BACKGROUND
rna interference  is a natural mechanism used by eukaryotes for gene silencing.
this mechanism uses small double-stranded rna, named micro  or small interfering  rnas, which are complementary to a target gene to degrade the corresponding mrna or block its translation.
the dsrna triggers the assembly of a ribonucleoprotein complex called the rna-induced silencing complex   <cit> .
the mechanism and complex composition has been best studied in drosophila melanogaster.
this is an enzymatic process that involves rnase iii-like proteins  and a dsrna binding protein   <cit> .
the second step, which leads to the cleavage of the target mrna, includes an argonaute  protein  <cit> .
mammalian cells have a single dicer protein, with a molecular weight of ~ <dig> kda.
dicer contains an atpase/rna helicase domain, a duf domain, a paz domain, two rnase iii domains and a dsrna binding domain   <cit> .
the dicer paz domain associates with the piwi domain of ago <dig>  <cit> .
dicer is responsible for cleaving the dsrna trigger  so it can be loaded into the risc.
ago <dig> is then recruited to the risc where it cleaves the target mrna or mediates translation inhibition after its association with the complementary strand from the mi/sirna  <cit> .
dicer knock-out  mice and cells are not viable indicating a major function for this protein during development and normal cell function  <cit> .
in human cells, the dsrbp that associates with dicer is the tar rna binding protein, trbp.
this protein is required for rnai function mediated by both sirnas and mirnas  <cit> , where it acts as a biosensor in the choice of dsrna loaded into the risc  <cit> .
furthermore, dicer, trbp and ago <dig> are necessary and sufficient for in vitro reconstitution of rnai activity  <cit> .
trbp <dig> and trbp <dig> are isoforms of the cellular protein trbp which was isolated by its ability to bind the human immunodeficiency - <dig> tar rna and characterized for its stimulation of the expression of the hiv long terminal repeat in human and murine cells  <cit> .
trbps have two dsrbds, a kr-helix motif within dsrbd <dig> and a medipal domain that mediates protein-protein interactions  <cit> .
trbps also bind to pkr and pact through their dsrbds and to merlin, dicer and pact through their medipal domain  <cit> .
trbps are encoded by the tarbp <dig> gene.
two adjacent promoters initiate transcription of alternative first exons for trbp <dig> and trbp <dig> mrnas and as a consequence, in comparison to trbp <dig>  trbp <dig> has  <dig> additional amino acids   <cit> .
trbps have functional activities in spermatogenesis, cell growth, oncogenesis and viral replication linked to their rna- and protein-binding abilities  <cit> .
among these, the trbp-dicer interaction and its function as part of the risc has been identified as an important component of the rnai pathway.
in this paper, we further characterize the specific domains in trbp and dicer that are required for their interaction and we analyze the consequences of this interaction in rnai function.
RESULTS
trbp medipal domain interacts with dicer through a unique domain in dicer located between the atpase and the helicase motifs
the trbp-dicer interaction was found by immunoprecipitation  with a dicer antibody followed by mass spectrometry analysis of the interacting proteins  <cit> .
we independently identified dicer in a two-hybrid screen using trbp as bait.
this screen resulted in the identification of six clones that interacted with trbp.
among these, the only clones which corresponded to an rbp mapped to dicer aa 173– <dig> or aa 267– <dig> .
because both trbp and dicer are dsrbps, and these corresponding domains often dimerize  <cit> , we also verified if the dicer dsrbd  could interact with trbp, but no interaction was found.
we next expressed the common sequence between the isolated two-hybrid clones, aa 267– <dig> called d <dig>  on pgbt <dig> and showed its interaction with trbp <dig> or trbp c  expressed in pgadgh.
this domain was further divided in two halves expressing aa 267– <dig> and 350– <dig>  neither domain interacted with trbp, indicating that the  <dig> aa d <dig> domain is the smallest trbp-interacting domain defined in this assay.
the medipal domain was previously defined as a protein-protein interacting domain in trbp  that binds merlin, dicer and pact  <cit> .
trbp interaction with pact in this domain has been mapped to aa 287– <dig> in trbp <dig>  to determine if trbp-dicer interaction can be limited to a shorter domain, we first expressed the subdomains as c <dig> and c <dig> expressing aa 228– <dig> and 340– <dig> respectively .
c <dig> did not show an interaction and the expression of c <dig> repeatedly induced yeast growth arrest indicating a cytotoxicity of this domain.
the domain was next divided into c <dig> and c <dig> expressing aa 228– <dig> and 298– <dig> respectively.
only c <dig> showed a specific interaction with d <dig>  indicating that this subdomain of the medipal is the active component for dicer binding.
the cellular distribution of dicer and trbp has not yet been fully elucidated.
we next examined their localization in the cell using immunofluorescence with antibodies directed against endogenous trbp and dicer.
interestingly, trbp localized mainly to the cytoplasm, while dicer was distributed throughout the cytoplasm and the nucleus.
we found significant colocalization between dicer and trbp in hela cells predominantly in the perinuclear space .
these data and previous results confirm that trbp and dicer can interact in a physiological environment.
the c <dig> sequence within the medipal domain of trbp is necessary and sufficient to bind dicer in mammalian cells
to determine the role of the c <dig> domain in the protein context, we used a complete trbp knock-down environment.
using murine tail embryonic fibroblasts  tarbp <dig> knock-out cells  <cit> , we evaluated the importance of the c <dig> domain in trbp on the interaction between trbp and dicer.
we expressed trbp <dig> and trbp <dig> as myc-tagged proteins as previously  <cit> , and myc-trbp1Δc <dig> and myc-trbp2Δc <dig> lacking this domain.
trbpsΔc <dig> were tested for their binding to dicer by ip .
in conditions where myc-trbp <dig> interacts with endogenous dicer, myc-trbp1Δc <dig> did not interact with dicer, whether the ip was performed with an anti-myc or an anti-dicer antibody .
results with myc-trbp <dig> and myc-trbp2Δc <dig> were similar .
we have previously excluded a binding of trbp-dicer through the dsrbds in trbp or through rna  <cit> .
the loss of trbp-dicer interaction in the absence of the c <dig> domain, but in the presence of rna, further argues against the involvement of rna in the interaction.
these combined results  indicate that the c <dig> domain is necessary and sufficient to mediate the interaction with dicer and that no other cellular protein or rna can rescue the lack of interaction between the mutant trbp and dicer in mammalian cells.
trbp, but not trbpΔc <dig> colocalizes with dicer in cells
to set up conditions to compare the cellular distribution of wild-type and truncated trbps, we first expressed trbp <dig> and trbp <dig> myc-tagged proteins in hela cells.
the cellular distribution of the myc-tagged proteins, as well as their colocalization with dicer was consistent with the endogenous localization of trbp .
importantly, we found the tagged trbp constructs co-localized with dicer in the perinuclear space.
to further define the importance of the c <dig> subdomain within trbp medipal, we expressed myc-trbp1Δc <dig> and myc-trbp2Δc <dig> in the same conditions as the wild type proteins .
with these mutants, we observed a marked change in cellular distribution as compared to the wild-type proteins.
of importance, the mutant proteins did not colocalize with dicer in the perinuclear space and for trbp2Δc <dig>  a clear relocalization to the plasma membrane was observed.
these results emphasize the importance of the c <dig> subdomain within the medipal domain as a major determinant in the cellular distribution of trbp.
to further verify the distributions of full-length trbp <dig> as compared to trbp2Δc <dig>  their localization was examined in relation to an egfp-tagged trbp <dig> construct.
egfp-trbp <dig> and myc-trbp <dig> or myc-trbp2Δc <dig> were coexpressed as previously in hela cells.
the colocalization of the egfp-tagged form of trbp <dig> with the myc-tagged form of trbp <dig> is shown by an extensive yellow coloration in the cytoplasm .
in comparison, colocalization of myc-trbp2Δc <dig> with egfp-trbp <dig> was significantly reduced, mainly due to a shift of the truncated protein to the cellular membrane.
this further supports the observation that the cellular distribution of truncated trbp <dig> significantly differs from that of the full-length protein.
trbp is required for rna interference mediated by shrna or exogenous mirna against egfp in murine cells
the requirement for trbp in the rnai pathway has been shown using cells that have been partially knocked down for trbp using sirnas  <cit> .
to determine rnai activity in a complete trbp knock-down environment, we used tarbp2-/- cells previously described  <cit>  compared to tarbp2+/- and wild type murine embryo fibroblasts .
to have a qualitative and a quantitative assay, we designed shrnas against egfp, and exogenous mirnas that have the sirna sequence against egfp in an mir <dig> backbone, as previously shown for another target  <cit>  .
although these constructs were both active in mef and tarbp2+/- cells as shown by egfp fluorescence, no significant activity could be detected in tarbp2-/- cells .
when quantified by flow cytometry, the activity of sh-egfp and mi-egfp was  <dig> and 60% in mef,  <dig> and 28% in tarbp2+/- cells but only  <dig> and 14% in tarbp2-/- cells .
to quantify rnai activity by another method, a western blot analysis was performed and showed no inhibition of egfp expression by either sh- or mirnas in tarbp2-/- cells .
trbp <dig> and trbp <dig>  but not trbpsΔc <dig> can rescue the rnai pathway in tarbp2-/- cells
to further determine if trbp could rescue rnai in tarbp2-/- murine cells, pcdna <dig> or pcdna <dig> constructs expressing trbp <dig> or trbp <dig> were cotransfected with egfp and shrna against egfp and verified for cell fluorescence and protein expression by western blot .
consistent with results in fig.
4d, pcdna <dig> empty vector did not restore the activity of the shrna in tarbp2-/- cells.
in contrast, we observed that rnai activity was recovered when trbp <dig> or, to a lesser extent, trbp <dig>  were expressed in the cells .
we next transfected the same cells with myc-trbp <dig>  myc-trbp <dig> and their Δc <dig> counterparts .
the full-length trbp proteins rescued the rnai function but the Δc <dig> mutants did not, indicating that binding of the c <dig> domain in trbp to the d <dig> domain in dicer is essential for efficient rnai activity.
trbp is required for rnai mediated by shrna or exogenous mirna against egfp in human cells
thus far, all human cell lines described express trbp, but we have shown previously that u251mg, an astrocytoma cell line, expresses an undetectable amount of trbp <dig> and a decreased amount of trbp <dig> compared to hela or jurkat cells  <cit>  .
to get more insight into the importance of trbp in human cell lines, we used the previous assays to evaluate rnai function in the relative absence of trbp.
by fluorescence analysis, we observed no decrease of egfp expression with the sh-egfp constructs and a weaker decrease of egfp with the mi-egfp constructs in the u251mg cells compared to hela cells .
quantification of this knock-down supported our initial observations.
to quantify these observations differently, we performed flow cytometry analysis and found similar differences with  <dig> and 45% decrease in hela, but only  <dig> and 32% decrease in u251mg for sh- and mi-egfp, respectively .
similarly, western blots indicated that u251mg cells poorly support rnai activity mediated by shrnas and mildly support rnai mediated by mirnas compared to hela cells .
trbp <dig> and trbp <dig>  but not trbpsΔc <dig>  can rescue the rnai pathway in human u251mg cells
to further confirm the role of trbp in rnai activity in human cells, u251mg cells were complemented with either pcdna <dig>  pcdna3-trbp <dig> or pcdna3-trbp <dig> constructs.
their ability to perform rnai was assessed as previously.
as in murine tarbp2-/- cells, trbps were able to effectively restore rnai activity in these cells , with trbp <dig> being more efficient than trbp <dig>  pcmv-myc-trbp <dig> and pcmv-myc-trbp <dig> constructs were equally effective in restoring rnai activity, however Δc <dig> mutants were unable to do so despite a similar expression .
these results confirm that the dicer-binding domain of trbp is required for efficient rnai activity in human cells.
discussion
the minimal active human risc is composed of three proteins, dicer, trbp and ago <dig>  <cit> .
within this complex, dicer and ago <dig> have rnase activities, whereas trbp is important in loading the appropriate dsrna into the complex  <cit> .
other proteins have been shown to associate with the risc, however their roles remain to be fully determined  <cit> .
we further defined the requirement for trbp in rnai using murine cells that are completely deficient for trbp .
using an shrna and an mirna targeting egfp, these cells displayed low or no rnai activity as compared to murine cells expressing trbp  , confirming that trbp is an essential protein for rnai activity in these cells.
the different approaches used to assess rnai activity yielded slightly different intensities of egfp knock-down, which can be attributed to the different sensitivities of each technique.
because no human cell has been shown to have a complete trbp deficit, we evaluated the rnai function in astrocytes, which have a weak expression of trbp.
this correlated with a low and moderate activity for the sh- and mirnas, respectively, compared to hela cells .
in this case, the difference in rnai activity between u251mg and hela cells follows the same trend in fluorescence, facs and western blotting.
the ability of u251mg cells to knock-down gfp expression using mirna-based silencing is likely explained by the residual trbp <dig> expression in this cell type.
our results demonstrate that in both murine and human cells, trbp is required for efficient rnai activity.
the interaction between dicer and trbp has been shown to be direct in vitro and in vivo with no requirement for ssrna, dsrna or another protein  <cit> .
although the site of interaction in trbp was determined to be in the c-terminal end, no distinct functional domain had been defined for dicer  <cit> .
two-hybrid assays further defined these domains as aa 267– <dig>  in dicer and aa 298– <dig> in trbp <dig>  .
d <dig> is located between the atpase and the helicase domains in dicer.
it was shown previously that atp is not required for dicer function in rnai  <cit> , suggesting that either this domain is not functional in the human protein or that it plays a role in other processes.
interestingly, although a complete knock-down of dicer is lethal, cells defective for only the atpase/helicase domain are viable with only a defect in mirna biogenesis  <cit> .
here we show that the functional importance of the atpase/helicase domain is in supporting the interaction with trbp.
these combined results suggest that the atpase/helicase domain is not functional in the human protein, and that the sole function of this region may be to recruit trbp through the d <dig> domain.
interestingly, when searched in blastp, d <dig> has no homology with any other protein in the databases, showing its unique properties.
further demonstration that the c <dig> domain is solely responsible for trbp binding to dicer is shown by the ip assays in the presence of complete cell extracts from tarbp2-/- cells .
in this assay, neither trbp1Δc <dig>  nor trbp2Δc <dig>  immunoprecipitated with dicer.
together with previous results that showed a direct interaction between trbp and dicer in the absence of rna or other proteins, these results confirm that no bridge between dicer and trbp occurs via dsrna or another protein.
trbpsΔc <dig> and dicer both have functional dsrbds that are able to bind dsrna.
the absence of interaction between the trbp mutants and dicer suggests that they do not bind the same rna at the same time.
dicer and trbp are cytoplasmic proteins but their colocalization has not been studied.
here, we show that endogenous dicer can be observed in both the nucleus and the cytoplasm, while trbp is distributed predominantly in the cytoplasm .
colocalization of the two proteins was found to occur predominantly in the perinuclear space.
overexpression of trbps did not affect their cellular distribution as immunofluorescence of myc-tagged constructs revealed similar staining to that of the endogenous protein .
as neither myc-trbp1Δc <dig>  nor myc-trbp2Δc <dig> colocalized with dicer in the perinuclear space, we conclude that binding of trbp to dicer is a major determinant of trbp cellular distribution and our results suggest that rnai takes place in the perinuclear space.
in contrast to myc-trbp1Δc <dig>  myc-trbp2Δc <dig> was partially associated to the cell membrane.
because trbp <dig> has only  <dig> more aa in its n-terminus than trbp <dig>  these aa might be responsible for the membrane association, which is only revealed when its interaction with dicer is abolished.
a closer analysis of the  <dig> aa sequence and the protein's posttranslational modifications will be necessary to elucidate this function.
this slight difference between trbp <dig> and trbp <dig> also correlates with the rescue of the rnai function mediated by shrnas against egfp .
in these assays, trbp <dig> completely rescued the shrna activity while trbp <dig> rescued it partially.
in contrast, we could not rescue shrna activity using the Δc <dig> mutants .
this demonstrates that the c <dig> domain, which mediates trbp interaction with dicer, is also required for rnai activity to occur.
these results are compatible with a role of dicer in the recognition and cleavage of precursor mirna and for trbp in loading small sirnas into the risc  <cit> .
this study provides new insights into the interaction between trbp and dicer in the risc.
these results open the way for further studies into differences between the specific roles of trbp <dig> and trbp <dig> in the rnai pathway.
CONCLUSIONS
this is the first study that maps dicer and trbp interaction domains precisely to  <dig> aa and  <dig> aa regions, respectively.
in dicer, the motif is located in a unique region between the atpase and the helicase domains.
in murine tarbp2-/- cells and in u251mg astrocytoma cells that do not support and partially support rnai, respectively, the trbp <dig> and trbp <dig> isoforms can rescue this pathway, whereas mutant forms lacking the interacting domain cannot.
trbp <dig> and trbp <dig> colocalize and immunoprecipitate with dicer whereas the mutants cannot, suggesting that trbp and dicer do not bind to the same rna at the same time during the rnai process.

 rna interference is a natural mechanism used by eukaryotes for gene silencing. this mechanism uses small double-stranded rna, named micro or small interfering rnas, which are complementary to a target gene to degrade the corresponding mrna or block its translation. the dsrna triggers the assembly of a ribonucleoprotein complex called the rna-induced silencing complex. the mechanism and complex composition has been best studied in drosophila melanogaster. this is an enzymatic process that involves rnase iii-like proteins and a dsrna binding protein. the second step, which leads to the cleavage of the target mrna, includes an argonaute protein. mammalian cells have a single dicer protein, with a molecular weight of ~ kda. dicer contains an atpase/rna helicase domain, a duf domain, a paz domain, two rnase iii domains and a dsrna binding domain. the dicer paz domain associates with the piwi domain of ago. dicer is responsible for cleaving the dsrna trigger so it can be loaded into the risc. ago is then recruited to the risc where it cleaves the target mrna or mediates translation inhibition after its association with the complementary strand from the mi/sirna. dicer knock-out mice and cells are not viable indicating a major function for this protein during development and normal cell function. in human cells, the dsrbp that associates with dicer is the tar rna binding protein, trbp. this protein is required for rnai function mediated by both sirnas and mirnas, where it acts as a biosensor in the choice of dsrna loaded into the risc. furthermore, dicer, trbp and ago are necessary and sufficient for in vitro reconstitution of rnai activity. trbp and trbp are isoforms of the cellular protein trbp which was isolated by its ability to bind the human immunodeficiency - tar rna and characterized for its stimulation of the expression of the hiv long terminal repeat in human and murine cells. trbps have two dsrbds, a kr-helix motif within dsrbd and a medipal domain that mediates protein-protein interactions. trbps also bind to pkr and pact through their dsrbds and to merlin, dicer and pact through their medipal domain. trbps are encoded by the tarbp gene. two adjacent promoters initiate transcription of alternative first exons for trbp and trbp mrnas and as a consequence, in comparison to trbp trbp has additional amino acids. trbps have functional activities in spermatogenesis, cell growth, oncogenesis and viral replication linked to their rna- and protein-binding abilities. among these, the trbp-dicer interaction and its function as part of the risc has been identified as an important component of the rnai pathway. in this paper, we further characterize the specific domains in trbp and dicer that are required for their interaction and we analyze the consequences of this interaction in rnai function.  trbp medipal domain interacts with dicer through a unique domain in dicer located between the atpase and the helicase motifs the trbp-dicer interaction was found by immunoprecipitation with a dicer antibody followed by mass spectrometry analysis of the interacting proteins. we independently identified dicer in a two-hybrid screen using trbp as bait. this screen resulted in the identification of six clones that interacted with trbp. among these, the only clones which corresponded to an rbp mapped to dicer aa 173– or aa 267–. because both trbp and dicer are dsrbps, and these corresponding domains often dimerize, we also verified if the dicer dsrbd could interact with trbp, but no interaction was found. we next expressed the common sequence between the isolated two-hybrid clones, aa 267– called d on pgbt and showed its interaction with trbp or trbp c expressed in pgadgh. this domain was further divided in two halves expressing aa 267– and 350– neither domain interacted with trbp, indicating that the aa d domain is the smallest trbp-interacting domain defined in this assay. the medipal domain was previously defined as a protein-protein interacting domain in trbp that binds merlin, dicer and pact. trbp interaction with pact in this domain has been mapped to aa 287– in trbp to determine if trbp-dicer interaction can be limited to a shorter domain, we first expressed the subdomains as c and c expressing aa 228– and 340– respectively. c did not show an interaction and the expression of c repeatedly induced yeast growth arrest indicating a cytotoxicity of this domain. the domain was next divided into c and c expressing aa 228– and 298– respectively. only c showed a specific interaction with d indicating that this subdomain of the medipal is the active component for dicer binding. the cellular distribution of dicer and trbp has not yet been fully elucidated. we next examined their localization in the cell using immunofluorescence with antibodies directed against endogenous trbp and dicer. interestingly, trbp localized mainly to the cytoplasm, while dicer was distributed throughout the cytoplasm and the nucleus. we found significant colocalization between dicer and trbp in hela cells predominantly in the perinuclear space. these data and previous results confirm that trbp and dicer can interact in a physiological environment. the c sequence within the medipal domain of trbp is necessary and sufficient to bind dicer in mammalian cells to determine the role of the c domain in the protein context, we used a complete trbp knock-down environment. using murine tail embryonic fibroblasts tarbp knock-out cells, we evaluated the importance of the c domain in trbp on the interaction between trbp and dicer. we expressed trbp and trbp as myc-tagged proteins as previously, and myc-trbp1Δc and myc-trbp2Δc lacking this domain. trbpsΔc were tested for their binding to dicer by ip. in conditions where myc-trbp interacts with endogenous dicer, myc-trbp1Δc did not interact with dicer, whether the ip was performed with an anti-myc or an anti-dicer antibody. results with myc-trbp and myc-trbp2Δc were similar. we have previously excluded a binding of trbp-dicer through the dsrbds in trbp or through rna. the loss of trbp-dicer interaction in the absence of the c domain, but in the presence of rna, further argues against the involvement of rna in the interaction. these combined results indicate that the c domain is necessary and sufficient to mediate the interaction with dicer and that no other cellular protein or rna can rescue the lack of interaction between the mutant trbp and dicer in mammalian cells. trbp, but not trbpΔc colocalizes with dicer in cells to set up conditions to compare the cellular distribution of wild-type and truncated trbps, we first expressed trbp and trbp myc-tagged proteins in hela cells. the cellular distribution of the myc-tagged proteins, as well as their colocalization with dicer was consistent with the endogenous localization of trbp. importantly, we found the tagged trbp constructs co-localized with dicer in the perinuclear space. to further define the importance of the c subdomain within trbp medipal, we expressed myc-trbp1Δc and myc-trbp2Δc in the same conditions as the wild type proteins. with these mutants, we observed a marked change in cellular distribution as compared to the wild-type proteins. of importance, the mutant proteins did not colocalize with dicer in the perinuclear space and for trbp2Δc a clear relocalization to the plasma membrane was observed. these results emphasize the importance of the c subdomain within the medipal domain as a major determinant in the cellular distribution of trbp. to further verify the distributions of full-length trbp as compared to trbp2Δc their localization was examined in relation to an egfp-tagged trbp construct. egfp-trbp and myc-trbp or myc-trbp2Δc were coexpressed as previously in hela cells. the colocalization of the egfp-tagged form of trbp with the myc-tagged form of trbp is shown by an extensive yellow coloration in the cytoplasm. in comparison, colocalization of myc-trbp2Δc with egfp-trbp was significantly reduced, mainly due to a shift of the truncated protein to the cellular membrane. this further supports the observation that the cellular distribution of truncated trbp significantly differs from that of the full-length protein. trbp is required for rna interference mediated by shrna or exogenous mirna against egfp in murine cells the requirement for trbp in the rnai pathway has been shown using cells that have been partially knocked down for trbp using sirnas. to determine rnai activity in a complete trbp knock-down environment, we used tarbp2-/- cells previously described compared to tarbp2+/- and wild type murine embryo fibroblasts. to have a qualitative and a quantitative assay, we designed shrnas against egfp, and exogenous mirnas that have the sirna sequence against egfp in an mir backbone, as previously shown for another target. although these constructs were both active in mef and tarbp2+/- cells as shown by egfp fluorescence, no significant activity could be detected in tarbp2-/- cells. when quantified by flow cytometry, the activity of sh-egfp and mi-egfp was and 60% in mef, and 28% in tarbp2+/- cells but only and 14% in tarbp2-/- cells. to quantify rnai activity by another method, a western blot analysis was performed and showed no inhibition of egfp expression by either sh- or mirnas in tarbp2-/- cells. trbp and trbp but not trbpsΔc can rescue the rnai pathway in tarbp2-/- cells to further determine if trbp could rescue rnai in tarbp2-/- murine cells, pcdna or pcdna constructs expressing trbp or trbp were cotransfected with egfp and shrna against egfp and verified for cell fluorescence and protein expression by western blot. consistent with results in fig. 4d, pcdna empty vector did not restore the activity of the shrna in tarbp2-/- cells. in contrast, we observed that rnai activity was recovered when trbp or, to a lesser extent, trbp were expressed in the cells. we next transfected the same cells with myc-trbp myc-trbp and their Δc counterparts. the full-length trbp proteins rescued the rnai function but the Δc mutants did not, indicating that binding of the c domain in trbp to the d domain in dicer is essential for efficient rnai activity. trbp is required for rnai mediated by shrna or exogenous mirna against egfp in human cells thus far, all human cell lines described express trbp, but we have shown previously that u251mg, an astrocytoma cell line, expresses an undetectable amount of trbp and a decreased amount of trbp compared to hela or jurkat cells. to get more insight into the importance of trbp in human cell lines, we used the previous assays to evaluate rnai function in the relative absence of trbp. by fluorescence analysis, we observed no decrease of egfp expression with the sh-egfp constructs and a weaker decrease of egfp with the mi-egfp constructs in the u251mg cells compared to hela cells. quantification of this knock-down supported our initial observations. to quantify these observations differently, we performed flow cytometry analysis and found similar differences with and 45% decrease in hela, but only and 32% decrease in u251mg for sh- and mi-egfp, respectively. similarly, western blots indicated that u251mg cells poorly support rnai activity mediated by shrnas and mildly support rnai mediated by mirnas compared to hela cells. trbp and trbp but not trbpsΔc can rescue the rnai pathway in human u251mg cells to further confirm the role of trbp in rnai activity in human cells, u251mg cells were complemented with either pcdna pcdna3-trbp or pcdna3-trbp constructs. their ability to perform rnai was assessed as previously. as in murine tarbp2-/- cells, trbps were able to effectively restore rnai activity in these cells, with trbp being more efficient than trbp pcmv-myc-trbp and pcmv-myc-trbp constructs were equally effective in restoring rnai activity, however Δc mutants were unable to do so despite a similar expression. these results confirm that the dicer-binding domain of trbp is required for efficient rnai activity in human cells. discussion the minimal active human risc is composed of three proteins, dicer, trbp and ago. within this complex, dicer and ago have rnase activities, whereas trbp is important in loading the appropriate dsrna into the complex. other proteins have been shown to associate with the risc, however their roles remain to be fully determined. we further defined the requirement for trbp in rnai using murine cells that are completely deficient for trbp. using an shrna and an mirna targeting egfp, these cells displayed low or no rnai activity as compared to murine cells expressing trbp, confirming that trbp is an essential protein for rnai activity in these cells. the different approaches used to assess rnai activity yielded slightly different intensities of egfp knock-down, which can be attributed to the different sensitivities of each technique. because no human cell has been shown to have a complete trbp deficit, we evaluated the rnai function in astrocytes, which have a weak expression of trbp. this correlated with a low and moderate activity for the sh- and mirnas, respectively, compared to hela cells. in this case, the difference in rnai activity between u251mg and hela cells follows the same trend in fluorescence, facs and western blotting. the ability of u251mg cells to knock-down gfp expression using mirna-based silencing is likely explained by the residual trbp expression in this cell type. our results demonstrate that in both murine and human cells, trbp is required for efficient rnai activity. the interaction between dicer and trbp has been shown to be direct in vitro and in vivo with no requirement for ssrna, dsrna or another protein. although the site of interaction in trbp was determined to be in the c-terminal end, no distinct functional domain had been defined for dicer. two-hybrid assays further defined these domains as aa 267– in dicer and aa 298– in trbp. d is located between the atpase and the helicase domains in dicer. it was shown previously that atp is not required for dicer function in rnai, suggesting that either this domain is not functional in the human protein or that it plays a role in other processes. interestingly, although a complete knock-down of dicer is lethal, cells defective for only the atpase/helicase domain are viable with only a defect in mirna biogenesis. here we show that the functional importance of the atpase/helicase domain is in supporting the interaction with trbp. these combined results suggest that the atpase/helicase domain is not functional in the human protein, and that the sole function of this region may be to recruit trbp through the d domain. interestingly, when searched in blastp, d has no homology with any other protein in the databases, showing its unique properties. further demonstration that the c domain is solely responsible for trbp binding to dicer is shown by the ip assays in the presence of complete cell extracts from tarbp2-/- cells. in this assay, neither trbp1Δc nor trbp2Δc immunoprecipitated with dicer. together with previous results that showed a direct interaction between trbp and dicer in the absence of rna or other proteins, these results confirm that no bridge between dicer and trbp occurs via dsrna or another protein. trbpsΔc and dicer both have functional dsrbds that are able to bind dsrna. the absence of interaction between the trbp mutants and dicer suggests that they do not bind the same rna at the same time. dicer and trbp are cytoplasmic proteins but their colocalization has not been studied. here, we show that endogenous dicer can be observed in both the nucleus and the cytoplasm, while trbp is distributed predominantly in the cytoplasm. colocalization of the two proteins was found to occur predominantly in the perinuclear space. overexpression of trbps did not affect their cellular distribution as immunofluorescence of myc-tagged constructs revealed similar staining to that of the endogenous protein. as neither myc-trbp1Δc nor myc-trbp2Δc colocalized with dicer in the perinuclear space, we conclude that binding of trbp to dicer is a major determinant of trbp cellular distribution and our results suggest that rnai takes place in the perinuclear space. in contrast to myc-trbp1Δc myc-trbp2Δc was partially associated to the cell membrane. because trbp has only more aa in its n-terminus than trbp these aa might be responsible for the membrane association, which is only revealed when its interaction with dicer is abolished. a closer analysis of the aa sequence and the protein's posttranslational modifications will be necessary to elucidate this function. this slight difference between trbp and trbp also correlates with the rescue of the rnai function mediated by shrnas against egfp. in these assays, trbp completely rescued the shrna activity while trbp rescued it partially. in contrast, we could not rescue shrna activity using the Δc mutants. this demonstrates that the c domain, which mediates trbp interaction with dicer, is also required for rnai activity to occur. these results are compatible with a role of dicer in the recognition and cleavage of precursor mirna and for trbp in loading small sirnas into the risc. this study provides new insights into the interaction between trbp and dicer in the risc. these results open the way for further studies into differences between the specific roles of trbp and trbp in the rnai pathway.  this is the first study that maps dicer and trbp interaction domains precisely to aa and aa regions, respectively. in dicer, the motif is located in a unique region between the atpase and the helicase domains. in murine tarbp2-/- cells and in u251mg astrocytoma cells that do not support and partially support rnai, respectively, the trbp and trbp isoforms can rescue this pathway, whereas mutant forms lacking the interacting domain cannot. trbp and trbp colocalize and immunoprecipitate with dicer whereas the mutants cannot, suggesting that trbp and dicer do not bind to the same rna at the same time during the rnai process. 
the binding site in trbp is a aa domain, called c located at the c-terminal end of trbp. tarbp2-/- cells, which do not express trbp, do not support rna interference mediated by short hairpin or micro rnas against egfp. we show that the trbp binding site in dicer is a amino acid region located between the atpase and the helicase domains. both trbps, but not trbpsΔc were able to rescue rnai function. in human cells with low rnai activity, addition of trbp or but not trbpsΔc rescued rnai function. trbps, but not trbpsΔc rescue rnai activity in rnai-compromised cells, indicating that the binding of dicer to trbp is critical for rnai function. the c domain is therefore necessary to bind dicer, irrespective of the presence of rna.
 dicer, ago and trbp are the minimum components of the human rna-induced silencing complex. while dicer and ago are rnases, trbp is the double-stranded rna binding protein that loads small interfering rna into the risc. trbp binds directly to dicer through its c-terminal domain.  we show that the trbp binding site in dicer is a amino acid region located between the atpase and the helicase domains. the binding site in trbp is a aa domain, called c located at the c-terminal end of trbp. the trbp and trbp isoforms, but not trbps lacking the c site, co-immunoprecipitated with dicer. the c domain is therefore necessary to bind dicer, irrespective of the presence of rna. immunofluorescence shows that while full-length trbps colocalize with dicer, trbpsΔc do not. tarbp2-/- cells, which do not express trbp, do not support rna interference mediated by short hairpin or micro rnas against egfp. both trbps, but not trbpsΔc were able to rescue rnai function. in human cells with low rnai activity, addition of trbp or but not trbpsΔc rescued rnai function.  the mapping of the interaction sites between trbp and dicer show unique domains that are required for their binding. since trbpsΔc do not interact or colocalize with dicer, we suggest that trbp and dicer, both dsrbps, do not interact through bound dsrna. trbps, but not trbpsΔc rescue rnai activity in rnai-compromised cells, indicating that the binding of dicer to trbp is critical for rnai function. 