the localization and subsequent translation of mrna are important means of regulating gene expression with high spatial and temporal control . in neurons , targeting mrna to specific sites and synthesizing proteins where and when they are needed is particularly beneficial because neurons have long dendrites and axons extending from a few hundred microns to up to a meter from the cell body .
the physiological roles of mrna localization in neurons have been implicated in various neuronal functions such as synaptic plasticity , axonal guidance and regeneration
. however , there are many remaining questions regarding how mrnas navigate through the complex neuronal arbors , how they are captured in specific regions , and how local translation is regulated ( buxbaum et al . , 2015 ; holt and schuman , 2013 ; hutten et al . , 2014 ; xing and bassell , 2013 ) .
observing the dynamics of mrna localization and translation will provide critical information to help understand how precise spatio - temporal regulation of gene expression is processed .
most previous studies have used in situ hybridization ( ish ) methods ( lawrence and singer , 1985 ) to visualize rna molecules inside a cell . in particular , fluorescence in situ hybridization ( fish )
enables the detection of individual rna molecules with various signal amplification procedures in fixed cells .
single - molecule fish ( smfish ) , which uses multiple fluorescent probes hybridized to a single mrna ( femino et al . , 1998 ;
, 2008 ) , is widely used to profile transcription , localization and degradation of rna .
however , fish experiments using fixed cells can not provide temporal information on rna regulation .
recent innovations in live - cell imaging technologies have made it possible to observe the sequence of molecular events in real time , which is critical to our understanding of mrna dynamics ( moon et al .
, 2016 ; spille and kubitscheck , 2015 ) . by imaging single mrna molecules in live cells , we can begin to understand the cause - and - effect relationship and to model the kinetics of rna regulation more quantitatively and predictably . in this review
, we provide a brief overview of recent progress in studies on mrna localization and translation in neurons .
in particular , we highlight several recent reports that have used single - molecule imaging techniques to contribute to our knowledge of mrna translocation in dendrites and axons .
additionally , we review new technical developments for multicolor imaging of single mrnas and their translational activities .
these approaches will provide a powerful toolkit to help understand the molecular mechanisms of rna localization and local translation in neurons , with unprecedented temporal and spatial resolution .
since the discovery of polysomes at the base of dendritic spines ( steward and levy , 1982 ) , many lines of evidence have indicated that de novo protein synthesis in dendrites is required for long - term synaptic plasticity ( hanus and schuman , 2013 ; jung et al . , 2014 ) .
deep rna sequencing revealed 2,550 mrnas that are present in dendrites and axons in the hippocampus ( cajigas et al . , 2012 ) .
a large fraction of these mrnas encode synaptic proteins such as signaling molecules , scaffolds and receptors . among these mrnas ,
activity - regulated cytoskeleton - associated protein ( arc ) , the -subunit of calcium / calmodulin - dependent protein kinase ii ( camkii ) and -actin mrnas have been extensively studied to unravel their regulation in live neurons . to understand how these mrnas are sorted and transported to dendrites , it is crucial to track the movement of individual mrna molecules ( fig .
single - mrna tracking has revealed the diverse and stochastic nature of mrna trafficking ( park et al . , 2010 ) .
transport and localization of mrna depend on the interplay of cis - acting rna elements , rna binding proteins ( rbps ) , and motor proteins ( czaplinski , 2014 ) .
various kinds of rbps bind to their target mrnas to form messenger ribonucleoprotein complexes ( mrnps ) . after recruiting motor proteins
, mrnps are transported along cytoskeletal tracks . a cis - acting rna sequence that confers dendritic localization , which is called a dendritic targeting element ( dte ) , is typically located in the 3 untranslated region ( 3 utr ) of the mrna .
, 2000 ) or the arc 3 utr ( dynes and steward , 2007 ; 2012 ) exhibit bidirectional transport in dendrites and localize at the base of dendritic spines .
a 54-nucleotide - long zipcode in the -actin 3 utr ( kislauskis et al . , 1994 ) and
zipcode binding protein 1 ( zbp1 ) are required for the localization of -actin mrna in dendritic filopodia and axonal growth cones ( eom et al . , 2003 ; zhang et al . , 2001 ) .
microinjected camkii and septin7 mrnas colocalize in the same particles , while map2 mrnas reside in different particles , suggesting that some rnas that share a common dte can be sorted in the same complexes and transported to distal dendrites ( tubing et al . ,
for example , there have been conflicting results for defining the dte in the camkii 3 utr ( doyle and kiebler , 2011 ) .
the cis - acting rna elements interact with a set of transacting factors or rbps to form mrnp complexes that are transported along microtubules by kinesin or dynein .
tracking reporter rnas in fmr1 knock - out ( ko ) neurons revealed the important roles of fragile x mental retardation protein ( fmrp ) as an adaptor that links mrna to the kinesin light chain ( dictenberg et al . , 2008 ) and for mrna delivery to dendritic spines in response to synaptic stimulation ( kao et al . , 2010 ) .
live imaging of neurons also showed that huntingtin ( htt ) , huntingtin - associated protein 1 ( hap1 ) , kif5a , and dynein intermediate chain are associated with -actin mrna during dendritic transport ( ma et al . , 2011 ) .
knocking down the levels of these proteins reduced the transport of -actin mrna , suggesting that they have roles in mrna localization in neurons .
an actin - based motor , myosin - va , contributes to the transport of tls ( translocated in liposarcoma)-containing mrnps in dendritic spines ( yoshimura et al . , 2006 ) .
most previous mrna tracking studies have been performed using transfection or injection of exogenous reporter mrnas .
however , reporter rnas usually do not contain all of the cisregulatory sequences and trans - acting rbps and thus may not represent the true characteristics of endogenous rnas . to label endogenous mrna ,
genetically engineered mice have been generated by inserting ms2 binding site ( mbs ) stemloops into a target rna and expressing ms2 capsid protein ( mcp)-gfp proteins ( lionnet et al .
all the endogenous -actin mrnas were labeled with the ms2-gfp system in live mice ( park et al . , 2014 ) .
tracking the labeled endogenous -actin mrnas revealed that mrna diffusion was much slower in neurons than in fibroblasts .
while most of the -actin mrnas in fibroblasts showed diffusive motion , a small fraction of the -actin mrnas in neurons showed bidirectional motion with a mean speed of 1.3 m / s .
recently , glutamate uncaging was used to demonstrate that -actin mrnas can localize in the stimulated dendritic region within 15 min of stimulation ( yoon et al . , 2016 ) .
nmda receptors and zbp1 have important roles in the localization process . in neurons cultured from zbp1 ko mice , -actin mrnas were transported to the stimulated region ; however , they did not maintain their localization .
the authors also showed that newly synthesized -actin proteins accumulated in the stimulated spine head and that this helped stabilize the expanded spines . taken together , tracking single - mrna movements enables us to reveal how rna transport and localization happen in live neurons .
the current working model is called the  sushi belt model  , in which mrnps stroll along axons and dendrites like sushi plates on a conveyor belt to serve potential customers ( doyle and kiebler 2011 ) . those mrnas may be captured by 
synaptic tags  that are relevant to the formation of long - term potentiation ( ltp ) and long - term depression ( ltd ) ( frey and morris , 1997 ) .
further studies using new developments in genetic engineering and in systems biology approaches will elucidate the precise regulatory mechanisms of mrna localization in dendrites .
while local translation in dendrites is well accepted , local protein synthesis in axons has been controversial over the years .
although a few early studies reported the presence of ribosomes in axons ( bunge , 1973 ; tennyson , 1970 ; zelena , 1970 ) , the low density of axonal mrnas and ribosomes supported the traditional view that axonal proteins are synthesized in the cell body . later , however , two studies showed evidence for local translation in the axons .
campbell and holt observed that growth cones of retinal ganglion neurons lose their ability to turn away from a source of sema3a when translation is inhibited ( campbell and holt , 2001 ) .
zhang and poo showed that localized synaptic potentiation by bdnf requires presynaptic protein translation in xenopus nerve - muscle cultures ( zhang and poo , 2002 ) .
since then , there have been many studies on mrna localization in axons and on the implications of those localized mrnas for axon guidance , survival and regeneration ( gumy et al . , 2014 ; jung et al . ,
bdnf- or netrin-1-induced growth cone turning is mediated by local translation of -actin mrna ( leung et al . , 2006 ;
bdnf induces src - mediated zbp1 phosphorylation , which releases zbp1 from -actin mrna ( huttelmaier et al . , 2005 ) .
after zbp1 is dissociated , -actin proteins are locally synthesized from the mrna and then direct growth cone turning . in a similar mechanism ,
map1b mrnas are also locally translated in response to sema3a ( li et al . , 2010 ) .
it has been suggested that fmrp has a role in protein synthesis - dependent collapse of growth cones induced by sema3a ( fig .
in addition , branching of sensory axons requires local protein synthesis and the stalling of mitochondria ( spillane et al . , 2013 ) .
using a gfp reporter construct containing the 3 utr of -actin mrna , the authors found that hot spots of axonal translation occur at the base of branches that also contained mitochondria .
zbp1 is colocalized with myosin - va in axons , and inhibition of myosin - va function increases the number of motile zbp1 particles ( nalavadi et al . , 2012 ) .
furthermore , some rbps can bind to several species of mrnas . in dorsal root ganglion neurons ,
sfpq , a splicing factor , works as an rbp binding to lmnb2 , bcl2l2 , impa1 and creb1 mrnas ( cosker et al . , 2016 ) .
in addition , sfpq plays a role in the axonal transport of these mrnas to promote axon survival . in central nervous system ( cns )
axons , studies on mrna localization have been facilitated by the use of microfluidic devices that separate axons from cell bodies and dendrites ( taylor et al . , 2005 ) . using a microfluidic device in conjunction with microarray analysis and fish ,
many species of mrnas have been detected in the axons of cns neurons as well as of peripheral nervous system ( pns ) neurons .
studies of mature cortical axons have identified mrnas that are involved in the translation machinery , transport , cytoskeletal components and mitochondrial maintenance ( taylor et al . , 2009 ) .
to date , there are only a few studies on live - cell imaging of axonal mrna transport . using the ms2-gfp system , bi et al . demonstrated that the 5 and 3 utrs of kor ( -opioid receptor ) mrna are required for its axonal transport in dorsal root ganglion ( drg ) neurons ( bi et al . , 2006 ) .
more recently , a molecular beacon method was used to visualize nefl ( neurofilament - l ) mrna in axons of primary mouse cortical neurons and human motor neurons derived from ips cells ( alami et al .
simultaneous imaging of nefl mrna and trans - active response dna - binding protein 43 ( tdp-43 ) , an rbp related to amyotrophic lateral sclerosis ( als ) , revealed that tdp-43 promotes anterograde transport of nefl mrnp granules .
this result suggests that tdp-43 has a role in the delivery of target mrna to distal axons . with the development of microfluidic devices , rna labeling techniques , and high - resolution live - cell imaging , it is now more feasible to study mrna transport in axons .
for instance , integrated microfluidic platforms are developed for investigating regeneration of injured axons ( kim et al . ,
neurons cultured from mcp  mbs knock - in mice now allow us to image single endogenous mrnas without transfection steps ( park et al . , 2014 ) . because local translation plays a crucial role in axon development and regeneration , it is important to understand the molecular mechanisms that govern mrna trafficking and localization in axons .
translation is a highly regulated , complex biological process in which mrna is decoded by a ribosome to produce a protein .
several whole - genome techniques for studying translation have been developed based on rna sequencing ( rna - seq ) or mass spectrometry ( ms ) .
ribosome profiling , for example , measures ribosome - protected regions of mrnas with next - generation sequencing ( ngs ) , providing position information on translating ribosomes throughout the cellular transcriptome ( ingolia et al . , 2009 ) .
global , averaged measurements of translation initiation , elongation , and termination rates can be made by ribosome profiling ( ingolia et al . , 2011 ) .
another sequencingbased technique called translating ribosome affinity purification ( trap ) ( heiman et al . , 2008 ) or ribotag ( sanz et al
. , 2009 ) relies on pulling down gfp- or ha - tagged ribosomal proteins and then the identification of mrnas associated with them .
ms - based methods such as pulsed stable isotope labeling by amino acids in cell culture ( silac ) ( schwanhausser et al . , 2009 ) and
biorthogonal noncanonical amino acid tagging ( boncat ) ( dieterich et al . , 2006 ) have been used to identify newly synthesized proteins after adding labeled amino acids .
because genome - wide methods can only detect the average behavior of many cells , imaging - based techniques have been developed to study the dynamics of translation in single cells ( chao et al . , 2012 ) .
fluorescent proteins have been used to detect local protein synthesis ( aakalu et al . ,
2001 ) ; however , the long maturation time of fluorescent proteins has been a major limitation of such methods . as an attempt to visualize translation from single mrnas , halstead et al .
developed an rna biosensor termed translating rna imaging by coat protein knock - off ( trick ) that can distinguish untranslated mrnas from those that have undergone at least one round of translation ( halstead et al .
orthogonal bacteriophage pp7 and ms2 stem - loops were used to label mrna with two different fluorescent proteins ( fig .
. simultaneous expression of the pp7 capsid protein fused with green fluorescent protein ( pcp - gfp ) and the ms2 capsid protein fused with red fluorescent protein ( mcp - rfp ) resulted in yellow fluorescent signals from untranslated mrnas . during the first round of translation , pcp - gfp is removed from the mrna as a ribosome traverses the coding region that contains the pp7 stemloops .
thus , translated mrnas are labeled only with mcp - rfp bound to the ms2 stem - loops in the 3 utr . while the trick technique provides information on the first round of mrna translation , recent studies by four independent groups ( morisaki et al .
, 2016 ; wang et al . , 2016 ; wu et al . , 2016 ;
yan et al . , 2016 ) have demonstrated two - color imaging of mrna and its nascent polypeptides .
these four studies used the ms2 or pp7 systems to label mrna and the suntag ( tanenbaum et al . , 2014 ) or flag tag ( viswanathan et al . , 2015 )
fluorescence - tagged ms2 or pp7 capsid proteins were expressed in cells , providing a strong fluorescence signal from each mrna .
the suntag and flag tag systems use fluorescently labeled antibody fragments that bind to multiple copies of a short epitope on each polypeptide .
an mrna undergoing active translation can be identified by the colocalization of two fluorescent signals from the mrna and nascent proteins .
wu et al . , 2016 ) used the suntag - ms2 technique not only in cell lines but also in hippocampal neurons .
they used ornithine decarboxylase ( odc ) or auxin - induced degron ( aid ) to facilitate rapid degradation of the epitope array after translation .
this allowed them to distinguish nascent polypeptides translated on the mrna from the background of freely diffusing polypeptides in the cytosol .
wang et al . found that the arc 3 utr increased the number of translation sites in the dendrites of neurons .
wu et al . showed that ~40% of the reporter mrnas were translated in the proximal dendrites ; however , only ~10% were translated in the distal dendrites . both studies
this result contradicts the previous speculation that translation is repressed during the transport of mrna to localization sites .
although these findings were obtained using exogenous reporters , the tagging method could be applied to endogenous genes with crispr / cas9 genome - editing techniques ( nelles et al . , 2016 ) .
if we could track endogenous mrna while measuring its translational output in response to diverse signaling in live tissues , we would be able to uncover many interesting details regarding the dynamics of gene expression .
further improvements in these imaging techniques ( pichon et al . , 2016 ) will certainly offer new insights into the complex regulation of translation at the single - molecule level in situ .
the elongated morphology of neurons requires directed transport of mrna and local translation of proteins for efficient gene expression in dendrites and axons .
because local protein synthesis has important roles in neuronal development and synaptic plasticity , it is crucial to understand how specific mrna molecules are targeted to the activated regions for local translation .
recent advances in live - cell single - rna imaging techniques have significantly increased our knowledge on the dynamics of rna in neurons
what are the cis - regulatory rna sequences for selective transport into dendrites and axons ?
how are mrnps delivered and captured at the stimulated synapses ? understanding the molecular mechanisms behind rna localization , translation and degradation in neurons will provide clearer answers to questions about the physiological relationships between gene expression and brain functions .
 local protein synthesis mediates precise spatio - temporal regulation of gene expression for neuronal functions such as long - term plasticity , axon guidance and regeneration . to reveal the underlying mechanisms of local translation , 
 it is crucial to understand mrna transport , localization and translation in live neurons . among various techniques for mrna analysis 
 , fluorescence microscopy has been widely used as the most direct method to study localization of mrna . 
 live - cell imaging of single rna molecules is particularly advantageous to dissect the highly heterogeneous and dynamic nature of messenger ribonucleoprotein ( mrnp ) complexes in neurons . here 
 , we review recent advances in the study of mrna localization and translation in live neurons using novel techniques for single - rna imaging . 
INTRODUCTION
SINGLE-mRNA TRAFFICKING IN DENDRITES
IMAGING mRNA LOCALIZATION IN AXONS
MONITORING TRANSLATION FROM A SINGLE mRNA IN LIVE NEURONS
CONCLUSION