the highersensitivity of RNA-seq, we were able to accommodate an additional 743 genes into thecascade-like transcriptome extending it to a total of 3110 genes , early trophozoites (r =0.93), late trophozoites (r = 0.91), schizonts(r = 0.89), and purified merozoites (r= 0.86). This indicates that when an mRNA is detected in one stage it isassociated proportionally with ribosomes within the same stage. An example pair ofgenes is shown in r = 0.98 and 0.93for PF14_0104 and PF14_0105, respectively), indicating that mRNA translation occursproportionally during the same stages at which these genes are transcribed (r ≥ 0.7) between mRNA abundance andtranslation  had a maximum difference of1.47-fold observed between early trophozoites and schizonts. Translationalefficiencies display a roughly 100-fold range in absolute values in each of thestages with the exception of the ring and merozoite stages, which exhibit moreextreme values. In these stages, the distribution of absolute TE values displays anapproximately fourfold larger spread than in early trophozoites, late trophozoites,or schizonts (2TE = 4.1) and the gene with the lowest TE is theFIKK family serine/threonine protein kinase . In merozoites the largest and lowest TE values correspond to the serinerepeat antigen 5  and the alphaadenylyl cyclase ,respectively.Ribosome profiling allows the monitoring of translation rates through thesimultaneous quantitative measure of mRNA abundance and ribosome density on mRNAs.The ratio of the footprint rpkM to the mRNA rpkM for any given gene represents itsrelative translational efficiency (TE) . To assechizonts . In ring2TE ≥ 2) across all stages irrespective of variations in itsmRNA abundance. In contrast TE values for the eukaryotic initiation factor 2alphakinase 1  are among the lowest measured despite high mRNA abundanceacross all stages  and reticulocytebinding protein homologue 3 , represent a clear example of lowtranslational efficiency. The PfRh3 pseudogene ribosome profile shows thattranslation of the 5′ end of this transcript occurs up until the encounter ofseveral in-frame stop codons, causing the reduction in ribosome density from thispoint on . This su2TE −3.2, −2.7, −2.9,respectively). Accordingly, the ribosome profile of this gene in merozoites shows ageneral depletion of ribosomes along the CDS  of reads mapping to this locus support asingle base deletion that creates a premature stop codon exactly at the site ofribosome footprint drop-off  was first described asa pseudogene based on the presence of an internal stop codon . Since t the CDS . In ringdrop-off . These d2TE = −3.6)despite relatively high transcript abundance across all stages demonstrating that post-transcriptional regulation at the level of translation is, atleast in part, responsible for its scarcity as a protein.In addition to expected instances of translational regulation, our data permit thediscovery of previously uncharacterized translational regulation, especially at theextremes of the TE distributions. One of the most notable examples of translationalsilencing is the eIF2α kinase IK1 (PF14_0423) for which ribosome footprintsaccumulate at the 5′ leader and 3′ UTR but not on the CDS, resulting inan extremely low translational efficiency  well-translated genesidentified here . Mass sp2TE = 3.8),tubulin beta chain , and early transcribedmembrane protein 2 .Among the top ten most highly translated genes are proteins involved in merozoiteegress and invasion MSP3, 6, 7, and 9 , serine repeat antigen 5 , andRAP1, 2, and 3  . InteresP. falciparum with few exceptions. These apply to a small subsetof translationally down- and up-regulated genes for which translational efficienciesappear to be inherent properties of the mRNA, independent of changes in mRNAabundance. Genes in this category, especially those that exhibit high translationalefficiencies, are enriched with functions associated with merozoite egress andinvasion during the transition from late stages into rings.Taken together these data demonstrate that transcription and translation are tightlycorrelated for the majority of genes expressed during the asexual life cycle ofP. falciparum transcriptome. We constructed a hiddenMarkov model (HMM) to automatically delineate the boundaries of both 5′leaders and 3′ UTRs for known gene models . Within the limits imposed by our data, we were able to describe5′ mRNA leaders and/or 3′ UTRs for 3569 genes in at least one of thestages  and displays alarge amount of 5′ leader ribosome occupancy. Half (49%) of the reads mapwithin the nine predicted uORFs on the 5′ leader of this gene, the other halfmaps outside these uORFs . These two genes represent exceptional cases for which uORFtranslation negatively correlates with translation of the downstream ORF.Nonetheless, there exist at least two clear exceptions. First, we were able tovalidate translation of the reported uORF present in the 5′ leader sequence ofthe var2csa mRNA which is expressed only in rings . The maj2TE = 3.8 and log25’RD = 2.9 in rings),and PFC0486c   for each gene expressed during the IDC, defined as upstream ribosomeoccupancy normalized for mRNA expression level and size of the leader sequence(5′ leader ribosome footprint rpkM/5′ leader mRNA rpkM) . No posin rings) .25′RD −0.03, and 0.11,respectively) relative to early trophozoites, late trophozoites, and schizonts , where therange of 5′RD values is also narrower (25′RD = 0.83) and depleted for translationallydown-regulated transcripts in all stages (mean log25′RD =−1.11), suggesting the possibility that 5′RD is a byproduct oftranslational efficiency itself . We focused on the subset of genes with highestand lowest 5′RD values (mean ± 2 SD) and found that only a fraction ofthe translationally up- and down-regulated genes overlap with this category ofextreme 5′RDs in each stage (25′RD= 5.1).In order to determine whether a direct relationship between 5′RD andtranslational efficiency of the downstream ORF exists, we compared these values foreach gene. 5′RD positively correlates, albeit moderately, with translationalefficiency in all stages, particularly in rings and merozoites   , nuclearn (SAGE) , and morn (SAGE) ,suggestn (SAGE) , yet then (SAGE) .5. Furthermore, our HMM mappingof 5′ leaders and 3′ UTRs facilitates the differentiation betweenindependently transcribed antisense RNA and transcripts that occur by virtue of beingpart of an adjacent gene. We took advantage of the nature of our data set to identifyantisense transcripts and looked for effects on sense mRNA translation.The 30 nt fragmentation and RNA-ligase-based library preparation method employed hereaffords exquisite strand specificity by minimizing artifacts associated with randompriming during reverse transcription. As evidence of this specificity, the highestexpressed gene in our data set, histone h2a (PFF0860c), yielded a total of 765,510reads on the sense strand, and only two reads on the antisense strand, correspondingto a sense:antisense ratio greater than 10For this analysis only, we relaxed our stringent coverage threshold from ≥32rM to ≥16 rM for inclusion of antisense transcripts. We based our threshold onthe presence of an antisense transcript to the sexual stage specific gene pfs16(PFD0310w) confirmed by strand-specific RT-PCR . This anWe next interrogated the impact of this set of antisense transcripts. Overall,antisense transcripts showed no effect on mRNA abundance and translationalefficiencies of the cognate sense transcript. These observations parallel thosedescribed for antisense transcripts in yeast . Thus, aP. falciparum IDC bydetermination of transcript abundance and architecture together with ribosomal densityand positioning. The quality of our data relies on several critical features: (1) hightemporal specificity and reproducibility of fully independent biological replicas offive strictly staged cultures; (2) purified merozoites to allow discrete measurements inthis stage without confounding contributions from schizonts or rings; (3) monosomeisolation from sucrose gradients to specifically enrich for ribosome-derived footprintsand avoid potential complications that can arise with methods like sucrose cushionswhich are prone to mRNA contamination; (4) sufficient sequencing depth of biologicalreplicates to set a statistical threshold for minimum read coverage and to demonstratereproducibility; (5) stringent strand specificity to facilitate an HMM for thedescription of transcript boundaries and the detection of antisense transcription.Herein we present, for the first time, a comprehensive view of the coupledtranscriptional and translational dynamics of the Previous studies of the transcript abundance in the malaria blood stages revealed aperiodic cascade of gene expression, whereby the majority of expressed genes exhibit onepeak of expression per cell cycle . The gloOur ribosome profiling results reveal a tight coupling of transcription and translationfor the majority of expressed genes, indicating that most protein products aretranslated with highly similar timing and in proportion to their corresponding mRNAtranscripts. Synthesized proteins are likely to exert their functions immediately upontranslation but post-translational regulation, not captured by our data, could still beat play. Direct correlations of translational efficiencies measured in this study alongwith proteomic data sets are hampered by the reduced sensitivity of the latter anddifferences in temporal resolution and staging of the parasites between data sets.However, the available proteomic evidence is largely consistent with the resultspresented here, particularly for highly translated proteins. The simultaneous capture ofmRNA abundance and translation is expected to be a more accurate proxy for proteinlevels than measurements of mRNA abundance alone  and provWhile no up- or down-regulation of global translation efficiencies were observed in anyparticular stage, more extreme translational efficiencies were measured in subsets ofgenes expressed in rings and merozoites. We find 177 translationally up-regulated geneswith functions predominantly related to merozoite egress and invasion, with peak mRNA inschizonts and peak translational efficiency in rings. It is likely that the genes withunknown functions, regulated in an analogous way during the merozoite to ringtransition, are also associated with this process. Our data support a model whereby thetranscripts of proteins necessary for merozoite structure and function are made in theprevious stage in large abundance, are translationally up-regulated during the invasionprocess, and remain highly translated well into the ring stage despite rapid mRNA decayduring this stage . WhetherP. falciparum.What mechanism could account for global ribosome accumulation in the 5′ leader?The presence of apparent 80S ribosomes within the 5′ leader sequence, regardlessof whether they cover uORFs or not, suggests an engagement mode in which the fidelity ofstart codon recognition is altered or suspended. Current models propose that the 43Spre-initiation complex loads onto the mRNA with the assistance of other initiationfactors near the 5′ cap and proceeds to scan down the length of the mRNA until itencounters an AUG codon. This is followed by the assembly of the 48S preinitiationcomplex and then finally the 80S complex  and frequent media changes(at least every 6–8 hr). Cells were synchronized by two consecutive sorbitoltreatments for three generations, for a total of six treatments. Maximum invasion,point at which half of the culture is either rings or schizonts, was defined as hourzero and independent time points containing ∼1010 parasites wereharvested 11, 21, 31, and 45 hr later.W2 strain cultures were maintained in Hyperflasks  in 500 mlRPMIc ,2 g/L sodium bicarbonate, 0.1 mM hypoxanthine, 25 mM HEPES (pH 7.4), and 50μg/L gentamycin), at 37°C, 5% Og at room temperature. An aliquot was removed andflash frozen in liquid nitrogen for total RNA purification, followed bypoly(A)-purification and chemical fragmentation with Zn2+ to∼30 nt for consistency in mRNA-Seq library preparation. The remaining culturewas treated with ice-cold 0.1% saponin in 1X PBS, 100 µg/ml cycloheximide, forRBC lysis. Parasites were resuspended in ice-cold parasite lysis buffer  and dripped into a conical tube filled with, and immersed in, liquidnitrogen. Frozen cells transferred placed in liquid nitrogen pre-chilled chambers andpulverized for 3 min at 15 Hz, on a Retsch MM301 mixer mill. Pulverized cells werethawed on ice, and cell debris was removed by centrifugation at 4°C,16,000×g for 10 min. The supernatant was treated with 2.88U/µg Micrococcal nuclease for 30 min at room temperature and immediately loadedonto sucrose gradients for ultracentrifugation at 35,000 rpm for 3 hr at 4°C ina L8-60 M Beckman centrifuge. Monosome fractions only, were collected to generateribosome footprint libraries for deep sequencing using the HiSeq 2000 , as described  and harvested by centrifugation for 8min at 3.65×escribed .Late stage schizonts (40–44 hpi) were magnetically purified using MACS LDcolumns  and resuspended in RPMIc without bloodaddition. After reaching maximum invasion (1:1 schizont to ring ratio), cultures wereharvested by centrifugation at 1500 rpm at room temperature for 5 min. Pelletedcultures were resuspended into fresh RPMIc and placed at 37°C. Merozoites in thesupernatant were treated with 100 µg/ml cycloheximide for 5 min at roomtemperature, harvested at 4000 rpm at 4°C for 5 min and resuspended in RPMIc andpassaged again through a MACS LD column. Parasite lysis buffer was added to themerozoite-enriched flow-through and flash frozen in liquid nitrogen. This procedurewas repeated three times every 45 min using the original culture. The same proceduredescribed above was used for RNA extraction and library preparation.P.falciparum PlasmoDB 3D7 version 7.1 genome using Bowtie 0.12.1  were detected and used to produce the SNP-corrected W2 genome based onthe 3D7 genome. Fastq files are available for download at NCBI SRA, accession#SRP042946.W2 strain genomic DNA was isolated from >90% synchronized ring stage cultures.Paired end libraries were constructed using the Nextera DNA Sample Prep Kit according to the manufacturer's instructionsreducing PCR cycles from nine to six and using 80% A/T dNTPs. Libraries weresequenced using the HiSeq 2000 (Illumina). Reads were aligned to the e 0.12.1  with parP. falciparum rRNA usingblast, and aligned uniquely to the W2 SNP-corrected genome using Bowtie 0.12.1  or intergenic (i) with threepossible emissions: (1) read present, (2) read not present but position isunmappable, (3) read not present but the position is mappable. Both state andemission probabilites were calculated using ∼30 kb training set of manuallyidentified transcript and non-transcript regions. The initial probabilities were setto 0.5. Transition probabilities were estimated from the median length of intergenicregions of (1252 nt) and median lengths of CDS regions (2545 nt), where thePt->i = (1/2545), Pt->t = (2544/2545),Pi->t = (1251/1252), andPi->i = (1/1252). We applied the Viterbialgorithm to predict the optimal path of transcript tracks per time point with a 10nt window resolution. HMM-defined 5′ leader and 3′ UTR coordinates areavailable for download at NCBI GEO, accession #GSE58402.The HMM was built using RNA-Seq data obtained in this study and two states:transcript  according to manufacturer's instructions,using either an antisense-specific primer to Pfs16 (PFD0310w) or a random nonamer.cDNA was amplified using the Pfs16 antisense-specific primer as a forward primer incombination with one of five reverse primers . 18S rRN reviewprocess). Similarly, the author response typically shows only responsesto the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of thepublished articles . An edited version of theletter sent to the authors after peer review is shown, indicating the substantiveconcerns or comments; minor concerns are not usually shown. Reviewers have theopportunity to discuss the decision before the letter is sent  and 3 reviewers, one of whom is a member of our Boardof Reviewing Editors.Thank you for sending your work entitled “Genome-wide Regulatory Dynamics ofTranslation in the The Reviewing editor and the other reviewers discussed their comments before we reachedthis decision, and the Reviewing editor has assembled the following comments to help youprepare a revised submission.P. falciparum. The use of such a regulatory strategy would help toexplain why there is a continuous flow of transcription from the majority of genes inP. falciparum throughout the development cycle in place of a moreregulated and dynamic gene expression strategy. In addition, the authors seek to enhanceour understanding of how this regulatory strategy is enacted in this organism. Thus,this is a timely and potentially and important manuscript. However, overall, themanuscript contains several important issues that require attention to help readersbetter appreciate the messages communicated.This manuscript presents genome-wide transcriptional maps and association of thedetected steady-state RNAs to ribosomes as a means of studying the possible use oftranslation as a post-transcriptional control mechanism during 5 stages of developmentin 1) Given the critiques of the riboseq approaches  it would be important to firmly establish that the RNAs isolated in the ribosomeprotocol are part of a productive polysome translation complex and instead of individualmonosomes. This concern is given support in P. falciparum IDC . It is also important to note that select noncodingRNAs show a translational efficiency similar to coding regions, even though they are nottranslated ; these noncodingRNAs can be distinguished from coding regions only by their ribosomal release score,which was not calculated in this study. Together, the translation efficiency measuredhere is not enough to indicate if a protein product is being made. The authors have toprovide additional evidence or analyses to conclusively state that there is a lack of“overt” translational control.2) Since the authors do not generate a proteomic dataset for their samples, it is notconvincing that the translation efficiency values that they infer from their ribosomaloccupancy profiles are direct indicators of protein synthesis. This is especially truegiven the delay in protein as compared to transcript detection observed during the3) Another reason for the detection of a transcript in the ribosomal occupancy profilecould be due to its presence in other ribonucleoprotein complexes that associate orco-sediment with the 80S monosome fractions. The authors infer the same when theydiscuss the 3' UTRs of transcripts . Could t4) Another concern is that the authors do not discuss their data in light of thepolysome occupancy study published by Bunnik et al., Genome Biology, 2013, and thedelayed translation phenomenon that has been observed by Le Roch et al., Genome Biol.,2004, and Foth et al., Mol. Cell. Proteomics, 2011.5) The antisense RNAs section is not 'up-to-date' with the literature. Threerecent studies have detected pervasive transcription of the parasite genome –López-Barragán et al., BMC Genomics, 2011; Siegel et al., BMC Genomics, 2014;Wei et al., PLOS One, 2014 – none of which are referenced and discussed here.6) The authors establish a threshold read count, in terms of reads per million mapped(rM), for downstream analysis. The threshold chosen here seems reasonable for this dataset, but I do think there is an important clarification to be made. There are twoseparate questions that might be addressed by a read count threshold: a minimumexpression level for biologically significant effects and a minimum count forstatistically reliable analysis. An rM threshold is reasonable for the former, while araw read count seems more appropriate for the latter. Doubling sequence coverage wouldpermit statistically reliable detection of lower expression levels.7) At several points the authors discuss a correlation between mRNA abundance andtranslation, or between transcription and translation; these seem to be usedinterchangeably. The authors seem to mean that the amount of protein produced correlateswell with the abundance of the mRNA that encodes it – but this phrase could alsosuggest that more abundant mRNAs are translated more highly. The authors should clarifythis point when it's first mentioned.8) The authors discuss relative translational efficiency (TE). Measures of overalltranslation and TE in different samples are all scaled by the per-million-mapped factorto correct for differences in sequencing depth, etc. This correction obscures changes inthe bulk mRNA content or translational activity between different stages. These effectsare discussed in e.g. Lovén et al., Cell 2012. The authors state that“Absolute mean translational efficiencies were the same in all stages” butthis conclusion is almost an arithmetic identity after applying per-million-mappednormalization. The polysome profiles presented in 9) In discussing RESA2 I was confused by 10) In discussing the eIF2alpha kinase IK1, the authors note that the gene istranslationally silenced, perhaps reflecting one of the few uORF-mediated translationalsilencing events in Plasmodium. As eIF2alpha phosphorylation can induce a bypass of suchsilencing, this seems like a potentially interesting auto regulatory system – inanimals, the eIF2alpha phosphatase GADD34 is regulated by uORF silencing in order toprovide feedback; here it would seem that positive feedback may act instead.11) The authors discuss qualitatively the observation that many abundant but poorlytranslated mRNAs are not detectable by proteomics, in contrast to well-translatedmessages. It would be valuable to quantify detectability, controlling for mRNAabundance, and test less anecdotally whether detectability, as a proxy for proteinabundance, follows translation better than mRNA abundance.12) It is striking that the 5' UTRs in Plasmodium were longer than the 3' UTRs– this differs from the situation in other eukaryotes, over a wide range ofabsolute UTR length. The authors should comment on this. 1) Given the critiques of the riboseq approaches  it would be important to firmly establish that the RNAsisolated in the ribosome protocol are part of a productive polysome translationcomplex and instead of individual monosomes. This concern is given supportinwhich shows considerable size 80s peaks in the untreated samples for the LateTroph, Schizonts and merozoites stages. In short, the detection of RNA protected byribosomes in these footprint experiments cannot always be with associated with RNAdestined for translation. Wilson & Masel, 2011 have reported that non-productivebinding of single ribosomes to mRNA do not undergo productive translation. Thus,RNAseq results obtained from the 80S peak might be compared to RNAseq resultsobtained from RNAs isolated from the polysome peak. This would act as a controlagainst this problematic possibility.We thank the reviewers for pointing out this important issue. Our response to this issueand #2 are linked; therefore please refer to the section below for ourresponse.2) Since the authors do not generate a proteomic dataset for their samples, itis not convincing that the translation efficiency values that they infer from theirribosomal occupancy profiles are direct indicators of protein synthesis. This isespecially true given the delay in protein as compared to transcript detectionobserved during the P. falciparum IDC . It is also important to note that select noncodingRNAs show a translational efficiency similar to coding regions, even though they arenot translated ; thesenoncoding RNAs can be distinguished from coding regions only by their ribosomalrelease score, which was not calculated in this study. Together, the translationefficiency measured here is not enough to indicate if a protein product is beingmade. The authors have to provide additional evidence or analyses to conclusivelystate that there is a lack of “overt” translationalcontrol.To summarize the questions raised by reviewer points #1 and #2:A) Do monosome fragments actually represent productive translating ribosomes?B) Given that there are footprints that apparently map to ncRNAs, which, by definition,are not expected to be translated, what is evidence that the TE values faithfullyreflect translational efficiency?C) What about the Wilson & Masel findings with regard to unproductive ribosomebinding?D) Given that we do not have a proteomic dataset to accompany this study, how do wereconcile our findings with the apparent delay in protein translation that is observedduring the IDC as published by Le Roch and Foth?Issues A & B are inherently linked. First, a number of methods have recently arisento assist in the discrimination of RNA fragments that derive from ribosome protection asopposed to anything else.Cell Rep., September2014), the authors propose a simple metric to distinguish true ribosome footprints fromeither spurious RNA fragments or non-ribosomal RNA binding proteins by comparing thecumulative read size distribution between protein coding genes and classical non-codingsequences (such as telomerase RNA or RNAse P). According to this study, comparison ofread size distribution alone is sufficient to identify true ribosome footprints.In a recently published study by Ingolia et al. .Thus, for each region with sufficient numbers of mapped fragments, we assess whether thecumulative distribution of fragment lengths is consistent with ribosome binding ornot.In another published paper by Guttman et al., the authors use a metric called theribosomal release score (RRS) which is similar to the disengagement score (DS) from Chewet al, to distinguish true coding regions from noncoding RNAs by determining whetherthere is evidence for a substantial decrease in the number of ribosome footprintsfollowing the stop codon, a feature that would be expected for a legitimately translatedregion.We have further analyzed our dataset using the ribosomal release score (RSS) metric fora number of non-coding RNAs for which we have derived TE values and compared thesencRNAs with the rest of our protein coding genes (below). We find that TE values forprotein coding genes are well correlated with the ribosome release scores (black dots),yet nearly all ncRNAs yield poor release scores, regardless of TE value (red dots).Thus, both the method using cumulative distributions of fragment lengths and the RRSmetric are consistent with ongoing translation in protein coding regions, as opposed toregions within classic ncRNAs (like tRNAs). However, it should be noted that not allregions fit neatly into one category or another. Regions such as the 5’ leadersequences reveal ribosome density with fragment length distributions that areindistinguishable from those within coding regions, yet in such regions, an RRS metriccannot be calculated. Ingolia et al. , argue that translationon transcripts outside of protein-coding regions can in fact produce polypeptides andtherefore a TE value is valid for many non-coding sequences such as 5’UTRs orlncRNAs.eLife 2013, Ingolia et al. Cell 2011, Smith et al., Cell Rep. 2014,reviewed in Ingolia, Nat. Rev Genet. 2014], yet clearly there is still some debate inthe field with regard to unexpected ribosome density outside of coding regions.Realizing the complexity of this issue, we have chosen a conservative path for ourmanuscript, whereby we use TE to only refer to protein coding regions, and RD  to refer to those outside of the coding regions (like 5’ RD).Within coding regions, the legitimacy of ribosome protected fragments and theirrelationship to translational efficiency is now widely supported in a variety oforganisms [Stern-Ginossar et al., Science 2012, Oh et al., Cell 2011, Dunn et al.,C) What about the Wilson & Masel findings with regard to unproductive ribosomebinding?With specific regard to the Wilson & Masel 2011 paper cited by the reviewer, theseauthors report ribosomal association to stable unannotated transcripts, yet did notactually assess whether these complexes were engaged in actual translation and synthesisof a peptide. In fact they state:“While we do not know the extent to which this ribosomal associationleads to translation, these SUTs, apart from RDT1, do not appear to encode functional,protein-coding genes.”de novo birth of protein-codinggenes.”The authors speculate that perhaps ribosome association to unannotated or short ORFs mayindeed lead to translation “by accident at least at low levels” and“provide raw material for D) How do we reconcile our findings with the apparent delay in protein translation thatis observed during the IDC as published by Le Roch and Foth?With regards to a comparison between the data presented in this manuscript and previousmass spectrometry based studies, the reviewer cites two articles which report a delay inmRNA abundance with respect to protein detection, Le Roch et al., Genome Biol., 2004 andFoth et al., Mol. Cell. Proteomics, 2011.previous stage. By excluding these genes, a delay betweentranscript abundance and the detection of its corresponding protein may be attributed toonly 129 transcripts.In Le Roch et al., the authors analyzed a total of 459 mRNA-protein pairs and found thatfor 171 genes transcript abundance in one stage correlated better with protein levels ofthe next stage (>0.5 increase in Spearman rank correlation). However, it should benoted that 45 of these 171 transcripts were also better correlated to the proteins inthe In Foth et al. the authors analyze 125 mRNA-protein pairs and detect multiple isoformsfor some of the proteins. A delay between mRNA abundance and protein detection isobserved for 63.7% of 237 protein isoforms .Here, we report 177 and 124 translationally up- and down-regulated genes, respectively.This represents 295 genes, which is more than twice the number detected in Le Roch orFoth. However, these 295 genes represent only 8% of the 3,605 genes present in ouranalysis, and for remaining 92%, we find that transcript abundance is proportional toribosome occupancy throughout the lifecycle.Thus, while we identify more instances of translational control than all prior studies,these instances appear to be the exception, and not the rule, as the vast majority ofgenes appear to lack overt translational control as measured by this assay.3) Another reason for the detection of a transcript in the ribosomal occupancyprofile could be due to its presence in other ribonucleoprotein complexes thatassociate or co-sediment with the 80S monosome fractions. The authors infer the samewhen they discuss the 3' UTRs of transcripts . Could this be the case forother regions of apparent ribosomal occupancy such as the 5' leader sequence?The authors should discuss this in more detail. A straightforward experiment would beto determine the protein composition of the 80S monosome fractions isolated forribosome profiling in different stages of the IDC. A related question: Is there alack of polysomes in merozoite stages? This appears to be the case inThe issue of distinguishing between fragments that derive from actual ribosome bindingas opposed to other RNA binding proteins is discussed above. In short, we use thecumulative distribution of read lengths in a manner identical to Ingolia et al. , as a discrimination method. Here, we find that the fragmentsderiving from the 5’ leaders show a perfectly overlapping cumulative distributionto coding regions. This is not the case for known ncRNAs, like tRNAs.Regardless, whether our 80S fractions contain binding activities other than ribosomescan be assessed by mass spectrometry, as the reviewer points out. During the course ofthis study, we in fact prepared schizont and merozoite monosome fractions and a pooledschizont polysome fraction for peptide sequencing by tandem mass spectrometry(LC-MS/MS). The results of this analysis are shown below:These results indicate that the monosomes and polysome fractions that we collectedcontain mostly ribosomal components (89–100%). The remainder 4.3-10.4% ofPlasmodium proteins that are non-ribosomal constitute highly expressed proteins such asheat shock proteins, exported proteins, membrane proteins, GAPDH, and other housekeepinggenes.Only 0.26% of spectral counts in the schizont monosome fraction mapped to putativenon-ribosomal RNA-binding proteins (such as the DNA/RNA-binding protein Alba). While wecannot exhaustively account for every protein that co-sediments with the 80S peak, thisproteomic evidence confirms that the vast majority of the 80S proteins areribosomal.With regard to the polysome profiles from merozoites, we agree that the relative peakheight of the polysome region compared to the 80S appears reduced. This could be theresult of a global reduction in overall translation at this stage. In this study, itshould be noted that we do not directly assess bulk or absolute levels of overalltranslation activity. Rather, this study examines the relative translational efficiencyof transcripts on a per-timepoint, per message basis. In merozoites, we do observe aslightly broader distribution of efficiencies, but not dramatically so.4) Another concern is that the authors do not discuss their data in light of thepolysome occupancy study published by Bunnik et al., Genome Biology, 2013, and thedelayed translation phenomenon that has been observed by Le Roch et al., GenomeBiol., 2004, and Foth et al., Mol. Cell. Proteomics, 2011.For a comparison with Le Roch et al., Genome Biol., 2004, and Foth et al., Mol. Cell.Proteomics, 2011 please see our response to points #1 & #2, part D.The reviewers ask for a comparison with the work published by Bunnik et al. wherepolysome-associated mRNA was compared to total steady state mRNA. A major conclusion ofthis paper is that there exists strong translational control for 471 genes, where thepeak of transcriptional abundance is in trophozoite or schizont stage, but they are mosthighly associated with polysomes in the ring stage.There are a number of confounding factors that obstruct a fair comparison on the workpresented here and that of Bunnik et al. These factors include the strain, the timepoints sampled, the methodology for isolation of polysomes, and most importantly, theoverall sequencing coverage. We discuss each of these factors below.P. falciparum toassess hours 0, 18, and 36 hours post invasion whereas we used the W2 strain to samplehours 11, 21, 31, 45 in addition to purified merozoites.In Bunnik et al., the authors used the 3D7 strain of A critical step in ribosome profiling is the isolation of intact polysomes. In ourhands, directly using sucrose gradients yields polysome profiles enriched in higherorder polysomes, with more than 3 ribosomes. Polysome profiles in Bunnik et al. areobtained by pelleting parasite lysates on sucrose cushions, and re-loading the ribosomalpellets on sucrose gradients. This additional step may cause the dissociation ofribosomes from mRNAs, and could explain the reduced amount of higher order polysomes intheir profiles, which in turn may skew their assessment of translation towards lowervalues. . Their deepest exome-widecoverage for polysomes is 12.7x , and their average coverage forpolysomes is 5.3x. The average exome-wide coverage of our ribosome footprint data is159.9x, which is, on average, 30 times greater than Bunnik et al. (tablebelow).More importantly, the major difference between the Bunnik dataset and our own is the setof 471 genes that the authors identify as being translationally up-regulated in rings.Approximately 76% of these genes fall below our threshold for minimum read coverage(32rM) for analysis, despite the fact that our ring stage coverage is 456-fold greaterthan the Bunnik zero hour polysome sample (315x coverage compared to Bunnik’s0.69x).Of the remaining genes , weidentified only five genes that we would consider to be translationally up-regulated inrings.Through our own biological replicates of each time point and our assessment ofreliability with respect to sequencing coverage , we beli5) The antisense RNAs section is not 'up-to-date' with the literature.Three recent studies have detected pervasive transcription of the parasite genome– López-Barragán et al., BMC Genomics, 2011; Siegel et al., BMCGenomics, 2014; Wei et al., PLOS One, 2014 – none of which are referenced anddiscussed here.We thank the reviewers for pointing this out. We updated our antisense section toreference López-Barragán et al., BMC Genomics, 2011 and Siegel et al., BMCGenomics, 2014. The text now reads “Previous serial analysis of gene expression(SAGE) , nuclearThe work by Wei et al. analyzes uncapped/non-adenylated intermediate size ncRNAs, whichwe do not focus on.6) The authors establish a threshold read count, in terms of reads per millionmapped (rM), for downstream analysis. The threshold chosen here seems reasonable forthis data set, but I do think there is an important clarification to be made. Thereare two separate questions that might be addressed by a read count threshold: aminimum expression level for biologically significant effects and a minimum count forstatistically reliable analysis. An rM threshold is reasonable for the former, whilea raw read count seems more appropriate for the latter. Doubling sequence coveragewould permit statistically reliable detection of lower expressionlevels.We took a conservative approach by choosing a minimum of rM of 32 per gene. We agreewith the reviewer that a reliable statistical analysis could still be obtained bylowering this threshold and we did so for the antisense analysis where we chose a≥16rM bar. Future studies into these low count reads will be necessary toestablish their biological significance.7) At several points the authors discuss a correlation between mRNA abundanceand translation, or between transcription and translation; these seem to be usedinterchangeably. The authors seem to mean that the amount of protein producedcorrelates well with the abundance of the mRNA that encodes it – but thisphrase could also suggest that more abundant mRNAs are translated more highly. Theauthors should clarify this point when it's first mentioned.We fully agree with the reviewers and clarified the ambiguity by changing the text from“To determine the exact level of correlation between mRNA abundance andtranslation we directly compared mRNA and ribosome footprint density measurements . In geneWe changed: “The simultaneous capture of transcription and translation isexpected to be…” to “The simultaneous capture of mRNA abundance andtranslation is expected to be…”We changed the phrase “… peak transcription occurring in rings andschizonts” to “…peak mRNA abundance occurring in rings andschizonts”.8) The authors discuss relative translational efficiency (TE). Measures ofoverall translation and TE in different samples are all scaled by theper-million-mapped factor to correct for differences in sequencing depth, etc. Thiscorrection obscures changes in the bulk mRNA content or translational activitybetween different stages. These effects are discussed in e.g. Lovén et al., Cell2012. The authors state that “Absolute mean translational efficiencies werethe same in all stages” but this conclusion is almost an arithmetic identityafter applying per-million-mapped normalization. The polysome profiles presentedinindicate large changes in overall translational activity at differentdevelopmental stages.This study measures the relative change in translational efficiency on a per-gene,per-time point basis, and does not measure absolute changes in bulk translation ortranscription, as the reviewer correctly points out. To clarify this for the reader, wehave added a statement in the Discussion highlighting this important point“However, we note that this dataset only provides a direct measure of relativechanges in translational efficiencies rather than changes in bulk transcription andtranslation.”With respect to the statement that the reviewer highlights regarding mean translationalefficiencies, we have corrected and amended this sentence, since the mean TEs are infact not identical in all stages . As shown in The text now reads:2TEµRings = -0.43, log2TEµE.trophs. = -0.56, log2TEµL.trophs. = -0.31, log2TEµSchizonts = -0.16 and log2TEµMerozoites = -0.68) had a maximum folddifference of 1.47-fold observed between early trophozoites and schizonts.”“Absolute mean translational efficiencies in all stages (log9) In discussing RESA2 I was confused by We thank the reviewers for indicating to us how to make the figure clearer. As advised,we added the annotated gene model described in PlasmoDB version 7.1, which is theversion used for reference in this work.10) In discussing the eIF2alpha kinase IK1, the authors note that the gene istranslationally silenced, perhaps reflecting one of the few uORF-mediatedtranslational silencing events in Plasmodium. As eIF2alpha phosphorylation can inducea bypass of such silencing, this seems like a potentially interesting auto regulatorysystem – in animals, the eIF2alpha phosphatase GADD34 is regulated by uORFsilencing in order to provide feedback; here it would seem that positive feedback mayact instead.We fully agree with the reviewers. Ribosome footprints partially overlap with 2 of the18 uORFs counted on the 5’ leader of the eIF2 alpha kinase transcript. Whetherthese uORFs or other 5’ leader motifs are responsible for the silencing of thedownstream coding region remains an important question. Additionally, this gene presentsthe lowest ribosomal release score calculated, which strongly suggests that it is notbeing translated.11) The authors discuss qualitatively the observation that many abundant butpoorly translated mRNAs are not detectable by proteomics, in contrast towell-translated messages. It would be valuable to quantify detectability, controllingfor mRNA abundance, and test less anecdotally whether detectability, as a proxy forprotein abundance, follows translation better than mRNA abundance.We did not wish to make the statement that genes that have high mRNA abundance would notbe detected by proteomic methods, and we thank reviewers for highlighting thismisunderstanding. We have therefore revised our manuscript as following:We changed the phrase from: “Thus, our ribosome profiling dataset highlightsinstances of translational control of genes for which, in the presence of abundant mRNA,the protein counterpart is unlikely to be detected by proteomic methods” to“Thus, our ribosome profiling dataset highlights instances of translationalcontrol of genes that may not be detected by proteomic methods.”12) It is striking that the 5' UTRs in Plasmodium were longer than the3' UTRs – this differs from the situation in other eukaryotes, over awide range of absolute UTR length. The authors should comment on this.P.falciparum truly lies at the extreme of the distribution.A review of the current literature reveals a wide distribution of different 5’UTRsizes in different types of organisms. Previous studies found that 5’UTRs ofmRNAs encoding genes which need to be strongly and finely regulated are often longerthan average . On the other hand, Lynch et al. (2005) proposed that thelength of the 5’UTR is driven mainly by random genetic drift and mutationalprocesses that cause stochastic turnover in transcription-initiation sites and prematurestart codons, combined with a selective constrain against the premature initiation oftranslation. Additional sequencing and mapping of transcript architecture in othereukaryotes will help establish in the future whether or not


Smyd1 in the first and second heart fields of mice using a knock-in (KI) Nkx2.5-cre driver. Robust deletion of floxed-Smyd1 in cardiomyocytes and the outflow tract (OFT) resulted in embryonic lethality at E9.5, truncation of the OFT and right ventricle, and additional defects consistent with impaired expansion and proliferation of the second heart field (SHF). Using a transgenic (Tg) Nkx2.5-cre driver previously shown to not delete in the SHF and OFT, early embryonic lethality was bypassed and both ventricular chambers were formed; however, reduced cardiomyocyte proliferation and other heart defects resulted in later embryonic death at E11.5-12.5. Proliferative impairment prior to both early and mid-gestational lethality was accompanied by dysregulation of transcripts critical for endoplasmic reticulum (ER) stress. Mid-gestational death was also associated with impairment of oxidative stress defense—a phenotype highly similar to the previously characterized knockout of the Smyd1-interacting transcription factor, skNAC. We describe a potential feedback mechanism in which the stress response factor Tribbles3/TRB3, when directly methylated by Smyd1, acts as a co-repressor of Smyd1-mediated transcription. Our findings suggest that Smyd1 is required for maintaining cardiomyocyte proliferation at minimally two different embryonic heart developmental stages, and its loss leads to linked stress responses that signal ensuing lethality.Smyd1/Bop is an evolutionary conserved histone methyltransferase previously shown by conventional knockout to be critical for embryonic heart development. To further explore the mechanism(s) in a cell autonomous context, we conditionally ablated  Cardiac organogenesis initiates in the cardiac crescent at embryonic day (E) 7.75 and gives rise to the linear heart tube by E8.0 . CardiacMb) and associated factors that contribute to oxygen storage and delivery  methionine  was included as a methyl donor. All reactions were carried out in 40μl HMT reaction buffer  at 30°C for 3 hours. A 12% SDS-PAGE gel was used to resolve the samples and fluorography was used to visualize positive methylation. Substrate loading was visualized by Coomassie blue staining.The escribed . BrieflyIn vivo methylation of TRB3 was performed with a procedure modified from the method of Liu et al. [methyl-3H]methionine was added to a concentration of 10 mCi per ml. Cells were then incubated for an additional 3 hr, and lysed in RIPA buffer . Endogenous TRB3 was immunoprecipitated with anti-TRB3 polyclonal antibody , and immunoprecipitated proteins were resolved on 8% SDS-PAGE. [3H]Methyl labeling was detected by fluorography in 22% PPO solution. Dried gels were exposed to Kodak MRX film for 1 week.u et al. . 48 hr pt test was used for statistical comparisons when appropriate. A p-value of < 0.05 was considered statistically significant. Global microarray analyses employed Nimblegen chips and were analyzed using Java Treeview and DAVID [A Student’s nd DAVID .Smyd1 during embryogenesis, we performed whole mount in situ hybridization on embryos recovered at E8.5 and E9.5. Results of this analysis demonstrated that at E8.5 and E9.5, robust expression of Smyd1 was observed throughout the looped heart tube, including the ventricles, OFT and venous pole ; Smyd1c utilizes an alternate first exon. Therefore, we designed this conditional allele with loxP sites flanking exons 2 and 3, the first 2 exons in common among all three isoforms and the same exons that were deleted in the conventional knockout [Smyd1flox/flox with EIIA-cre [In order to analyze the role of Smyd1 specifically in the heart, we generated a knockout . Thus, iEIIA-cre , which iSmyd1 CKO using the Ki-Nkx2.5cre/+ strain previously shown to robustly delete in the first and SHF from E7.5 [Ki-Nkx2.5cre/+; Smyd1+/flox x Smyd1flox/flox revealed that 100% of Ki-Nkx2.5cre/+; Smyd1flox/flox (henceforth referred to as Smyd1 Ki-CKO) embryos died prior to E10.5. In contrast, control littermates of all other genotypes survived with no apparent abnormalities. No transcripts from the deleted exons were detectable from Smyd1 Ki-CKO hearts at E9.5 . The length of the OFT and width of the RV appeared truncated  and two genes crucial for establishing atrial-ventricle morphology and sidedness, Shh and Pitx2 [Smyd1 Ki-CKO , Activation Transcription Factor 5 (Atf5), and Chop1/GADD153. Simultaneously, genes canonically down-regulated during autophagy induced by ER-stress [Idh1) and Cell Cycle and Apoptosis Regulatory Protein 1 (Ccar1), were down-regulated. Furthermore, genes known to be unregulated in cardiomyocytes by both ER stress and hypoxia, such as Prolyl hydroxylase domain 1 and 3 (PHD/Egln1/3), Jun dimerization protein 2 (Jundm2/JDP2), adrenomedullin (Adm), and Tribbles 3 (TRB3) [Smyd1 Ki-CKO  ,39,40,411 Ki-CKO .Smyd1 using a transgenic (Tg)-Nkx2.5-cre line that initiates deletion primarily in differentiated ventricular cardiomyocytes [Smyd1 expression, likely the result of Smyd1 expression in cells outside of the cre-expressing zone, such as OFT myocardium  exhibited pericardial edema, thinned pericardium and decreased trabeculation in similar heart sections  is similar to that resulting from the knockout of skNAC, a Smyd1-interacting transcription factor exclusively expressed in striated muscle and initially characterized as a positive regulator of Mb [Smyd1 Tg-CKO and skNAC KO exhibit similar dysregulation of gene expression. To this end, we analyzed expression of genes previously shown to be deregulated in skNAC knockouts at E11.5 [skNAC knockout were also down-regulated in the Smyd1 Tg-CKO. Of the 3 genes upregulated in the skNAC knockout, only Metallothionein (Mt2) was significantly upregulated. The most significantly deregulated category was oxidative stress response [Mt2 in the Smyd1 Tg-CKO may be in response to reduced expression of Mb and Vnn1. High upregulation of Mt2 in both animal models suggests that impaired oxidative stress defense systems may have contributed to the similar phenotype and timing of death. Expression of skNAC was decreased in the Smyd1 Tg-CKO, but loss of skNAC did not affect expression of Smyd1. Thus, Smyd1 is necessary for proper expression of skNAC, but not vice versa. Hand2 was down-regulated in the Smyd1 Tg-CKO, but unaffected in the skNAC-/-. This result supports the idea that Smyd1 and skNAC work in both complementary and independent pathways [The observed mid-gestation or of Mb . Smyd1 dor of Mb ,12. Thusresponse ,5,8,9,12Smyd1 Tg-CKO of the ER stress program activated in Ki-CKO hearts  associated with Tg-CKO proliferative loss, and accordingly, Ccar1 was down-regulated (p<.061) [Atf5, Chac1, Idh1 and Ndrg1 was unaffected. These data support the idea that both the ER and the oxidative stress response is initiated in the absence of Smyd1 at both gestational stages.Based on previously observed similarities in genes modulated by stress response to both ER and oxidative stress ,40,41,42O hearts . As show(p<.061) ,37. In cSmyd1 Ki-CKO and Tg-Smyd1 CKO  [TRB3 is a kinase domain-containing multifunctional protein expressed broadly within the cytoplasm and nucleus of numerous cell types. While it was first characterized as a suppressor of AKT signaling ,27, TRB3CKO Fig.  and 3H. CKO Fig.  and thatCKO Fig. . This prX/L-P49) ,14,15. AX/L-P49) .in vitro methylation assays, employing bacterially-expressed and purified 6X-His-Smyd1 and 6XHis-TRB3 along with several TRB truncations using 3H-S-adenosyl-methionine as a methyl donor. Smyd1, but not an HMTase SET domain catalytic mutant (Smyd1-Y234F), methylated full-length TRB3(1–355) as well as the TRB3(1–80) N-terminus but not further C-terminal truncations  or primarily in differentiated ventricular cardiomyocytes (Tg-Nkx2.5-cre). Our findings indicate that Smyd1 is a critical regulator of cardiac development at both the stage at which the SHF gene program is initiated and at later stages of cardiac morphogenesis. Both CKOs exhibit impaired cell proliferation and activation of stress pathways, which may be consequence of proliferative failure. A component of the underlying molecular mechanism of Smyd1 is upregulation and subsequent methylation of TRB3, a sensor for both ER and oxidative stress and suppressor of Akt signaling [To gain insight into the physiological functions of ignaling ,27. SmydSmyd1 CKO phenotypes using two different Nkx2.5-cre drivers were dissimilar. Ki-CKO embryos died by E10.5 with impaired expansion of the SHF. Tg-CKO embryos survived until E11.5 or later, showed normal development of SHF-derived structures and exhibited myocardial abnormalities in both ventricles. The knock-in Nkx2.5-cre disrupts the Nkx2.5 open reading frame [Smyd1 Ki-CKO is also heterozygous for Nkx2.5. It is possible that loss of one Nkx2.5 allele amplifies the loss of Smyd1 phenotype; however, the difference in phenotype between the two CKOs is more likely due to differences in spatial and temporal expression of the two Nkx2.5-cre drivers. The transgenic Nkx2.5-cre [Nkx2.5-cre employs the full range of endogenous enhancer/promoter elements and is active as early as E7.5 in the cardiac crescent [Tg-Nkx2.5-cre has a more restricted expression pattern than the endogenous Nkx2.5 gene [Smyd1 or lack of deletion in SHF progenitors and OFT myocardium.It is of particular interest that the ng frame . As a cox2.5-cre  is fusedcrescent . Since t2.5 gene , it is lSmyd1 Ki-CKO hearts exhibited similarities with those of the conventional Smyd1 knockout [Smyd1 Tg-CKO phenotype was more similar to that of the skNAC knockout [Smyd1 knockout failed to form a RV, showed reduced expression of SHF genes and died before E10.5. Likewise, the Smyd1 Ki-CKO showed reduced size of SHF-derived structures (OFT and RV), a reduction is SHF gene expression and lethality by E10.5. The striated muscle-restricted transcription factor skNAC is one of the few known Smyd1-interacting proteins [skNAC knockout and Smyd1 Tg-CKO both exhibited myocardial defects in both ventricles, including thinning of the compact myocardium and decreased trabeculation. Furthermore, many of the same genes dysregulated in the skNAC knockout were similarly dysregulated in the Smyd1 Tg-CKO. These observations support the idea that the Smyd1-skNAC interaction regulates cardiomyocyte gene expression and is important for proper heart development.knockout , whereasknockout . The conproteins ,11. The Smyd1 elimination at E9.5, 324 were down-regulated, while 499 were upregulated. Smyd1 loss altered gene expression of a number of pathways  that are also upregulated in adult cardiac hypertrophy and/or heart failure [Smyd1 in adult hearts leads to marked hypertrophy with upregulation of the same markers .Although expression of  failure ,51,52,53Smyd1 Ki-CKO, three key trans-activators of the autophagic pathway were upregulated, including BCL2/adenovirus E1B 19-kDa interacting protein 3 (Bnip3), Activation Transcription Factor 5 (Atf5), and Chop1/GADD153 [Smyd1 Ki-CKO, the hallmark autophagy marker, cation transport regulator homolog 1 (Chac1) [Chac1 and Idh1 are known to be differentially expressed in response to autophagic vs. apoptotic signals [Isocitrate Dehydrogenase 1 (Idh1) and Cell Cycle and Apoptosis Regulatory Protein 1 (Ccar1) were down-regulated in the Smyd1 Ki-CKO. These genes are canonically down-regulated during autophagy induced by ER-stress, but are upregulated in ER stress-induced apoptosis [Smyd1 Ki-CKO. Prolyl hydroxylase domain 1 and -3 (PHD/Egln1/3) are known to be upregulated in cardiomyocytes by both ER stress and hypoxia [Smyd1 Ki-CKO including, Jun dimerization protein 2 (Jundm2/JDP2), adrenomedullin (Adm), and Tribbles 3 (TRB3) [In the absence of Smyd1, cardiomyocyte proliferation is reduced concurrent with upregulation of genes involved in ER and oxidative stress responses. Gene expression profiling at E9.5 uncovered an apparent attempt to resolve ER stress via autophagy. Cells exposed to ER stress undergo the unfolded protein response in an attempt to avoid apoptosis, but may also activate autophagy ,7. While/GADD153 ,7,56. At signals ,37. Isocpoptosis ,37. This hypoxia , as are 3 (TRB3) ,39,40,41Smyd1 Ki-CKO cardiomyocytes did not show morphological signs of cellular degradation characteristic of the execution phase of this mechanism. This second stage is mediated by the autophagic PERK/eIF2α axis which, at least by transcript profiling, was unaffected in Smyd1 Ki-CKO (data not shown) [N-myc down-regulated gene 1 (Ndrg1) [Smyd1 Ki-CKO. Ndrg1 over-expression inhibits the ER stress-mediated autophagic pathway by suppressing PERK/eIF2α leading to a pro-survival phenotype [Idh1 reduction as well as Egln1 and Egln3 upregulation leads to decreased intracellular NADPH and, thus, to an increase in intracellular ROS [Although the above gene expression deregulation suggests initiation of autophagic resolution to ER stress, t shown) . This ap (Ndrg1)  in the Shenotype . As a fuular ROS . Such trNdrg1 and Adm, upregulated targets discussed above in the context of ER stress, are also strong inducers of angiogenesis. Several additional genes upregulated by Smyd1 Ki-CKO belong to the angiogenesis pathway. These include VegFα, Plek, and CxCl4 , ER stress, oxidative stress and autophagy . ConsistTRB3 transcription, perhaps indirectly via activation of Chop1, which encodes an established trans-activator of TRB3 [Collectively these data indicate that Smyd1 regulates oxidative and ER stress responses at two levels. First, Smyd1 activates  of TRB3 . Second, of TRB3 . It remaSmyd1 is essential for proper expression of the cardiac gene program. Smyd1 null mutants exhibit malformation of SHF derivatives owing, at least in part, to reduced expansion of the progenitor pool. Smyd1 also regulates response to ER and oxidative stress. In the absence of Smyd1, a gene expression program consistent with a non-apoptotic autophagic response is initiated. These gene expression changes are consistent with observed reduction in cell proliferation and the absence of apoptosis. We also have shown that later in development, Smyd1 continues to play an essential role in response to oxidative stress, through both interaction with skNAC and dysregulation of non-skNAC target genes. As Smyd1 is continuously expressed into adulthood, it is likely involved with these pathways in both normal and pathologic conditions. For example, during ischemia as well as development and progression of maladaptive myocardial remodeling and failure, there is significant nutrient and oxygen starvation which leads to the induction of a number of ER and oxidative stress response genes—several of whom we observed here to be deregulated by Smyd1 deficiency [In summary, we have shown that ficiency ,62,63. FS1 FigA. Illustration of the gene targeting strategy to generate the Smyd1 conditional knockout allele. The neo cassette was subsequently removed via breeding to EIIA-cre and selecting for loss of neo and retention of exons 2–3 . B. Confirmation of correct Smyd1 gene targeting by Southern blot. Southern blots were performed using probes flanking the left and right homology arms (probes 2 and 3) to distinguish the 13.7 kb and 4.5 kb wild type (WT) allele fragments from the targeted allele (7.5 and a 7.0 kb), respectively. C.Smyd1 flox and WT alleles were distinguished by PCR. D, E. No Smyd1 RNA (D) or protein (E) was detectable in Smyd1flox/flox;Ki-Nkx2.5-cre (Ki-CKO) hearts at E9.5.(TIF)Click here for additional data file.S2 FigC-terminal MYC-Trb3 deletion constructs were tested by co-immunoprecipitation with FLAG-Smyd1 to map the region of Trb3 that interacts with Smyd1. Cos7 cells were co-transfected FLAG-Smyd1 and the MYC-Trb3 deletion constructs, incubated for 30 h and lysed. Cell lysates were immunoprecipitated using a monoclonal anti-FLAG M2 antibody. The immunoprecipitates were separated by SDS-PAGE and analyzed by western blot using a polyclonal anti-MYC A14 antibody. Input is shown in the lower panels. The far N-terminal deletion (1–80) was sufficient for interaction with Smyd1.(PDF)Click here for additional data file.S3 Fig6X-His-tagged wild-type Trb3(1–355) and the indicated truncation mutants were tested as substrates for methylation by full-length Smyd1. Methods are detailed in Materials and Methods section and legend to (TIF)Click here for additional data file.S1 TableNkx2.5+/+; Smyd1Flox/Flox) and mutant  hearts at E9.5. Down-regulated transcripts indicated in yellow.Annotated genes were organized based on gene ontology using DAVID software . These d(PDF)Click here for additional data file.


Prdx6 was significantly associated with pig meat quality, especially meat tenderness. However, the transcriptional regulation of porcine Prdx6 remains unclear. In this study, we determined the transcription start site (TSS) of porcine Prdx6 gene by 5′ rapid-amplification of cDNA ends (5′ RACE). Several regulatory elements including CCAAT/enhancer-binding proteinβ (C/EBPβ), Myogenic Differentiation (MyoD), cAMP response element binding protein (CREB), stimulating protein1 (Sp1) and heat shock factor (HSF) binding sites were found by computational analyses together with luciferase reporter system. Overexpression and RNA interference experiments showed that C/EBPβ or CREB could up-regulate the expression of porcine Prdx6 gene at both mRNA and protein level. Electrophoretic mobility shift assays (EMSA) and chromatin immunoprecipitation assays (ChIP) confirmed that C/EBPβ and CREB could interact with Prdx6 promoter. Immuoprecipitation results also showed that C/EBPβ could interact with Prdx6 in vivo. Taken together, our findings identified C/EBPβ and CREB as the important regulators of porcine Prdx6 gene expression, and offered clues for further investigation of Prdx6 gene function.Peroxiredoxin6 (Prdx6) is one of the peroxiredoxin (Prdxs) family members that play an important role in maintaining cell homeostasis. Our previous studies demonstrated that  Six eroxides , 4. Prdxeroxides –8.2+ independent phospholipase A2 activities that could participate in many physiological processes [2 activity, Prdx6 can regulate phospholipid metabolism. And the GSH peroxidase activity allows Prdx6 to protect cells against oxidative stress by removing reactive oxygen species (ROSs) and limiting ROS levels. ROS are generated in cells during several normal physiologic processes and will be immediately removed by antioxidants like Prdx6. When the equilibrium status between the production and elimination of reactive oxygen species is broken, oxidative stress occur [Prdx6 transcription regulation, many researchers have identified the mainly redox-active regulator of this gene in human, rat or mouse, like Nrf2 [Prdx6 is widely distributed in all major organs with high expression levels in lung, kidney, liver, eye and testes –12. It irocesses . With thss occur . Excess ss occur , 14. Prdss occur , Parkinsss occur , diabetess occur , cancer ss occur –21, catass occur . The forike Nrf2 –25, NF-κike Nrf2 , 27, Sp1ike Nrf2 , AP1 [18ike Nrf2 .Prdx6 gene was differentially expressed in longissimus dorsi between different pig breeds (data unpublished). Besides, two SNPs  were detected in the fourth exon of porcine Prdx6 gene, and were significantly associated with meat quality, especially meat tenderness [Prdx6 gene is regulated is still unclear in pigs until now. In this study, the porcine Prdx6 promoter region and its transcription start site were identified and the possible transcription factors for the Prdx6 gene regulation were also investigated. We found that the transcription factor C/EBPβ and CREB could bind to their binding sites in Prdx6 promoter and regulate the expression of target genes. This study provided the insight into the basal regulation mechanisms of porcine Prdx6 gene, and the possible signaling pathway for the participation of Prdx6 gene in the adipogenesis and myogenesis.In animal production, research has shown that the Prdx6 might be a potential protein marker for meat tenderness in bovine biopsies and samples collected shortly after slaughter . Meat tenderness . The aboTM RT Reagent Kit .The animal experimental procedures were approved by the Huazhong Agricultural University Institutional Animal Care and Use Committee, Wuhan, China. All pigs used in this study were from the Pig Farm of Huazhong agricultural university. The pigs were slaughtered after low voltage electrical stunning. Ten tissues including kidney, small intestine, stomach, longissimus dorsi, fat, brain, heart, liver, lung and spleen were collected from three Large White pigs at age of 4 month and then immediately frozen in liquid nitrogen and stored at -80°C. The DNA was extracted using the phenol–chloroform . Total RNA was extracted using the TRIzol reagent  and was reverse transcribed to cDNA using the PrimeScriptMouse myoblast cell line C2C12, mouse embryonic fibroblast-adipose like cell line 3T3-L1 and porcine kidney cell line PK15 were purchased from the Institutes for Biological Sciences Cell Resource Center, Chinese Academy of Sciences, Shanghai, China. Porcine intramuscular fat (IMF) cells were obtained according to the description in previous report .Prdx6 gene. RT-PCR was performed using the CFX96 System  in a total volume of 20 μl containing SusPr6DL-F/R or β-actin-F/R primers (-△△Ct) method was used to analyze the relative gene expression data.Using RT-PCR, ten tissues including heart, liver, spleen, lung, kidney, stomach, brain, longissimus dorsi, fat and small intestine from three 4-month-old Large White pigs were used to analyze the expression profile of  primers  and SYBR5′ RACE was performed using a Smarter™ RACE cDNA Amplification Kit , according to the manufacturer’s user manual. Total RNA of liver tissue from the same 4 month Large White pig was chosen as template for synthesis of Prdx6 cDNA. The gene specific primer (GSP) was synthesized by Sangon (China) as following sequence, GSP: 5′-GACCATCACGCTGTCTCCATTCTTCC-3′. The PCR products were purified and cloned into pMD-18T vector (Takara) and transformed into DH5α bacteria. Eighteen positive colonies were selected and commercially sequenced by Shanghai Sangon.http://www.cbrc.jp/research/db/TFSEARCH.html) and TESS (http://www.cbil.upenn.edu/tess). Multiple sequence alignment was performed using the ClustalW2.0 .Transcription factor binding sites were predicted using the TFsearch (Prdx6 promoter deletion fragments (D1-D8) were amplified with primers listed in NcoI and BglⅡ , and then subcloned into luciferase reporter vector pGL3-Basic  respectively.Ten DpnⅠ, following the manufacturer’s protocol. The pGL3-D2 vectors containing the C/EBPβ and MyoD binding sites were chosen as template. The mutation primers M-C/EBPβ-F/R and M-MyoD-F/R were listed in The C/EBPβ mutated vector and MyoD mutated vector were constructed by the PCR-based site-directed mutagenesis using the KOD-Plus-Neo and HSF1 gene (NCBI No: NM_001243819.1) was amplified with hsf1-F/R primers (KpnI and XbaI (Thermo), and cloned into pcDNA3.1 vector (Promega) using T4 DNA Ligase (Takara). Other eukaryotic expression vectors  used in over-expression experiment were from our laboratory.The HSF1 eukaryotic expression vector (pcDNA3.1-HSF1) was constructed. Using the cDNA (from pig liver tissue) as template, the coding sequence of  primers . The PCR2. Cells were seeded into 24-well plate and after 24 h incubation, each well was transfected with 1.0 μg of DNA constructs and 50 ng of Renilla luciferase reporter plasmid  using 2 μl lipofectamine 2000 . For the co-transfection groups, each well was transfected with 0.8 μg of Prdx6 promoter constructs, 0.5 μg of over-expression vector and 50 ng pRL-TK using 2 μl lipofectamine 2000. At 24–36 h after transfection, cells were lysed and assayed for the luciferase activities using dual-luciferase reporter assay system (Promega). The luciferase activity was measured using PerkinElmer 2030 Multilabel Reader (PerkinElmer) and normalized on the basis of Renilla activity.The C2C12, 3T3-L1 and PK15 cells were cultured in DMEM medium  supplemented with 10% fetal bovine serum  at 37°C and 5% CO2. After 24 h incubation, cells of each well were transfected with 4μg of over-expression vector or pcDNA3.1 empty vector using 9 μl lipofectamine 2000. C2C12 cells were seeded into 6-well plate and cultured at 37°C and 5% CO2. When reaching 70% confluence, cells of each well were transfected with 100 pmol of siRNA using 9 μl lipofectamine 2000. The C/EBPβ and CREB siRNAs . Equal amounts of protein samples were loaded into a 10% SDS-PAGE and transferred to PVDF membrane . After 1.5 h blockading within 5% nonfat dried milk, membranes were incubated overnight with primary antibodies at 4°C. After washing for 3 times, membranes were incubated with secondary antibodies for 1.5 h at room temperature. The results were visualized using the ECL Western Blotting Detection System . Primary antibodies contain anti-Prdx6 , anti-C/EBPβ , anti-CREB , anti-HSF1 and β-actin antibody . Goat anti-rabbit IgG-HRP and goat anti-mouse IgG-HRP secondary antibodies  were used in this study.Nuclear extracts were prepared from C2C12 cells or porcine intramuscular fat (IMF) cells with the Nuclear Extraction Kit . EMSA was performed according to the manufacturer’s instruction of Chemiluminescent EMSA Kit (Beyotime). The biotin-labeled double-stranded oligonucleotides (Sangon) containing predicted CREB or C/EBPβ binding sites were incubated with 10 μg nuclear extracts for 20 min at room temperature and were subjected to electrophoresis on 6% polyacrylamide gels. Then transferred to a nylon membrane and results were visualized using the ECL (Bio-Rad). In competition group, a 1-fold molar excess of unlabeled oligonucleotides was added to the reaction mixture prior to the addition of biotin labeled probe. For the supershift group, 2 μg C/EBPβ (Santa Cruz) or CREB  antibodies was added to verify the specificity of DNA-protein interaction. The EMSAs were performed according to previous reports , 34.Prdx6 promoter (ChIP assay was carried out using the ChIP assay Kit (Beyotime) following the manufacturer’s protocol. The PK15 cells cultured in 15 cm-diameter dishes were fixed in formaldehyde for 20 min at room temperature and neutralized with glycine for 5 min. After washing with cold PBS, scraped and harvested cell lysates were sonicated to produce chromatin fragments about 200–750 bp in size. An equal amount of chromatin was immunoprecipitated overnight at 4°C with 4 μg of the following antibodies: anti-CREB, anti-C/EBPβ and Normal Rabbit IgG, and added the ProteinA+G Agarose beads (Beyotime) for further 5 h incubation. Then the beads were washed, and the bound chromatin was eluted in ChIP Elution Buffer. The DNA was treated with proteinase K for 4 h at 45°C. After purified, PCR was carried out using 3 μl DNA sample with primers specific to the promoter . The firPK15 cells cultured in 10 cm-diameter dishes were collected and lysed in nondenaturing lysis buffer  supplemented with protease and phosphatase inhibitor cocktails. Well equal mass of lysate was incubated overnight with 2 μg either anti-Prdx6 or anti-IgG together with 25 μl Protein A+G Agarose beads. After centrifuge the beads were washed with 1ml lysates buffer for three or four times. Then the beads were added with 15 μl 2×SDS loading buffer and boiled for 10 minutes, then were loaded onto an SDS-PAGE gel for western blot analysis.t test. Data are expressed as mean ± S.D., and statistical significance was considered as p < 0.05. **P<0.01; *P<0.05; NS, not significant.Statistical analysis was performed using Student’s Prdx6 gene, we determined the exact TSS of Prdx6 by 5′ RACE. Firstly, the tissue expression profile of Prdx6 was analyzed by real time-PCR. The Prdx6 was expressed in almost all the tissues, especially with the highest expression in liver tissue; the kidney, heart, longissimus dorsi (LD), fat and lung tissues also showed higher expression level; while a very low expression level was detected in brain tissue , MyoD (-1068/-1063), CREB . Besides, four GC-boxes (Sp1-response element) were found at the proximal region . By alignment of multiple sequences, higher similarities of those factor binding sites were found among pig, bovine and human  to further analyze the transcriptional activity of Prdx6 promoter. The results showed that the minimal transcription activity region was located at -16bp~+15bp containing one HSF binding site  were linked to the pGL3-basic vector, and their luciferase activity was analyzed in PK15, C2C12 and 3T3-L1 cell lines . The proing site .Prdx6 gene transcription. As shown in Prdx6 transcription at mRNA level, five transcription factors were transfected into PK15 cells. The RT-PCR results revealed that C/EBPβ, CREB and HSF could improve Prdx6 transcription level, while MyoD and Sp1 had no effects  was significantly higher than that of D4 deletion (with only the second CRE site) detected in C2C12 cells (P = 0.003) (ChIP assay was preformed to confirm the binding of CREB to the trol IgG . The imm= 0.003) . No signin vitro and in vivo  were significantly higher than those of D3 deletion (no C/EBPβ binding site)  , which wPrdx6 gene by CREB and C/EBPβ have been illustrated, the interaction of Prdx6 protein with CREB and C/EBPβ proteins were still not clear in vivo. To address this, the immunoprecipitation was performed in PK15 cells. Proteins from the PK15 cells were collected and immunoprecipitated by Prdx6 antibody or IgG antibody. The results showed that compared with IgG group, C/EBPβ protein was detected in the immunoprecipitates of anti-Prdx6 group  level. Previous reports indicated that oxidative stress and the generation of ROS could regulate the adipogenesis. For example, the hypoxia-inducible factor HIF1 which can inhibit PPARγ was regulated by ROS [Prdx6 and thereby leads to the down-regulation of ROS levels. Besides, C/EBPβ could interact with Prdx6 protein (The CCAAT/enhancer-binding proteinβ (C/EBPβ) is a basic leucine zipper DNA-binding factor and could recognize a consensus DNA sequence (TTNNGNAAT) in target gene . In prese vector . Over-exy C/EBPβ . Many std by ROS . ROS havd by ROS . So it i protein , suggestPrdx6 promoter region contains two CREB binding sites , and only the first one (-601/-588) could bind with CREB and affect the Prdx6 promoter activity by EMSA, ChIP and over-expression experiments (myostatin expression and further inhibits muscle differentiation [The cAMP response element binding protein (CREB) is a cellular transcription factor that recognizes and binds to the cAMP response element  of target gene promoter . By regueriments . Previountiation . Stewartntiation . CREB-inntiation . The prentiation , 49. SinPrdx6 gene, such as Sp1, MyoD and HSF. Prdx6 promoter contains four conserved GC-boxes but no TATA-box. According to the previous study, the TATA-less promoter is likely to be regulated by GC-box and its response element Sp1 [Prdx6 promoter. Although the HSF binding site  of Prdx6 promoter was not conserved among species, it showed remarkable effect in initiating promoter activity  The porcine PK15 cells used in this paper were different from previous study, in which the human LEC cells were used and the Sp1 had been proved to be an activator of Prdx6 [Prdx6 gene regulated by Sp1 is different among species.The present study also analyzed several other potential regulators of porcine ment Sp1 . A conseactivity . Co-tranof Prdx6 . HSF1 coof Prdx6 . The posof Prdx6 . Therefocis elements of porcine Prdx6 and studied the transcription regulators affecting Prdx6 transcription. The CREB and C/EBPβ were found for the first time to transactivate Prdx6 gene expression by binding to the relevant promoter region. We also found the C/EBPβ could interact with Prdx6. A hypothetical model was proposed that Prdx6 might participate in adipogenesis or myogenesis by interacting with C/EBPβ or scavenging ROS level (Prdx6 gene function.In conclusion, we have identified the transcription start site and important OS level . The pre