BACKGROUND
the life cycle of trypanosoma cruzi involves two intermediary hosts and at least three well-defined developmental stages that are based on morphology and biological characteristics  <cit> .
the metacyclogenesis process, in which replicating epimastigotes transform into infective and non-replicating metacyclic trypomastigotes, occurs in the midgut of the invertebrate triatomine host and can be mimicked in vitro under chemically defined conditions  <cit> .
interestingly, the differentiation of epimastigotes is triggered by nutritional stress, and different sets of genes are expressed in normally growing epimastigotes and in nutritionally stressed epimastigotes  <cit> .
the mechanisms of gene expression regulation in t. cruzi present some peculiarities.
in the absence of typical rna polymerase ii promoter regions, transcription begins within the divergent strand-switch region and occurs in both directions.
as a result, dozens to hundreds of genes with unrelated functions are transcribed in the same polycistronic unit, which upon processing results in different levels of protein production  <cit> .
these features demonstrate that the regulation of gene expression in these organisms is mainly post-transcriptional and is based on mechanisms involving the localization, translation and degradation of mrnas  <cit> .
in recent years, several mechanisms have been described as possible strategies for gene expression regulation.
the translation is a very important step of gene expression regulation.
the ribosome profiling was developed in order to have a better view of the mrnas that are actually engaged to translation and how is the relevance of the translational control  <cit> .
for trypanosoma brucei and plasmodium falciparum, it has been shown that translation has a very important role in gene expression regulation in these parasites .
another mechanism is the organization of mrnas into post-transcriptional operons or regulons, where mrnas with related functions are associated with similar sets of proteins in mrnps, thereby enabling their coordinated expression .
another possible mechanism is the association of rnas into rna granules  that ultimately determine the fate of mrnas within the cell .
elongation factor 1α  binds gtp, and the gtp-bound form interacts with aminoacyl-trna and carries it to the a site of the ribosome  <cit> .
control of ef-1α protein levels is important for normal cell function, as ef-1α acts as a regulator of the cell cycle.
accordingly, ef-1α overexpression is associated with many tumor types  <cit> .
ef-1α has also been described as an actin-binding protein in several species  <cit> .
in addition, other non-canonical functions have been assigned to ef-1α because of its involvement in various cellular processes, including metabolism  <cit> , cytoskeletal organization  <cit> , oncogenic transformation  <cit> , apoptosis and anti-apoptosis  <cit> , and gene expression  <cit> .
it has also been demonstrated that ef-1α plays a role in connecting the translation machinery with the cytoskeleton, thus permitting mrna localization in chicken embryo fibroblasts.
ef-1α associates with f-actin; the resulting complex binds to beta-actin mrna and anchors it in the cellular protrusions of the fibroblast  <cit> .
ef-1α is also found in rna granules, which are transported by microtubules in neurons  <cit> .
in t. cruzi, ef-1α  <cit>  was previously shown to bind mrnas not only in polysomes but also in polysome-free fractions, both in exponentially growing epimastigotes and in parasites under nutritional stress  <cit> .
the association of ef-1α with mrna complexes that are not associated with the protein translation apparatus permitted us to better characterize ef-1α mrnps by identifying their associated transcripts.
this approach yielded further insights into the role of ef-1α mrnps in the regulation of t. cruzi gene expression.
RESULTS
t. cruzi ef-1α genomic organization and protein characteristics
t. cruzi ef-1α is organized as a multicopy gene family that includes  <dig> tandem copies on chromosome  <dig>  <cit> .
the ef-1α protein has three domains.
domain i is responsible for gtp binding, domain ii is responsible for recognizing the 3' end of the trna, and domain iii connects the aminoacyl-trna to the ribosome   <cit> .
the t. cruzi ef-1α protein has an estimated molecular weight of 43 kda, and it is highly conserved among trypanosomes and other eukaryotes, with  <dig> % identity when compared to trypanosomes and  <dig> % identity when compared to homo sapiens.fig.
<dig> scheme of the ef-1α protein from t. cruzi.
the domains are indicated; the gtp-binding domain is dark grey, the second domain is black and the third domain is light grey.
the numbers indicate the positions of the domains within the protein.
western-blot analysis of t. cruzi extracts with antisera against ef-1α and actin.
epi: exponentially growing normal epimastigotes.
stress: epimastigotes under nutritional stress.
ad 24 h: epimastigotes adhered to the substrate.
meta: metacyclic trypomastigotes.
ten μg of protein was loaded into each lane.
top panel: anti-ef-1α antibody signal .
bottom panel: anti-β-actin used for normalization .
the relative densities of the bands  are indicated.
the molecular mass standards are indicated
ef-1α protein expression is controlled throughout the parasite life cycle .
accordingly, proteomic data have shown that ef-1α protein levels decrease in metacyclic trypomastigotes compared to epimastigotes  <cit> .
all analyzed forms of ef-1α showed a granular distribution throughout the cytoplasm of the parasite, with a decrease of signal in metacyclic trypomastigotes that corroborated the proteomic and western blot results .
the immunofluorescence controls are shown in the additional file 4: figure s <dig> fig.
<dig> immunolocalization assay.
cells were incubated with anti-ef-1α antibody .
immune complexes were detected by reaction with alexa 488-labelled goat anti-rabbit antibody .
kinetoplasts and nuclei were stained with dapi, and the obtained image was merged with the ef-1α image.
bar = 10 μm
ef-1α is present in large mrnp complexes that are not associated with translation machinery
to investigate the distribution of the ef-1α protein in complexes, we analyzed its sedimentation profile in sucrose density gradients .
protein extracts of fractions from unstressed and stressed epimastigotes treated with cycloheximide or puromycin were analyzed by western blot using antisera against the ef-1α protein and the ribosomal protein s <dig> as a control .fig.
<dig> polysome fractionation.
unstressed epimastigote polysome profile.
stressed epimastigote polysome profile.
fractions were analyzed by western blotting with antiserum against ef-1α  and s <dig> ribosomal protein .
the numbers in the fractions are related to:  <dig> and  <dig> – free fraction,  <dig> – 40 s,  <dig> – 60 s, 9- <dig> monosomes to heavy polysomes
a typical polysome sedimentation profile was obtained from epimastigotes treated with cycloheximide, a drug that inhibits the function of the translation factor eef <dig>  eef <dig> acts in the translocation of peptidyl-trna from the a site to the p site of the ribosome.
thus, cycloheximide treatment blocks translation elongation and maintains the association between ribosomes and mrna.
the ef-1α protein was detected in light fractions and in fractions corresponding to as many as  <dig> ribosomes .
alternatively, for polysome dissociation analysis, parasites were treated with puromycin, a drug that causes the premature release of the polypeptide chain from the ribosome.
interestingly, even after polysome dissociation by puromycin, the distribution of ef-1α in the gradient was unchanged.
ef-1α migrated with the heaviest fractions under these conditions, indicating a translation machinery-independent association with large complexes .
nutritional stress is a trigger for t. cruzi differentiation  <cit> .
in nutritionally stressed epimastigotes treated with cycloheximide, the polysome profile was characteristic of stressed cells.
the polysomes were smaller and less abundant than in normal culture conditions.
ef-1α protein was also present over a range of lighter to heavier fractions, but much less ef-1α was detected in the heavier fractions in stressed conditions than in unstressed epimastigotes, a finding that reflects the lower translational activity in stressed conditions .
the ef-1α migration pattern in stressed parasites after puromycin treatment was similar to that of unstressed puromycin-treated epimastigotes .
the results of the polysome sedimentation analysis suggested that, in addition to its association with ribosomes during translation, ef-1α is associated with heavier complexes that co-sediment with polysomes but are not necessarily associated with the translation machinery.
this type of profile is typical of rna granules that consist of mrnp complexes that are not involved in translation.
one example is the mrnp complex involving the t. cruzi protein tcdhh <dig>  which forms large complexes associated with mrnas that co-sediment with polysomes but that are connected to the storage or degradation of associated transcripts  <cit> .
ef-1α has a possible non-canonical function that very likely involves the formation of non-translatable mrnp complexes.
mrnas associated with ef-1α mrnps
next, we aimed to investigate whether ef-1α was involved in mrnp complexes and to characterize the associated mrnas in unstressed and nutritionally stressed epimastigotes.
the ribonomic assay was performed with biological triplicates and was followed by rna-seq.
the ef-1α immunoprecipitation was confirmed by western blot showing the protein in the elution fraction .
rabbit pre-immune serum under the same conditions was used as a control.
the sequencing data were deposited in the ncbi sequence read archive  database under accession number srp <dig>  we used an rpkm  value of at least  <dig>  an fdr  ≤  <dig>  and a minimum of a four-fold change with respect to the control  as a threshold for selecting mrnas that were associated with ef-1α-containing mrnps.
using these criteria we identified  <dig> transcripts that were enriched for ef-1α mrnp by at least fourfold over the control  .
in stressed parasites,  <dig> mrnas were identified as enriched in mrnps in comparison to the control  .
the rna-seq data was validated by qrt-pcr .
we observed specific subsets of mrnas that were associated with ef-1α mrnps in unstressed or stressed epimastigotes.
some transcripts were identified in both physiological conditions, while others were specific to a given condition.
a possible functional correlation among the enriched transcripts was investigated using the gene ontology  categories of the david functional annotation and blast2go tools  <cit>  .
upon grouping the mrnas on the basis of the go terms, we noticed the enrichment of terms specific to biological processes in epimastigotes and stressed epimastigotes.
in unstressed epimastigotes, there was a slight enrichment in terms related to atp binding and metal ion binding .
however, for nutritionally stressed epimastigotes, it was possible to identify a greater number of go categories, such as single-organism metabolic processes, amino acid metabolic processes, glycolysis, glutamine metabolic processes, cobalt ion binding and iron ion binding .
the glycolysis pathway in trypanosomatids is very similar to other eukaryotes, with the exception of two main differences: some enzymes in the pathway are compartmentalized within the glycosome, and inhibitory control of hexokinase and phosphofructokinase is absent .
the compartmentalization of the enzymes seems to be important for parasite survival  <cit> .
in t. brucei, expression of phosphoglycerate kinase and triose phosphate isomerase in the cytoplasm rather than in the glycosome leads to parasite death  <cit> .
interestingly, the mrnas that encode the glycolytic enzymes enclosed in glycosomes were found to associate with ef-1α.
enolase, fructose-biphosphate aldolase, glucose-6-phosphate isomerase, aldolase- <dig> epimerase, hexokinase and alcohol dehydrogenase transcripts were also found associated with ef-1α-containing mrnps .
it is tempting to speculate that ef-1α associates with a subset of transcripts to transport them to the vicinity of glycosomes.
such transport would allow localized translation and more efficient transport into the organelle, hence minimizing the number of active enzymes in the cytoplasm.
we also analyzed the expression patterns of some transcripts that were associated with ef-1α by using the transcriptome data of the t. cruzi life cycle available at tritrypdb.org  <cit>  .
an observable pattern of expression was shared by all the transcripts; namely, they are upregulated in epimastigotes , and they all encode enzymes associated with carbohydrate and lipid metabolism .fig.
<dig> gene ontology pie chart.
the percentage indicates the enrichment for the go terms in the transcripts associated to ef-1α in epimastigotes and stressed epimastigotes
epimastigote
stressed epimastigote
fig.
<dig> mrna expression levels of the t. cruzi life cycle stages  determined by microarray analysis  <cit> .
six hybridizations were performed and consisted of three dye-swap experiments from three independent samples .
in each case, the experimental sample was from a single life-cycle stage and the control sample was an equal mixture of all four life-cycle stages  <cit> .
expression values for 2-channel microarray experiments are shown as log ratios   <cit> .
the numbers refer to different mrnas: 1- tcclb.
<dig>  ; 2- tcclb.
<dig>  ; 3- tcclb.
<dig>  ; 4- tcclb.
<dig>  ; 5- tcclb.
<dig>  ; 6- tcclb.
<dig>  ; 7- tcclb.
<dig>  ; 8- tcclb.
<dig>  ; 9- tcclb.
<dig>  ; 10- tcclb.
<dig>  ; 11- tcclb.
<dig>  ; 12- tcclb.
<dig>  ; 13- tcclb.
<dig>
the rna regulon theory, which states that mrnas encoding proteins with related functions are associated with specific mrnp complexes  <cit>  could explain the results obtained for ef-1α in t. cruzi, however further experiments need to be performed in order to validate this hypothesis for ef-1α.
one example of a regulon in t. cruzi is the tczc3h39-mrnp, which binds a specific subset of mrnas that encode the cytochrome oxidase c complex.
interestingly, this association occurs in response to stress  <cit> .
a comparison of transcripts associated with ef-1α and with tczc3h <dig> show no target overlap, indicating that the transcripts associated with each protein form distinct mrnps in the cell.
the stress triggers a global rearrangement of the metabolism in the cell, including the translation machinery.
some rnas shift from the polysomes to rna granules as p-bodies or stress granules while others remain associated to polysomes and are actively translated, for example heat shock proteins and chaperones, as a translational reprogramming necessary in order to rapidly respond to the stress condition  <cit> .
the shifts observed on the targets associated to ef1-α protein suggest that this protein might play a non-canonical role in t.cruzi through participation in distinct mrnp complexes, such as its potential role in localized translation in epimastigotes or translation repression in parasites under nutritional stress conditions .
the mrna and protein levels in a cell are not necessarily correlated.
recent data using polysome-profiling techniques allow a more precise quantification of the mrnas actively engaged in translation .
the transcripts stability is not sufficient to explain the level of the encoded proteins since this is a reflex of translation efficiency.
however, the role of rna binding proteins associated to mrnp complexes cannot be underestimated since they ultimately define the fate of a given mrna as being translated, stored or degraded.
CONCLUSIONS
specific subsets of mrnas were enriched in ef-1α-containing mrnps, indicating a non-canonical role for this protein in t. cruzi.
we are aware that ef-1α is a rather abundant and positively charged cytoplasmic protein that might bind nonspecifically to mrnas.
however, the experimental conditions used in the previous work  minimize spurious and non-specific binding between proteins and mrnas  <cit> .
sucrose gradient sedimentation analysis showed that ef-1α migrated to the heavier fractions of the gradient irrespective of whether polysomes were dissociated with puromycin, indicating its association with large mrnp complexes.
the mrnas bound to ef-1α mrnps were enriched for metabolic processes; notably, six of these transcripts encoded glycosomal proteins that are involved in the glycolysis pathway.
this result indicates that in t. cruzi, as well as in other eukaryotes, ef-1α might have additional functions other than in translation.
the results presented here allow us to hypothesize that ef-1α acts by associating with transcripts that must be localized to specific cellular sites for translation and during the stress response might be acting in mrnps that associates to translation repression.
our findings shed new light on the understanding of post-transcriptional regulation in t. cruzi.

 the life cycle of trypanosoma cruzi involves two intermediary hosts and at least three well-defined developmental stages that are based on morphology and biological characteristics. the metacyclogenesis process, in which replicating epimastigotes transform into infective and non-replicating metacyclic trypomastigotes, occurs in the midgut of the invertebrate triatomine host and can be mimicked in vitro under chemically defined conditions. interestingly, the differentiation of epimastigotes is triggered by nutritional stress, and different sets of genes are expressed in normally growing epimastigotes and in nutritionally stressed epimastigotes. the mechanisms of gene expression regulation in t. cruzi present some peculiarities. in the absence of typical rna polymerase ii promoter regions, transcription begins within the divergent strand-switch region and occurs in both directions. as a result, dozens to hundreds of genes with unrelated functions are transcribed in the same polycistronic unit, which upon processing results in different levels of protein production. these features demonstrate that the regulation of gene expression in these organisms is mainly post-transcriptional and is based on mechanisms involving the localization, translation and degradation of mrnas. in recent years, several mechanisms have been described as possible strategies for gene expression regulation. the translation is a very important step of gene expression regulation. the ribosome profiling was developed in order to have a better view of the mrnas that are actually engaged to translation and how is the relevance of the translational control. for trypanosoma brucei and plasmodium falciparum, it has been shown that translation has a very important role in gene expression regulation in these parasites. another mechanism is the organization of mrnas into post-transcriptional operons or regulons, where mrnas with related functions are associated with similar sets of proteins in mrnps, thereby enabling their coordinated expression. another possible mechanism is the association of rnas into rna granules that ultimately determine the fate of mrnas within the cell. elongation factor 1α binds gtp, and the gtp-bound form interacts with aminoacyl-trna and carries it to the a site of the ribosome. control of ef-1α protein levels is important for normal cell function, as ef-1α acts as a regulator of the cell cycle. accordingly, ef-1α overexpression is associated with many tumor types. ef-1α has also been described as an actin-binding protein in several species. in addition, other non-canonical functions have been assigned to ef-1α because of its involvement in various cellular processes, including metabolism, cytoskeletal organization, oncogenic transformation, apoptosis and anti-apoptosis, and gene expression. it has also been demonstrated that ef-1α plays a role in connecting the translation machinery with the cytoskeleton, thus permitting mrna localization in chicken embryo fibroblasts. ef-1α associates with f-actin; the resulting complex binds to beta-actin mrna and anchors it in the cellular protrusions of the fibroblast. ef-1α is also found in rna granules, which are transported by microtubules in neurons. in t. cruzi, ef-1α was previously shown to bind mrnas not only in polysomes but also in polysome-free fractions, both in exponentially growing epimastigotes and in parasites under nutritional stress. the association of ef-1α with mrna complexes that are not associated with the protein translation apparatus permitted us to better characterize ef-1α mrnps by identifying their associated transcripts. this approach yielded further insights into the role of ef-1α mrnps in the regulation of t. cruzi gene expression.  t. cruzi ef-1α genomic organization and protein characteristics t. cruzi ef-1α is organized as a multicopy gene family that includes tandem copies on chromosome. the ef-1α protein has three domains. domain i is responsible for gtp binding, domain ii is responsible for recognizing the 3' end of the trna, and domain iii connects the aminoacyl-trna to the ribosome. the t. cruzi ef-1α protein has an estimated molecular weight of 43 kda, and it is highly conserved among trypanosomes and other eukaryotes, with % identity when compared to trypanosomes and % identity when compared to homo sapiens.fig. scheme of the ef-1α protein from t. cruzi. the domains are indicated; the gtp-binding domain is dark grey, the second domain is black and the third domain is light grey. the numbers indicate the positions of the domains within the protein. western-blot analysis of t. cruzi extracts with antisera against ef-1α and actin. epi: exponentially growing normal epimastigotes. stress: epimastigotes under nutritional stress. ad 24 h: epimastigotes adhered to the substrate. meta: metacyclic trypomastigotes. ten μg of protein was loaded into each lane. top panel: anti-ef-1α antibody signal. bottom panel: anti-β-actin used for normalization. the relative densities of the bands are indicated. the molecular mass standards are indicated ef-1α protein expression is controlled throughout the parasite life cycle. accordingly, proteomic data have shown that ef-1α protein levels decrease in metacyclic trypomastigotes compared to epimastigotes. all analyzed forms of ef-1α showed a granular distribution throughout the cytoplasm of the parasite, with a decrease of signal in metacyclic trypomastigotes that corroborated the proteomic and western blot results. the immunofluorescence controls are shown in the additional file 4: figure s fig. immunolocalization assay. cells were incubated with anti-ef-1α antibody. immune complexes were detected by reaction with alexa 488-labelled goat anti-rabbit antibody. kinetoplasts and nuclei were stained with dapi, and the obtained image was merged with the ef-1α image. bar = 10 μm ef-1α is present in large mrnp complexes that are not associated with translation machinery to investigate the distribution of the ef-1α protein in complexes, we analyzed its sedimentation profile in sucrose density gradients. protein extracts of fractions from unstressed and stressed epimastigotes treated with cycloheximide or puromycin were analyzed by western blot using antisera against the ef-1α protein and the ribosomal protein s as a control.fig. polysome fractionation. unstressed epimastigote polysome profile. stressed epimastigote polysome profile. fractions were analyzed by western blotting with antiserum against ef-1α and s ribosomal protein. the numbers in the fractions are related to: and – free fraction, – 40 s, – 60 s, 9- monosomes to heavy polysomes a typical polysome sedimentation profile was obtained from epimastigotes treated with cycloheximide, a drug that inhibits the function of the translation factor eef eef acts in the translocation of peptidyl-trna from the a site to the p site of the ribosome. thus, cycloheximide treatment blocks translation elongation and maintains the association between ribosomes and mrna. the ef-1α protein was detected in light fractions and in fractions corresponding to as many as ribosomes. alternatively, for polysome dissociation analysis, parasites were treated with puromycin, a drug that causes the premature release of the polypeptide chain from the ribosome. interestingly, even after polysome dissociation by puromycin, the distribution of ef-1α in the gradient was unchanged. ef-1α migrated with the heaviest fractions under these conditions, indicating a translation machinery-independent association with large complexes. nutritional stress is a trigger for t. cruzi differentiation. in nutritionally stressed epimastigotes treated with cycloheximide, the polysome profile was characteristic of stressed cells. the polysomes were smaller and less abundant than in normal culture conditions. ef-1α protein was also present over a range of lighter to heavier fractions, but much less ef-1α was detected in the heavier fractions in stressed conditions than in unstressed epimastigotes, a finding that reflects the lower translational activity in stressed conditions. the ef-1α migration pattern in stressed parasites after puromycin treatment was similar to that of unstressed puromycin-treated epimastigotes. the results of the polysome sedimentation analysis suggested that, in addition to its association with ribosomes during translation, ef-1α is associated with heavier complexes that co-sediment with polysomes but are not necessarily associated with the translation machinery. this type of profile is typical of rna granules that consist of mrnp complexes that are not involved in translation. one example is the mrnp complex involving the t. cruzi protein tcdhh which forms large complexes associated with mrnas that co-sediment with polysomes but that are connected to the storage or degradation of associated transcripts. ef-1α has a possible non-canonical function that very likely involves the formation of non-translatable mrnp complexes. mrnas associated with ef-1α mrnps next, we aimed to investigate whether ef-1α was involved in mrnp complexes and to characterize the associated mrnas in unstressed and nutritionally stressed epimastigotes. the ribonomic assay was performed with biological triplicates and was followed by rna-seq. the ef-1α immunoprecipitation was confirmed by western blot showing the protein in the elution fraction. rabbit pre-immune serum under the same conditions was used as a control. the sequencing data were deposited in the ncbi sequence read archive database under accession number srp we used an rpkm value of at least an fdr  ≤  and a minimum of a four-fold change with respect to the control as a threshold for selecting mrnas that were associated with ef-1α-containing mrnps. using these criteria we identified transcripts that were enriched for ef-1α mrnp by at least fourfold over the control. in stressed parasites, mrnas were identified as enriched in mrnps in comparison to the control. the rna-seq data was validated by qrt-pcr. we observed specific subsets of mrnas that were associated with ef-1α mrnps in unstressed or stressed epimastigotes. some transcripts were identified in both physiological conditions, while others were specific to a given condition. a possible functional correlation among the enriched transcripts was investigated using the gene ontology categories of the david functional annotation and blast2go tools. upon grouping the mrnas on the basis of the go terms, we noticed the enrichment of terms specific to biological processes in epimastigotes and stressed epimastigotes. in unstressed epimastigotes, there was a slight enrichment in terms related to atp binding and metal ion binding. however, for nutritionally stressed epimastigotes, it was possible to identify a greater number of go categories, such as single-organism metabolic processes, amino acid metabolic processes, glycolysis, glutamine metabolic processes, cobalt ion binding and iron ion binding. the glycolysis pathway in trypanosomatids is very similar to other eukaryotes, with the exception of two main differences: some enzymes in the pathway are compartmentalized within the glycosome, and inhibitory control of hexokinase and phosphofructokinase is absent. the compartmentalization of the enzymes seems to be important for parasite survival. in t. brucei, expression of phosphoglycerate kinase and triose phosphate isomerase in the cytoplasm rather than in the glycosome leads to parasite death. interestingly, the mrnas that encode the glycolytic enzymes enclosed in glycosomes were found to associate with ef-1α. enolase, fructose-biphosphate aldolase, glucose-6-phosphate isomerase, aldolase- epimerase, hexokinase and alcohol dehydrogenase transcripts were also found associated with ef-1α-containing mrnps. it is tempting to speculate that ef-1α associates with a subset of transcripts to transport them to the vicinity of glycosomes. such transport would allow localized translation and more efficient transport into the organelle, hence minimizing the number of active enzymes in the cytoplasm. we also analyzed the expression patterns of some transcripts that were associated with ef-1α by using the transcriptome data of the t. cruzi life cycle available at tritrypdb.org. an observable pattern of expression was shared by all the transcripts; namely, they are upregulated in epimastigotes, and they all encode enzymes associated with carbohydrate and lipid metabolism.fig. gene ontology pie chart. the percentage indicates the enrichment for the go terms in the transcripts associated to ef-1α in epimastigotes and stressed epimastigotes epimastigote stressed epimastigote fig. mrna expression levels of the t. cruzi life cycle stages determined by microarray analysis. six hybridizations were performed and consisted of three dye-swap experiments from three independent samples. in each case, the experimental sample was from a single life-cycle stage and the control sample was an equal mixture of all four life-cycle stages. expression values for 2-channel microarray experiments are shown as log ratios. the numbers refer to different mrnas: 1- tcclb.; 2- tcclb.; 3- tcclb.; 4- tcclb.; 5- tcclb.; 6- tcclb.; 7- tcclb.; 8- tcclb.; 9- tcclb.; 10- tcclb.; 11- tcclb.; 12- tcclb.; 13- tcclb. the rna regulon theory, which states that mrnas encoding proteins with related functions are associated with specific mrnp complexes could explain the results obtained for ef-1α in t. cruzi, however further experiments need to be performed in order to validate this hypothesis for ef-1α. one example of a regulon in t. cruzi is the tczc3h39-mrnp, which binds a specific subset of mrnas that encode the cytochrome oxidase c complex. interestingly, this association occurs in response to stress. a comparison of transcripts associated with ef-1α and with tczc3h show no target overlap, indicating that the transcripts associated with each protein form distinct mrnps in the cell. the stress triggers a global rearrangement of the metabolism in the cell, including the translation machinery. some rnas shift from the polysomes to rna granules as p-bodies or stress granules while others remain associated to polysomes and are actively translated, for example heat shock proteins and chaperones, as a translational reprogramming necessary in order to rapidly respond to the stress condition. the shifts observed on the targets associated to ef1-α protein suggest that this protein might play a non-canonical role in t.cruzi through participation in distinct mrnp complexes, such as its potential role in localized translation in epimastigotes or translation repression in parasites under nutritional stress conditions. the mrna and protein levels in a cell are not necessarily correlated. recent data using polysome-profiling techniques allow a more precise quantification of the mrnas actively engaged in translation. the transcripts stability is not sufficient to explain the level of the encoded proteins since this is a reflex of translation efficiency. however, the role of rna binding proteins associated to mrnp complexes cannot be underestimated since they ultimately define the fate of a given mrna as being translated, stored or degraded.  specific subsets of mrnas were enriched in ef-1α-containing mrnps, indicating a non-canonical role for this protein in t. cruzi. we are aware that ef-1α is a rather abundant and positively charged cytoplasmic protein that might bind nonspecifically to mrnas. however, the experimental conditions used in the previous work minimize spurious and non-specific binding between proteins and mrnas. sucrose gradient sedimentation analysis showed that ef-1α migrated to the heavier fractions of the gradient irrespective of whether polysomes were dissociated with puromycin, indicating its association with large mrnp complexes. the mrnas bound to ef-1α mrnps were enriched for metabolic processes; notably, six of these transcripts encoded glycosomal proteins that are involved in the glycolysis pathway. this result indicates that in t. cruzi, as well as in other eukaryotes, ef-1α might have additional functions other than in translation. the results presented here allow us to hypothesize that ef-1α acts by associating with transcripts that must be localized to specific cellular sites for translation and during the stress response might be acting in mrnps that associates to translation repression. our findings shed new light on the understanding of post-transcriptional regulation in t. cruzi. 
we observed that specific subsets of mrnas were associated with ef-1α-mrnps in unstressed or stressed epimastigotes. these results indicate that in t. cruzi, as in other eukaryotes, ef-1α may play a non-canonical cellular role. following puromycin treatment, ef-1α migrated with the heaviest gradient fractions in a sucrose polysome profile, indicating that its association with large protein complexes was independent of the translation machinery. gene ontology analysis identified enrichment of gene sets involved in single-organism metabolic processes, amino acid metabolic processes, atp and metal ion binding, glycolysis, glutamine metabolic processes, and cobalt and iron ion binding. we observed the enrichment of functionally related transcripts bound to ef-1α in normal growth conditions as well as in nutritionally stressed cell indicating a potential role of ef-1α mrnp in stress response. ef-1α is expressed throughout the parasite life cycle, but it shows a slight decrease in protein levels in the metacyclic trypomastigote form. in a previous study, ef-1α was found associated with mrnp-forming mrnas in polysome-free fractions both in epimastigotes growing under normal conditions and in nutritionally stressed parasites. regulation of gene expression in trypanosomatids is mainly posttranscriptional. trypanosoma cruzigene expressionmrnpsef- alphanon-canonical functionissue-copyright-statement© the author 2015 ef-1α is conserved in many organisms, and it plays roles in many cellular processes other than translation, including rna transport, the cell cycle, and apoptosis. posttranscriptional regulation requires association between mrnas and specific proteins to form mrnp complexes. some mrnas were identified in both physiological conditions, whereas others were condition-specific. tight regulation of mrna stability and access to polysomes allows trypanosoma cruzi to adapt to different environmental conditions during its life cycle. thus, we investigated the dynamics of ef-1α in association with t. cruzi epimastigote mrnas under normal and stressed nutritional conditions.
 regulation of gene expression in trypanosomatids is mainly posttranscriptional. tight regulation of mrna stability and access to polysomes allows trypanosoma cruzi to adapt to different environmental conditions during its life cycle. posttranscriptional regulation requires association between mrnas and specific proteins to form mrnp complexes. proteins that lack a canonical rna-binding domain, such as eukaryotic elongation factor-1α, may also associate with mrnps. ef-1α is conserved in many organisms, and it plays roles in many cellular processes other than translation, including rna transport, the cell cycle, and apoptosis.  in a previous study, ef-1α was found associated with mrnp-forming mrnas in polysome-free fractions both in epimastigotes growing under normal conditions and in nutritionally stressed parasites. this finding suggested the possibility that ef-1α has a non-canonical function. thus, we investigated the dynamics of ef-1α in association with t. cruzi epimastigote mrnas under normal and stressed nutritional conditions. ef-1α is expressed throughout the parasite life cycle, but it shows a slight decrease in protein levels in the metacyclic trypomastigote form. the protein is cytoplasmically localized with a granular pattern in all forms analyzed. following puromycin treatment, ef-1α migrated with the heaviest gradient fractions in a sucrose polysome profile, indicating that its association with large protein complexes was independent of the translation machinery. we next characterized the ef-1α-associated mrnas in unstressed and stressed epimastigotes. we observed that specific subsets of mrnas were associated with ef-1α-mrnps in unstressed or stressed epimastigotes. some mrnas were identified in both physiological conditions, whereas others were condition-specific. gene ontology analysis identified enrichment of gene sets involved in single-organism metabolic processes, amino acid metabolic processes, atp and metal ion binding, glycolysis, glutamine metabolic processes, and cobalt and iron ion binding.  these results indicate that in t. cruzi, as in other eukaryotes, ef-1α may play a non-canonical cellular role. we observed the enrichment of functionally related transcripts bound to ef-1α in normal growth conditions as well as in nutritionally stressed cell indicating a potential role of ef-1α mrnp in stress response. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords trypanosoma cruzigene expressionmrnpsef- alphanon-canonical functionissue-copyright-statement© the author 2015 