BACKGROUND
micrornas  are small  noncoding rnas that associate with argonaute proteins in ribonucleoprotein complexes   <cit> .
micrornas play diverse regulatory roles in development and physiological cellular functions  <cit> .
in addition, mirnas are involved in a wide spectrum of human diseases including cancer, cardiovascular and autoimmune inflammatory conditions  <cit> .
post-transcriptional nuclear processing of primary mirna transcripts  by the rnase iii enzyme drosha and its rna-binding partner, digeorge syndrome critical region gene  <dig> protein   <cit> , generates ~ 65- <dig> nucleotide  hairpin-structured mirna precursors .
pre-mirnas are subsequently bound to the ran-gtp protein exportin- <dig> and are transported to the cytoplasm  <cit>  where they undergo processing by the rnase iii enzyme dicer into ~  <dig> nt rna duplexes  <cit> .
tar rna binding protein  facilitates dicer processing of pre-mirnas  <cit> .
ago proteins act as rna chaperones facilitating unwinding of rna duplexes and loading of single-stranded mirnas into ago complexes  <cit> .
the mechanisms involved in mirna strand selection in mammals are poorly understood.
we previously reported that endogenous ago2-pre-mirna  complexes are detected in cytoplasmic and nuclear extracts of human cells, and that pre-mirnps are enriched in dicer-null cells  <cit> .
we showed that recombinant pre-mirnps function as riscs in vitro.
this finding complicated our in vitro risc reconstitution studies since recombinant ago <dig> in complex with pre-mirnas cleaves target rnas bypassing the requirement for dicer activity.
in this study, we identified rna targets that are not cleaved by ago2:pre-mirna complexes.
we demonstrated that dicer and ago <dig> are sufficient for in vitro recapitulation of the cytoplasmic mirnp assembly and that trbp is dispensable.
we also showed that select ago2:pre-mirna complexes are active against 5'-as well as 3'-arm rna targets, suggesting that ago <dig> binds primarily to the 5'- and alternatively, to the 3'-end of select pre-mirnas.
RESULTS
recombinant ago2:pre-mir-24- <dig> complex directs cleavage of a 5'- but not of a 3'-arm target
we previously reported that mammalian ago <dig> directly binds to pre-mirnas and forms active, non-canonical, risc  in vitro  <cit> .
ago <dig> can only use the hairpin-like pre-mirnas as guides for target cleavage when spontaneous or ago2-mediated dissociation of the hairpin arms permits target annealing to pre-mirnas.
interestingly, two recent studies demonstrated rna chaperone activity of human ago proteins against rna duplexes  <cit> .
although the function and biological significance of endogenous pre-mirnps is unclear, non-canonical risc activity of pre-mirnps complicates studies of risc reconstitution.
to bypass this alternative in vitro pathway, we first asked whether non-canonical risc is also active against targets complementary to the 3'-arm of pre-mirnas.
to test our hypothesis, we preloaded purified gst-ago <dig> with pre-mir-24- <dig> in near stoichiometric amounts and added a 5'-p32-radiolabeled target complementary to either the 5'- or the 3'-arm of pre-mir-24- <dig>  as expected, efficient 5'-arm target cleavage was detected in the absence of dicer, whereas 3'-arm target cleavage was not observed .
this suggests preferential activity of pre-mir-24-1:ago <dig> complex against targets complementary to the proximal end of pre-mir-24- <dig>
ago <dig> and dicer are sufficient for in vitro recapitulation of cytoplasmic steps of mirna biogenesis
although in vitro reconstitution of mammalian risc with recombinant ago <dig>  dicer and trbp has been previously reported, it is unclear whether in this study risc was formed by ago loaded with a dicer-generated mirna or unprocessed pre-mirna  <cit> .
having demonstrated inactivity of ago2-pre-mir-24- <dig> against a 3'-arm target, we were in a position to test risc loading, by circumventing the non-canonical pathway.
ago <dig> was incubated with 5'-p-pre-mir-24- <dig> and dicer, but not trbp, then provided with a 5'-radiolabeled 3'-arm rna target .
reaction products were analyzed by urea-page.
efficient cleavage of the radiolabeled ta- <dig> was observed , indicating trbp-independent loading of the 3'-arm product of pre-mir-24- <dig> into ago <dig>  inclusion of trbp to the reconstitution reactions did not affect risc activity .
these results indicate that ago <dig> and dicer alone were sufficient for canonical risc loading in vitro.
in order to demonstrate that the 5'-arm product of pre-mir-24- <dig> was also loaded into ago <dig>  in addition to the 3'-arm product, the same reactions were repeated using 5'-radiolabeled pre-mir-24- <dig> and unlabeled rna target.
labeled rnas were analyzed by denaturing and native page .
as expected, dicer, but not ago <dig> or trbp, processed pre-mir-24- <dig> to ~ <dig> nucleotide mir-24-1:mir-24-1* duplex, which was then dissociated by ago <dig> and formed canonical risc loaded with either mir-24- <dig> or mir-24-1* .
although when these reactions were repeated with labeled pre-mir-24- <dig> and unlabeled target rna, trbp appeared to have a mild inhibitory effect on dicer , such effect was not observed when pre-mir- <dig> was used instead of pre-mir-24- <dig>  or when risc activity was tested .
irrespective of regulatory activity, the presence of trbp was also not essential for pre-mir-30a risc loading reconstitution .
together, our results indicate that recombinant ago <dig> and dicer are sufficient to recapitulate risc assembly in vitro.
we demonstrated unwinding of dicer-generated mirna duplexes in the presence of ago <dig> and risc loading with single-stranded mirnas deriving either from the 3'-arm , or the 5'- arm of pre-mir-24- <dig> .
trbp is not essential for pre-mirna processing by dicer, unwinding of the rna duplex or loading of mirna or mirna* into ago <dig>
risc activity of select pre-mirnps is guided against 5'-and 3'-arm targets
we tested several pre-mirnas to demonstrate that a non-canonical dicer-free ago pre-mirnp is preferentially active against a 5'-arm target.
surprisingly, we observed 3'-arm target cleavage by select pre-mirna-guided riscs.
pre-mir-24- <dig> and pre-mir-30a consistently demonstrated 5'- but not 3'-arm target cleavage, while pre-mir-138- <dig> and pre-mir-103- <dig> guided cleavage of targets complementary to both arms.
in the case of pre-mir-138- <dig>  the 17nt 5'- cleavage product of target tb- <dig> , corresponds, as expected, to cleavage across the nucleotide in position  <dig> from the 5'-end of pre-mir-138- <dig> .
the 5'- product of the 3'-arm target tb- <dig>  corresponds to cleavage across the nucleotide in position  <dig> from the 3'-end of pre-mir-138- <dig> .
an additional 18nt 5'-product is seen, indicating cleavage across position  <dig> from the 3'-end.
these findings suggest that, in vitro, in addition to binding to the 5'-phosphorylated end, recombinant ago <dig> likely binds to the 3'-unphosphorylated end of pre-mir-138- <dig>  imprecise cleavage of the 3'-arm target resulting in two different size products suggests weak ago <dig> binding to the unphosphorylated base of the 3'-end.
an alternative explanation of these findings would be that ago <dig> processes pre-mirnas and is then loaded with guide rnas deriving from their 3'- arm.
however, we have previously  <cit>  and currently demonstrated that such processing is not detected in vitro  at least with the conditions and pre-mirnas we tested using purified, dicer-free, recombinant ago <dig>  ruling out this alternative scenario.
pre-mir-103- <dig>  like pre-mir-138- <dig>  directed cleavage of targets complementary to its 5'- and 3'- arms .
imprecise cleavage of the 3'-arm target was noted again, as with pre-mir-138- <dig>  in pre-mir-103-2-guided cleavage, addition of recombinant trbp was not crucial for the use of the 5'- or 3'- pre-mirna arm by ago <dig> .
when ago <dig> was loaded with a 73-nucleotide single-stranded, unstructured rna, both 5'- and 3'- end targets were cleaved, although 3'-end target cleavage was significantly less efficient .
this again suggests that ago <dig> binds primarily, as expected, to the 5'-phosphorylated base of the guide rna and can also bind to a small extent to the 3'- unphosphorylated base of the single-stranded guide rna.
endogenous ago2-pre-mirna complexes are detected in nuclear and cytoplasmic extracts of mammalian cells  <cit> .
we have previously shown that in dicer-null mouse embryonic fibroblasts , endogenous agos are loaded with unprocessed pre-mirnas  <cit> .
to test whether ago <dig> binds to the 3'- end of endogenous pre-mirnas, we immunoprecipitated ago complexes from dicer-/- mefs using the 2a <dig> antibody <cit>  and performed in vitro risc assays.
in this inducible cell line a small amount of let-7a is detected  even when dicer is undetectable .
however, let-7a*, which normally originates from the 3'-arm of pre-let-7a, is not detected in total rna from dicer +/- or dicer-/- mefs .
to test the risc activity of endogenous pre-let7-a rnps, we provided rna targets complementary to the 5'- and 3'-arm of pre-let-7a.
in addition to the 5'- arm cleavage, a 3'-arm target cleavage product was observed, likely generated by risc activity of pre-let-7a rnps, rather than by canonical, let-7a* mirnps , suggesting that endogenous pre-let-7a associates to ago <dig> via either its 5'-phosphorylated or its 3'- unphosphorylated end.
discussion
most endogenous pre-mirnas are normally processed by dicer and consequently functional mirnps are more abundant than pre-mirnps  <cit> .
we have previously shown that canonical pre-mirnas associate with ago <dig> and that in vitro, dicer-free ago <dig> does not process pre-mirnas into mature mirnas or any intermediate products  <cit> .
it was subsequently shown that ago <dig> binds to pre-mir- <dig> and mediates its processing via a non-canonical, dicer-independent pathway  <cit> .
unlike most mammalian mirnas, pre-mir- <dig> has a unique stem structure, with a perfect sense:antisense complementarity, resembling a 17nt sirna duplex.
our present and previous studies  <cit>  indicate that pre-mirnas with stem bulges are not processed by ago <dig>  but are used as guides for risc activity in vitro.
our unexpected findings, also reported by yoda et al  <cit> , complicate in vitro dissection of risc biogenesis and function.
in this study, we identified pre-mirnps inactive against 3'-arm targets and this finding allowed us to demonstrate that dicer and ago <dig> are sufficient to process pre-mirnas, unwind mirna duplexes and load single-stranded mirnas into ago <dig>  trbp was dispensable for all three steps in vitro, arguing against an essential role for this protein in risc reconstitution or mirna strand selection but not excluding a regulatory function.
several studies have shown regulation of mirna biogenesis by auxiliary factors .
it has been previously shown that trbp regulates pre-mirna processing by dicer  <cit> ; furthermore, it has been proposed that trbp is necessary to recruit ago <dig> to the dicer/substrate complexes  <cit> .
earlier reports suggested that trbp in complex with ago and dicer is required for risc loading reconstitution in vitro  <cit> .
our studies indicate that trbp is not essential for this process.
apart from clarifying the minimal factors required for mammalian risc loading, our results suggest that pre-mirnas bind to ago <dig> via either their 5'- or 3'- end.
crystal structure studies have shown that the 3'-  <cit>  and the 5'- end  <cit>  of mirnas bind to argonaute proteins and that there is a strong preference for u or a as a first 5'- end base  <cit> .
it is likely that pre-mirnas are anchored within ago <dig> in a similar fashion.
we tested four differently structured pre-mirnas and the size of the 5'-arm target product consistently corresponded to the expected cleavage across the nucleotide in position  <dig> from the 5'-end of the pre-mirna.
most of the 3'-arm target products corresponded also to cleavage across the 10th nucleotide from the 3'-end.
however, additional products were detected suggesting imprecise cleavage, likely due to weak ago <dig> binding to the 3'-unphosphorylated pre-mirna end.
it is unlikely that these rna products represent degradation artifacts, as all the radiolabeled rna oligos were gel-purified.
CONCLUSIONS
our results indicate that ago <dig> and dicer are sufficient for processing and loading of mirnas into risc.
furthermore, our studies suggest that ago <dig> binds primarily to the 5'-and alternatively, to the 3'-end of select pre-mirnas.
results from our experiments using immunopurified pre-mirnps from dicer-null cells also support this alternative mode of endogenous pre-mirna binding to ago <dig>  endogenous pre-mirnps accumulate in dicer-deficient cells, likely because the secondary structure of pre-mirnas and/or their binding by ago <dig> protects them from degradation; normally, ago <dig> containing pre-mirnps are not abundant  <cit> .
since it is unlikely that mirnps and pre-mirnps exert redundant regulatory functions on gene expression, our in vitro findings should be cautiously interpreted, until the function of pre-mirnps and biological significance of their risc activity are elucidated.

 micrornas are small noncoding rnas that associate with argonaute proteins in ribonucleoprotein complexes. micrornas play diverse regulatory roles in development and physiological cellular functions. in addition, mirnas are involved in a wide spectrum of human diseases including cancer, cardiovascular and autoimmune inflammatory conditions. post-transcriptional nuclear processing of primary mirna transcripts by the rnase iii enzyme drosha and its rna-binding partner, digeorge syndrome critical region gene protein, generates ~ 65- nucleotide hairpin-structured mirna precursors. pre-mirnas are subsequently bound to the ran-gtp protein exportin- and are transported to the cytoplasm where they undergo processing by the rnase iii enzyme dicer into ~ nt rna duplexes. tar rna binding protein facilitates dicer processing of pre-mirnas. ago proteins act as rna chaperones facilitating unwinding of rna duplexes and loading of single-stranded mirnas into ago complexes. the mechanisms involved in mirna strand selection in mammals are poorly understood. we previously reported that endogenous ago2-pre-mirna complexes are detected in cytoplasmic and nuclear extracts of human cells, and that pre-mirnps are enriched in dicer-null cells. we showed that recombinant pre-mirnps function as riscs in vitro. this finding complicated our in vitro risc reconstitution studies since recombinant ago in complex with pre-mirnas cleaves target rnas bypassing the requirement for dicer activity. in this study, we identified rna targets that are not cleaved by ago2:pre-mirna complexes. we demonstrated that dicer and ago are sufficient for in vitro recapitulation of the cytoplasmic mirnp assembly and that trbp is dispensable. we also showed that select ago2:pre-mirna complexes are active against 5'-as well as 3'-arm rna targets, suggesting that ago binds primarily to the 5'- and alternatively, to the 3'-end of select pre-mirnas.  recombinant ago2:pre-mir-24- complex directs cleavage of a 5'- but not of a 3'-arm target we previously reported that mammalian ago directly binds to pre-mirnas and forms active, non-canonical, risc in vitro. ago can only use the hairpin-like pre-mirnas as guides for target cleavage when spontaneous or ago2-mediated dissociation of the hairpin arms permits target annealing to pre-mirnas. interestingly, two recent studies demonstrated rna chaperone activity of human ago proteins against rna duplexes. although the function and biological significance of endogenous pre-mirnps is unclear, non-canonical risc activity of pre-mirnps complicates studies of risc reconstitution. to bypass this alternative in vitro pathway, we first asked whether non-canonical risc is also active against targets complementary to the 3'-arm of pre-mirnas. to test our hypothesis, we preloaded purified gst-ago with pre-mir-24- in near stoichiometric amounts and added a 5'-p32-radiolabeled target complementary to either the 5'- or the 3'-arm of pre-mir-24- as expected, efficient 5'-arm target cleavage was detected in the absence of dicer, whereas 3'-arm target cleavage was not observed. this suggests preferential activity of pre-mir-24-1:ago complex against targets complementary to the proximal end of pre-mir-24- ago and dicer are sufficient for in vitro recapitulation of cytoplasmic steps of mirna biogenesis although in vitro reconstitution of mammalian risc with recombinant ago dicer and trbp has been previously reported, it is unclear whether in this study risc was formed by ago loaded with a dicer-generated mirna or unprocessed pre-mirna. having demonstrated inactivity of ago2-pre-mir-24- against a 3'-arm target, we were in a position to test risc loading, by circumventing the non-canonical pathway. ago was incubated with 5'-p-pre-mir-24- and dicer, but not trbp, then provided with a 5'-radiolabeled 3'-arm rna target. reaction products were analyzed by urea-page. efficient cleavage of the radiolabeled ta- was observed, indicating trbp-independent loading of the 3'-arm product of pre-mir-24- into ago inclusion of trbp to the reconstitution reactions did not affect risc activity. these results indicate that ago and dicer alone were sufficient for canonical risc loading in vitro. in order to demonstrate that the 5'-arm product of pre-mir-24- was also loaded into ago in addition to the 3'-arm product, the same reactions were repeated using 5'-radiolabeled pre-mir-24- and unlabeled rna target. labeled rnas were analyzed by denaturing and native page. as expected, dicer, but not ago or trbp, processed pre-mir-24- to ~ nucleotide mir-24-1:mir-24-1* duplex, which was then dissociated by ago and formed canonical risc loaded with either mir-24- or mir-24-1*. although when these reactions were repeated with labeled pre-mir-24- and unlabeled target rna, trbp appeared to have a mild inhibitory effect on dicer, such effect was not observed when pre-mir- was used instead of pre-mir-24- or when risc activity was tested. irrespective of regulatory activity, the presence of trbp was also not essential for pre-mir-30a risc loading reconstitution. together, our results indicate that recombinant ago and dicer are sufficient to recapitulate risc assembly in vitro. we demonstrated unwinding of dicer-generated mirna duplexes in the presence of ago and risc loading with single-stranded mirnas deriving either from the 3'-arm, or the 5'- arm of pre-mir-24-. trbp is not essential for pre-mirna processing by dicer, unwinding of the rna duplex or loading of mirna or mirna* into ago risc activity of select pre-mirnps is guided against 5'-and 3'-arm targets we tested several pre-mirnas to demonstrate that a non-canonical dicer-free ago pre-mirnp is preferentially active against a 5'-arm target. surprisingly, we observed 3'-arm target cleavage by select pre-mirna-guided riscs. pre-mir-24- and pre-mir-30a consistently demonstrated 5'- but not 3'-arm target cleavage, while pre-mir-138- and pre-mir-103- guided cleavage of targets complementary to both arms. in the case of pre-mir-138- the 17nt 5'- cleavage product of target tb-, corresponds, as expected, to cleavage across the nucleotide in position from the 5'-end of pre-mir-138-. the 5'- product of the 3'-arm target tb- corresponds to cleavage across the nucleotide in position from the 3'-end of pre-mir-138-. an additional 18nt 5'-product is seen, indicating cleavage across position from the 3'-end. these findings suggest that, in vitro, in addition to binding to the 5'-phosphorylated end, recombinant ago likely binds to the 3'-unphosphorylated end of pre-mir-138- imprecise cleavage of the 3'-arm target resulting in two different size products suggests weak ago binding to the unphosphorylated base of the 3'-end. an alternative explanation of these findings would be that ago processes pre-mirnas and is then loaded with guide rnas deriving from their 3'- arm. however, we have previously and currently demonstrated that such processing is not detected in vitro at least with the conditions and pre-mirnas we tested using purified, dicer-free, recombinant ago ruling out this alternative scenario. pre-mir-103- like pre-mir-138- directed cleavage of targets complementary to its 5'- and 3'- arms. imprecise cleavage of the 3'-arm target was noted again, as with pre-mir-138- in pre-mir-103-2-guided cleavage, addition of recombinant trbp was not crucial for the use of the 5'- or 3'- pre-mirna arm by ago. when ago was loaded with a 73-nucleotide single-stranded, unstructured rna, both 5'- and 3'- end targets were cleaved, although 3'-end target cleavage was significantly less efficient. this again suggests that ago binds primarily, as expected, to the 5'-phosphorylated base of the guide rna and can also bind to a small extent to the 3'- unphosphorylated base of the single-stranded guide rna. endogenous ago2-pre-mirna complexes are detected in nuclear and cytoplasmic extracts of mammalian cells. we have previously shown that in dicer-null mouse embryonic fibroblasts, endogenous agos are loaded with unprocessed pre-mirnas. to test whether ago binds to the 3'- end of endogenous pre-mirnas, we immunoprecipitated ago complexes from dicer-/- mefs using the 2a antibody and performed in vitro risc assays. in this inducible cell line a small amount of let-7a is detected even when dicer is undetectable. however, let-7a*, which normally originates from the 3'-arm of pre-let-7a, is not detected in total rna from dicer +/- or dicer-/- mefs. to test the risc activity of endogenous pre-let7-a rnps, we provided rna targets complementary to the 5'- and 3'-arm of pre-let-7a. in addition to the 5'- arm cleavage, a 3'-arm target cleavage product was observed, likely generated by risc activity of pre-let-7a rnps, rather than by canonical, let-7a* mirnps, suggesting that endogenous pre-let-7a associates to ago via either its 5'-phosphorylated or its 3'- unphosphorylated end. discussion most endogenous pre-mirnas are normally processed by dicer and consequently functional mirnps are more abundant than pre-mirnps. we have previously shown that canonical pre-mirnas associate with ago and that in vitro, dicer-free ago does not process pre-mirnas into mature mirnas or any intermediate products. it was subsequently shown that ago binds to pre-mir- and mediates its processing via a non-canonical, dicer-independent pathway. unlike most mammalian mirnas, pre-mir- has a unique stem structure, with a perfect sense:antisense complementarity, resembling a 17nt sirna duplex. our present and previous studies indicate that pre-mirnas with stem bulges are not processed by ago but are used as guides for risc activity in vitro. our unexpected findings, also reported by yoda et al, complicate in vitro dissection of risc biogenesis and function. in this study, we identified pre-mirnps inactive against 3'-arm targets and this finding allowed us to demonstrate that dicer and ago are sufficient to process pre-mirnas, unwind mirna duplexes and load single-stranded mirnas into ago trbp was dispensable for all three steps in vitro, arguing against an essential role for this protein in risc reconstitution or mirna strand selection but not excluding a regulatory function. several studies have shown regulation of mirna biogenesis by auxiliary factors. it has been previously shown that trbp regulates pre-mirna processing by dicer; furthermore, it has been proposed that trbp is necessary to recruit ago to the dicer/substrate complexes. earlier reports suggested that trbp in complex with ago and dicer is required for risc loading reconstitution in vitro. our studies indicate that trbp is not essential for this process. apart from clarifying the minimal factors required for mammalian risc loading, our results suggest that pre-mirnas bind to ago via either their 5'- or 3'- end. crystal structure studies have shown that the 3'- and the 5'- end of mirnas bind to argonaute proteins and that there is a strong preference for u or a as a first 5'- end base. it is likely that pre-mirnas are anchored within ago in a similar fashion. we tested four differently structured pre-mirnas and the size of the 5'-arm target product consistently corresponded to the expected cleavage across the nucleotide in position from the 5'-end of the pre-mirna. most of the 3'-arm target products corresponded also to cleavage across the 10th nucleotide from the 3'-end. however, additional products were detected suggesting imprecise cleavage, likely due to weak ago binding to the 3'-unphosphorylated pre-mirna end. it is unlikely that these rna products represent degradation artifacts, as all the radiolabeled rna oligos were gel-purified.  our results indicate that ago and dicer are sufficient for processing and loading of mirnas into risc. furthermore, our studies suggest that ago binds primarily to the 5'-and alternatively, to the 3'-end of select pre-mirnas. results from our experiments using immunopurified pre-mirnps from dicer-null cells also support this alternative mode of endogenous pre-mirna binding to ago endogenous pre-mirnps accumulate in dicer-deficient cells, likely because the secondary structure of pre-mirnas and/or their binding by ago protects them from degradation; normally, ago containing pre-mirnps are not abundant. since it is unlikely that mirnps and pre-mirnps exert redundant regulatory functions on gene expression, our in vitro findings should be cautiously interpreted, until the function of pre-mirnps and biological significance of their risc activity are elucidated. 
using these target rnas we were able to recapitulate in vitro pre-mirna processing and canonical risc loading, and define the minimal factors required for these processes. these ago2:pre-mirna complexes are catalytically active in vitro and constitute non-canonical riscs. our results indicate that ago and dicer are sufficient for processing and loading of mirnas into risc. we recently reported that argonaute also assembles into complexes with mirna precursors. argonaute, the core component of the rna induced silencing complex, binds to mature mirnas and regulates gene expression at transcriptional or post-transcriptional level. in this work, we characterized ago2:pre-mirna complexes and identified rnas that are targeted by mirnas but not their corresponding pre-mirnas. furthermore, our studies suggest that ago binds primarily to the 5'- and alternatively, to the 3'-end of select pre-mirnas.
 argonaute, the core component of the rna induced silencing complex, binds to mature mirnas and regulates gene expression at transcriptional or post-transcriptional level. we recently reported that argonaute also assembles into complexes with mirna precursors. these ago2:pre-mirna complexes are catalytically active in vitro and constitute non-canonical riscs.  the use of pre-mirnas as guides by ago bypasses dicer activity and complicates in vitro risc reconstitution. in this work, we characterized ago2:pre-mirna complexes and identified rnas that are targeted by mirnas but not their corresponding pre-mirnas. using these target rnas we were able to recapitulate in vitro pre-mirna processing and canonical risc loading, and define the minimal factors required for these processes.  our results indicate that ago and dicer are sufficient for processing and loading of mirnas into risc. furthermore, our studies suggest that ago binds primarily to the 5'- and alternatively, to the 3'-end of select pre-mirnas. 