micrornas ( mirnas ) are single stranded non - coding rna molecules of 22 nucleotides ( nt ) that regulate gene expression via post - transcriptional and/or translational repression ( ambros , 2004 ) .
primary mirna ( pri - mirnas ) are transcribed in the nucleus by rna polymerase ii or iii , where 70 nt stem - loop mirna precursors ( pre - mirnas ) are subsequently excised by the microprocessor complex containing the rnase iii enzyme drosha , and exported to the cytoplasm via exportin-5 ( kim et al . , 2009 ) .
dicer , another rnase iii enzyme , further processes pre - mirnas to produce mature mirnas , the final product being a 22 base - pair duplex with 2 nt - long 3 overhangs ( he and hannon , 2004 ) .
then , one strand of the mature mirna is loaded onto argonaute ( ago , also known as eif2c ) , a core protein of the rna - induced silencing complex ( risc ) .
mirna forms base pairs with a target mrna as a guide for ago binding and to direct the specificity of the risc effector , decreasing target mrna levels and/or its translation ( fabian et al . ,
2010 ) , where mrna destabilization is the dominant mechanism ( eichhorn et al . , 2014 ; guo et al . ,
mirnas are abundant in the mammalian genome ( more than 2000 human mirnas are currently reported in mirbase ) ( kozomara and griffiths - jones , 2014 ) and their regulatory role is essential , affecting various biological phenomena ( kim , 2005 ; sim et al . , 2014 ) . supporting evidence derives from the fact that a lethal phenotype during early development was observed in dicer1-null ( bernstein et al . , 2003 ) or ago2-null ( liu et al . ,
2004 ) mice , and various biological defects were also reported after losses of individual mirnas ( park et al . , 2010 ) .
in addition , alterations of mirna regulation are related to many diseases such as neurological disorders ( hebert and de strooper , 2009 ) , various types of cancer ( croce , 2009 ) , and cardiovascular diseases ( olson , 2014 ) .
importantly , all of these defects were ultimately caused by a dysregulation in target gene expression .
however , the limitation here is our ability to delineate a general principle for identification of specific rna targets upon which mirnas act .
the problem stems from the observation that most of mirna target sites have partial complementarity ( ambros , 2004 ) .
in contrast to plants , a near - perfect base pairing of mirna to its target is rare in animals , making it a challenge to predict the target sites ( bartel , 2009 ) .
however , initial prediction attempts provided evidence that local short stretches (  6 nt ) of consecutive base - pairing significantly contribute to target recognition ( john et al . , 2004 ; krek et al . , 2005 ; lewis et al .
conceptually termed as the  nucleus ,  the short consecutive matches could initiate a mirna - target duplex , followed by the propagation of partial annealing that may further stabilize mirna - target hybridization ( filipowicz , 2005 ; rajewsky , 2006 ) .
intriguingly , nuclei were further found to be typically located in the 5 end region of mirnas called the 
seed  , enabling the prediction of mirna target sites ( lewis et al . ,
functional mirna - target interactions are known to majorly require as few as 6-nt matches within the seed region ( position 2 - 8 , fig .
there are possible 6-mers ( positions 16 , 27 , and 38 ) , 7-mers ( positions 28 and 17 ) , and 8-mer ( position 18 ) matches in the seed . otherwise ,
 offset 6-mer seed  because of its position and a marginal effect on repression ( friedman et al . , 2009 ) .
such canonical seed sites were initially known by early biological studies ( lee et al . , 1993 ; poy et al . , 2004 ; wightman et al . ,
1993 ) , which were further validated by microarray experiments that detected enrichment of seed matches in mirna - dependent transcripts showing repression ( grimson , 2007 ; lim , 2005 ) , and also by bioinformatics analyses , which found widespread conservation of seed sites in 3 untranslated regions ( 3 utrs ) in multi - genome sequences ( lewis et al . , 2005 ; xie et al . , 2005 ) .
seed - pairing rules have been informative in prediction and analysis of canonical seed sites , often in combination with evolutionary conservation ( friedman et al . , 2009 ;
lewis et al . , 2005 ) , secondary structure ( long et al . ,
however , since a 6 nt match presents on average every 4,000 nt , likely to be occurred often by chance , such strategies still suffer from both false - positive ( 4066% ) and false - negative predictions ( 5070% ) ( mourelatos , 2008 ) even in the usage of microarray or proteomic approaches ( baek et al .
, seed - pairing rules can not identify non - canonical target sites , which have been reported as functional ( brodersen and voinnet , 2009 ) .
since seed - pairing rules are widely adopted , there has been an unintentional bias to study only the canonical seed matches , overlooking the non - canonical targets .
nevertheless , several biological studies have functionally validated that perfectly matched mirna seeds are neither necessary nor sufficient for all functional mirna - target interactions ( brodersen and voinnet , 2009 ) .
for example , supplementary components in near - perfect sites compensate for imperfect seed matches and are functional for target cleavage ( mir-196 for hoxb8 , fig .
1b ) as well as the wobble ( g : u pairing ) in seed sites of lin-41 with 3 compensatory pairings ( vella et al . ,
lsy-6 can also tolerate wobble paring in the seed to downregulate its target , cog-1 ( didiano and hobert , 2006 ) . in mammals ,
nanog , oct4 and sox2 genes , well - known regulators generating induced pluripotent stem ( ips ) cells , have been shown to contain functional wobble ( mir-296 for nanog , fig .
1b ) and bulge pairing sites for cognate mirna seeds ( mir-134 , mir-296 , and mir-470 ) , harboring a few cases in their coding sequences ( cds ) ( tay et al . ,
in contrast to a fruit fly genetic study where 3 compensatory sites were shown to be as functional as the 5 dominant canonical seed sites ( brennecke et al . , 2005 ) , such non - canonical mirna target sites were revealed to be rare in mammals ( less than 5% ) ( friedman et al . , 2009 ) and
their effects were modest ( grimson , 2007 ; wee et al . , 2012 ) .
when seed - pairing rules were applied , putative mirna targets from microarrays that showed mirna - dependent repression were often demonstrated to have high false - negatives , implicating prevalent usage of non - canonical target sites . in lieu of this
, microarray analysis of mir-24-transfected k562 cells found that several mir-24 targets are repressed through non - canonical sites , named  seedless 
1b ) ( lal et al . , 2009 ) . in addition ,  centered sites  , comprising 1112 consecutive base - pairing to the center of mirna , were also identified by the analysis of microarray data where neither perfect seed nor 3 compensatory pairing was observed ( fig .
however , the limitation of such studies is that , lacking information on precise binding sites , they are unable to distinguish between direct and indirect mirna targets .
uncertainty in direct mirna target sites necessitates the development of experimental methods capable of recovering mirnas physically associated with their targets ( easow et al . , 2007 ) .
initially , biochemical isolation of mirna - mrna complexes via ago protein - specific immunoprecipitation was attempted in order to purify mrnas bound by ago - mirna ( easow et al .
, 2007 ; hammell , 2008 ; hendrickson et al . , 2008 ; karginov , 2007 ) .
however , the integrity of the approach was questioned because of the possible high background caused by nonspecific rna - protein interactions , especially mediated by in vitro rearrangements in the ago - rna complex ( mili and steitz , 2004 ; riley et al . , 2012b ) .
however , the cross - linking and immunoprecipitation ( clip ) method that uses ultraviolet ( uv ) irradiation to crosslink rna - protein complexes in direct contact within approximately single angstrom distances in living cells , can secure specific rna - protein interactions by allowing stringent purification ( ule , 2003 ) . combined with high - throughput sequencing ( hits - clip ) ( licatalosi , 2008 ) ,
it was successfully applied to ago ( ago hits - clip ) to produce transcriptome - wide information of mirna binding sites ( chi et al . , 2009 ) .
the novelty lies not only in providing direct mirna target sites , but also in accomplishing high target specificity ( 93% ) , low false - positives ( 1327% ) , and low false - negatives ( 1525% ) ( chi et al . ,
furthermore , by analyzing crosslinking - induced mutation sites ( cims ) , footprints of ago - mirna binding regions ( 4562 nt ) became available at single nucleotide resolution ( zhang and darnell , 2011 ) .
a modification of this method , par - clip ( photoactivatable - ribonucleoside - enhanced crosslinking and immuno - precipitation ) , used a nucleoside analog that improved the efficiency of uv crosslinking and subsequent mutations ( hafner et al . ,
2010 ) , and iclip ( individual - nucleotide resolution uv crosslinking and immunoprecipitation ) analyzed truncated cdnas at the position of the protein - rna crosslink sites ( konig et al . , 2010 ) .
ago hits - clip was the first to offer a general means of mapping precise mirna target sites and has been widely applied to cultured cells ( haecker et al . , 2012 ; hafner et al .
, 2010 ; kim et al . , 2015 ; kishore et al . , 2011 ;
leung et al . ; loeb et al . , 2012 ; riley et al . , 2012a ; xue et al . , 2013 ) , tissues ( boudreau et al . , 2014 ; chi et al .
, 2009 ; kameswaran et al . , 2014 ) , and even to a whole organism ( c. elegans ) ( grosswendt et al . , 2014 ; zisoulis et al . , 2010 )
it also provides an opportunity to analyze general properties of mirna - target interactions in an unbiased and transcriptome - wide manner .
intriguingly , ago hits - clip analysis revealed that not all identified direct ago - target interactions follow canonical seed - pairing rules ( chi et al .
indeed , seed matches for 90% of ago - bound mirnas only explain 73% of ago - mrna interactions in the mouse brain , and the rest of 27% are orphans with no predicted seed matches , strongly suggesting that non - canonical mirna target sites might be prevalent in vivo .
non - canonical mirna - target sites called  nucleation bulges  were identified by analyzing ago hits - clip  orphan clusters  ( chi et al . , 2012 ) .
initially , g - bulge sites for mir-124 were found to be abundant in the mouse brain , where the target sites matched to the seed ( positions 27 ) contained a bulged - out g nucleotide corresponding to position between 5 and 6 of the mirna ( mir-124 for mink1 , fig .
upon further analysis , a general rule governing such non - canonical interactions , called the  pivot pairing rule  , was proposed ( fig .
this rule dictates that the nucleotide composition in the bulge position should be determined by the base - pairing competency to a nucleotide in position 6 of the mirna , named 
pivot  to confer the thermodynamic stability on the consecutive 5 base pairs of nucleation ( termed  nucleation bulge  ; figs .
otherwise , the base - pair - mediated interaction of only the 4 nt resulting from the non - competent nucleotide in the pivot ( non - nucleation bulge ) is unstable and not functional .
well accommodates a canonical interaction mode through nucleation pairing in position 26 , which is followed by the propagation to position 68 of the mirna ( fig .
application of the pivot pairing rule successfully decoded the non - canonical nucleation bulge sites , comprising 15% of all ago - mirna - mrna interactions in the mouse brain ( chi et al . ,
nucleation bulge sites were also observed in ago hits - clip analyses performed in the human brain ( boudreau et al . , 2014 ) and several cell lines ( hafner et al . , 2010 ) .
in addition , their sequences are evolutionally conserved ( chi et al . , 2012 ) .
the pivot pairing rule improved both quality and quantity of mirna target sites in their identification ( stefani and slack , 2012 ) since it can serve as a general rule that can be incorporated in any computational analysis ( kim et al . , 2013 ) .
in addition to nucleation bulges ,  seed - like motifs  that contain mismatches in seed pairing were found by examining differential ago hits - clip binding sites in mir-155 deficient t cells ( mir-155 for gimap3 , fig .
1c ) , indicating that 20% of ago - mir-155 binding sites contain seed - like motifs ( loeb et al . , 2012 ) .
recent variants of ago hits - clip , which can directly sequence the binding sites together with mirnas by inducing ligation between mirnas and their target rna sites , also confirmed the widespread occurrence of non - canonical  seed - like motifs  including some  nucleation bulges 
initially , clash ( cross - linking , ligation , and sequencing of hybrids ) identified that 60% of seed interactions are  seed - like motifs  in hek293 t cells ( helwak et al . ,
soon after , modified par - clip and analyses of mirna - target chimeras in ago hits - clip showed that 30% of mirna - target chimeras were also  seed - like motifs 
more recently , clear - clip ( covalent ligation of endogenous argonaute - bound rnas ) also reported the same observation ( moore et al . , 2015 ) .
although most of the non - canonical interactions were identified by ago hits - clip , it would be difficult to explain their occurrence through globally applicable rules .
however , non - canonical  nucleation bulges  could be governed by a general rule of  pivot pairing  , leading to further insights into mirna - target recognition within other similar transcriptome - wide non - canonical interactions .
2 ) ( chi et al . , 2012 ) . combining the concepts of a  nucleus 
( filipowicz , 2005 ; rajewsky , 2006 ; tomari and zamore , 2005 ) and findings from structural studies for recognition mechanisms of ago silencing complexes - nucleation , propagation and cleavage of target rnas ( schirle et al . , 2014 ; wang et al . , 2009 ) , transitional nucleation is defined as a transient mirna - target duplex with a 5-base - paired nucleation ( position 26 ) ( fig .
if the transitional nucleation becomes sufficiently stable to form , this state may be further transformed into a bulge formation where the originally matched pivot nucleotide in position 6 becomes bulged - out and subsequently extended to hybridization towards the 3 end of the mirna ( further than position 6 , fig .
this model is also well supported by several structural studies of ago ( elkayam et al . , 2012 ; nakanishi et al . , 2012 ; schirle and macrae , 2012 ) where nucleotides poised for transitional nucleation ( position 26 ) are particularly prearranged becoming a - form helical structures , which are susceptible for base pairing .
intriguingly , such a - form - like helical geometry is disrupted after a pivot ( between position 6 and 7 ) formed by a kink resulting from the insertion of the amino acid isoleucine ( i365 ) from the human ago2 protein ( elkayam et al .
thus , in theory , any target site pairing to the seed region ( either a seed match or a nucleation bulge ) requires a shift of this nuclear helix to overcome the kink . in support of this model ,
single - molecule analysis showed such stepwise processes whereby ago2 initially scans for target sites using a small region ( position 24 ) ( chandradoss et al . , 2015 ) and subsequently mediates a rapid and stable binding to the seed region of a mirna ( jo et al .
, 2015 ; salomon et al . , 2015 ) , serving as a proofreading procedure for target recognition ( yao et al . , 2015 ) .
mirna is reshaped by loading onto ago , being divided into several functional domains - the anchor , seed , central , 3 supplementary , and tail regions ( fig .
3 ) ( salomon et al . , 2015 ; schirle et al . , 2014 ; wee et al . , 2012 ) .
importantly , the seed region has two prearranged continuous base stacking configurations ( positions 26 and 79 ) caused by kinks at nucleotides 67 and 910 ( fig .
3a ) ( elkayam et al . , 2012 ; schirle and macrae , 2012 ; schirle et al . , 2014 ) .
therefore , transitional nucleation starts pairing through helix 26 and subsequently propagates to helices 79 , overcoming the kink at 67 for cases of 5 dominant interactions ( fig .
3b ) , such as the seed ( bartel , 2009 ) or nucleation bulge sites ( chi et al . ,
the opposite may also happen for central dominant interactions ( shin et al . , 2010 ) -
the interaction could be initiated by paring through helix 79 along with the central region ( positions 1012 ) and further expand up to the 3 supplementary region ( positions 1316 ) ( fig .
3c ) . in this case , crossing the barrier of the kink at position 910 may be required . for 
seed - like motifs  , where seed sites contain mismatches , deletions , or wobble pairings ( grosswendt et al .
, 2012 ; moore et al . , 2015 ) , transitional nucleation may require 3 compensatory interactions ( fig .
3d ) , which could be a general determinant of additional specificity for ago binding as shown by clear - clip ( moore et al . , 2015 ) .
biological systems initially generate marginally effective non - canonical regulations when existing biological mechanisms require alternative strategies to compensate for what major canonical pathways have been unable to accomplish .
followed by this notion , majority of non - canonical mirna - target sites were shown to mediate gene repression at a modest level ( chi et al . , 2012 ;
, 2009 ; loeb et al . , 2012 ; moore et al . , 2015 ) only except for centered sites ( shin et al . , 2010 ) , which can trigger slicing activity of ago but only exist as few in whole transcriptome
. however , such modest repression , shown by which the most of non - canonical target sites including nucleation bulges for mirnas , was often observed as insignificant in large - scale gene expression analyses ( agarwal et al . , 2015 ) . these were possibly because the marginal repression was confounded by the issue of cellular heterogeneity , variability derived from secondary effects of target repression , and differences in sensitivity and threshold used in the analyses , or non - canonical sites identified by ago clip based methods
could be the true interaction but may not be always functional as the consequence of transient bindings for searching targets ( chandradoss et al . , 2015 ) or as requiring combinatorial occurrences of target sites ( krek et al .
in fact , evaluation of target repression at individual single cell level ( moore et al . , 2015 ) and
gene expression analyses in combination with clip data ( chi et al . , 2012 ;
, 2013 ; loeb et al . , 2012 ; moore et al . , 2015 ) did observe significant repression mediated by non - canonical sites albeit the effect is still marginal .
since clip data only indicate the direct bindings , they should be analyzed together with gene expression data to access the functionality .
future studies should be performed carefully to clear out such issues whenever they analyze marginal effects from non - canonical interactions .
the modest effects from the widespread non - canonical sites are likely to be caused by reduced numbers of targets bound by ago - mirna ( chi et al . , 2012 ; grosswendt et al . , 2014 ;
, 2012 ; moore et al . , 2015 ) probably due to low binding affinity . in support of this , any mismatches or wobbles in the seed region decrease target binding but enhance the turnover of the risc complex ( wee et al . , 2012 ) , suggesting that non - canonical binding can induce intermediate affinity without affecting the concentration of the ago complex .
additionally , this may be a mechanism that provides an unbound ago - mirna complex to adjacent target sites , as shown by the single - molecule analysis where the lateral diffusion from weakly bound ago promoted cooperation between neighboring target sites ( chandradoss et al . , 2015 ) . as a result , combination of canonical and non - canonical sites
may provide a variety of spectra in the regulation of gene expression , enabling a fine - tuning of repression activity .
moreover , relative to canonical seed sites , non - canonical sites have modest sequence conservation across species ( chi et al . , 2012 ;
, 2012 ; moore et al . , 2015 ) , suggesting that they may be evolutionary intermediates under selective pressure for a shift towards high affinity seed sites .
in addition , gene ontology analysis of ago hits - clip showed that the majority of non - canonical targets have similar functions to the canonical ones , although they are slightly different in detail ( chi et al . , 2012 ; loeb et al . , 2012 ;
this indicates that non - canonical targets may have a different biological function that needs to be acquired to improve or compensate for the canonical targets .
although several non - canonical mirna - target sites were reported as functional , they did not receive much intention since they could not be definitely defined ( brodersen and voinnet , 2009 ) .
however , ago hits - clip method , which can generate a precise transcriptome - wide map of mirna target sites ( chi et al . , 2009 )
, unexpectedly revealed that large portion of mirna - target interactions are non - canonical ( chi et al . , 2012 ) .
advances in ago hits - clip analyses further identified non - canonical  nucleation bulges 
, 2012 ; moore et al . , 2015 ) , expanding our knowledge in the understanding of mirna targets and their functions .
moreover , the transitional nucleation model , yielded by the analytic process of explaining the pattern of nucleation bulges , offers a general molecular model that can be used to understand the mechanism of mirna target recognition through seed regions ( chi et al . , 2012 ) . extending this knowledge to applications of rna silencing ,
modified sirnas that contain abasic substitution in the pivot ( position 6 ) were recently developed to completely eliminate mirna - like off - target repression ( lee et al . , 2015 ; seok et al . ,
. biological significance of the non - canonical interactions could be interpreted as evolutional intermediates with slightly distinct functions .
 micrornas ( mirnas ) are small non - coding rnas ( 22 nucleotides ) regulating gene expression at the post - transcriptional level . by directing the rna - induced silencing complex ( risc ) to bind specific target mrnas 
 , mirna can repress target genes and affect various biological phenotypes . 
 functional mirna target recognition is known to majorly attribute specificity to consecutive pairing with seed region ( position 28 ) of mirna . 
 recent advances in a transcriptome - wide method of mapping mirna binding sites ( ago hits - clip ) elucidated that a large portion of mirna - target interactions in vivo are mediated not only through the canonical  seed sites  but also via non - canonical sites ( 1580% ) , setting the stage to expand and determine their properties . here 
 we focus on recent findings from transcriptome - wide non - canonical mirna - target interactions , specifically regarding  nucleation bulges  and  seed - like motifs  . 
 we also discuss insights from ago hits - clip data alongside structural and biochemical studies , which highlight putative mechanisms of mirna target recognition , and the biological significance of these non - canonical sites mediating marginal repression . 
INTRODUCTION
CANONICAL TARGET SITES: SEED PAIRING RULES
EVIDENCE OF NON-CANONICAL TARGET SITES
TRANSCRIPTOME-WIDE MIRNA BINDING SITES 
WIDESPREAD NON-CANONICAL INTERACTIONS: NUCLEATION BULGES AND SEED-LIKE MOTIFS
TRANSITIONAL NUCLEATION MODEL
INSIGHT FOR MARGINAL REPRESSION AND SIGNIFICANCE OF NON-CANONICAL INTERACTIONS
CONCLUSION