Update on Targeting of mRNAs

mRNA Localization in Plant Cells1[OPEN]

Li Tian,a,2,3 Hong-Li Chou,b Masako Fukuda,a,c Toshihiro Kumamaru,c and Thomas W. Okitaa

aInstitute of Biological Chemistry, Washington State University, Pullman, Washington 99164-6340bHuck Institutes of the Life Sciences, Pennsylvania State University, University Park, Pennsylvania 16802cPlant Genetics Laboratory, Institute of Genetic Resources, Faculty of Agriculture, Kyushu University, Fukuoka819-0395, Japan

ORCID IDs: 0000-0003-1497-7923 (L.T.); 0000-0002-6355-9034 (H.-L.C.); 0000-0003-4870-1247 (T.K.); 0000-0002-2246-0599 (T.W.O.).

Localization of mRNAs at the subcellular level is an essential mechanism for specific protein targeting and local control ofprotein synthesis in both eukaryotes and bacteria. While mRNA localization is well documented in metazoans, somatic cells, andmicroorganisms, only a handful of well-defined mRNA localization examples have been reported in vascular plants and algae.This review summarizes the function and mechanism of mRNA localization and highlights recent studies of mRNA localizationin vascular plants. While the emphasis focuses on storage protein mRNA localization in rice endosperm cells, information ontargeting of RNAs to organelles (chloroplasts and mitochondria) and plasmodesmata is also discussed.

WHAT IS MRNA LOCALIZATION?

Localization of mRNAs was initially discovered inthe 1980s, when b-actin mRNA was found to beasymmetrically distributed in ascidian eggs and em-bryos (Jeffery et al., 1983). This nonuniform spatialdistribution pattern was later observed for maternalmRNAs in Xenopus (Rebagliati et al., 1985) and Dro-sophila oocytes (Frigerio et al., 1986; Berleth et al.,1988) and supported the proposal of prelocalizedRNA during early development (Davidson, 1971;Kandler-Singer and Kalthoff, 1976). Subsequently,mRNA localization was observed in a variety of so-matic cells such as fibroblasts (Lawrence and Singer,1986), oligodendrocytes (Trapp et al., 1997), and neu-rons (Garner et al., 1988). Today, mRNA localization isprevalent in bacteria, yeast, algae, vascular plants, andmetazoans and, therefore, is an ancient, universal,evolutionarily conserved mechanism.Our understanding of mRNA localization stems

mainly from research in Xenopus, Drosophila, buddingyeast, fungi, and structurally polarized animal cells(Holt and Bullock, 2009; Martin and Ephrussi, 2009;Medioni et al., 2012; Shahbabian and Chartrand, 2012;Weatheritt et al., 2014; Weil, 2014; Chin and Lécuyer,

2017; Lazzaretti and Bono, 2017; Teimouri et al., 2017).By contrast, only a handful of examples for mRNA lo-calization have been observed in plants. Nevertheless,substantial progress has been made over the years.Here, we discuss the importance and mechanism ofmRNA localization, summarize mRNA localizationstudies in land plants, and provide suggestions for fu-ture research in plants.

ADVANCES

• Proteomic analyses have identified more than 200 cytoskeleton-associated RNA-binding proteins (RBPs) or associated factors from rice endosperm, many of them involved in transporting RNAs to specific intracellular locations.

• Analysis of TILLING mutants and T-DNA insertion lines in genes for the RNA-binding proteins Tudor-SN, RBP-P, and RBP-L indicate that these zipcode-binding proteins are required for storage protein mRNA localization in rice endosperm cells.

• Mitochondrial targeting requires the N-terminal leader and 3’UTR sequences.

• Recent advances of fluorescence-based mRNA labeling systems combined with high-resolution confocal microscopy provide promising approaches to visualize the real-time trafficking of mRNAs in plant cells.

1This work was supported by grants from the National ScienceFoundation (MCB-1444610 and IOS-1701061), from the Japan Societyfor the Promotion of Science (to M.F. and T.K.), and the U.S. Depart-ment of Agriculture National Institute of Food and Agriculture(Hatch Umbrella Project 1015621 and Multistate Research ProjectNC1200).

2Author for contact: li.tian@wsu.edu.3Senior author.L.T. and T.W.O. developed the ideas and the initial framework;

L.T. prepared the figures; the article was written by L.T., H.-L.C.,M.F., T.K., and T.W.O.

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WHY DOES MRNA LOCALIZATION MATTER?

The importance of mRNA localization is best exem-plified in early metazoan development, where morethan 70% of the expressed mRNAs are asymmetricallydistributed in the Drosophila embryo (Lécuyer et al.,2007). As one of the key steps in posttranscriptionalgene regulation and protein synthesis, localization ofmRNA serves several functions during cell growth anddevelopment. First, the direct consequence of mRNAlocalization is to save cellular energy. As local transla-tion from a single mRNA molecule can yield manycopies of protein molecules at strategic locations in thecells, mRNA localization undoubtedly provides a moreenergy-efficient way to achieve specific targeting andasymmetrical distribution of proteins. Second, mRNAlocalization is an effective mechanism of concentratingproteins at specific intracellular locations. This is espe-cially true for the rice (Oryza sativa) prolamine mRNAs,which are concentrated on the endoplasmic reticulum(ER) membrane that delimits the prolamine intracis-ternal granules, to form a spherical protein body (Liet al., 1993; Choi et al., 2000; Hamada et al., 2003a,2003b). Third, local translation of specific proteins at thesite of action can prevent proteins from forming protein-protein interactions that impede function or are harmfulto the cell. One of the best examples is the localization ofmyelin basic protein (MBP) mRNA to the myelinatingcompartments of oligodendrocytes (Shan et al., 2003;Boggs, 2006). Due to its nonspecific membrane-bindingproperties, randomly distributed MBP in the cytoplasmwould disrupt the integrity of the endomembrane sys-tem (Du et al., 2007). By transporting its mRNA to thecell’s myelin compartment, local translation at this siteavoids this potential problem. Lastly, mRNA localiza-tion aids in the assembly and formation of multiproteincomplexes (Kramer and McLennan, 2019). Stochasticmodeling of protein-protein interactions indicates thatthe rate of assembly of interacting proteins randomlydistributed in the cell would be very low unless theproteins were colocalized during translation. Ribosome-profiling studies readily demonstrate that the initialsteps leading to the formation of many multiproteincomplexes occur by the coassembly of nascent poly-peptides during translation (Shiber et al., 2018).

Coupling with the precise spatiotemporal controlof gene expression and protein synthesis at the siteof action, mRNA localization plays a critical role inregulating many diverse processes during normalcell physiology and development, in disease, host-pathogen responses, and cellular adaptation to stress.More information about the effects of dysregulation ofmRNA localization in animal and fungi systems isavailable in other reviews (Cody et al., 2013; Falliniet al., 2016; Wang et al., 2016; Chin and Lécuyer, 2017).

HOW DOES MRNA LOCALIZATION WORK?

The localization of mRNAs is a multistep processinvolving events in both the nucleus and cytoplasm.

The initial step is the recognition and binding of cis-acting zipcode elements by trans-acting factors, usu-ally RNA-binding proteins (RBPs), during transcriptionin the nucleus, forming heterogenous nuclear ribonu-cleoprotein complexes. These RBPs may also serve inmRNA processing (i.e. splicing and polyadenylation)during posttranscription in the nucleus. The associationof a set of RBPs with mature mRNAs forms the primarymessenger ribonucleoprotein (mRNP) complex, medi-ating nucleic export of mRNAs. After export into thecytoplasm, the primary mRNP complexes undergoextensive remodeling where one or more proteins areremoved and others are added (Lewis and Mowry,2007; Niedner et al., 2013). For example, once in thecytoplasm, themRNP is modified to include an adaptorprotein, which links the mRNP complex to a cytoskel-etal motor protein enabling transport on the cytoskel-eton. At its destination, the mRNP undergoes furtherremodeling where it is anchored and subsequently ac-tivated for translation (Jansen et al., 2014). Alterna-tively, the mRNP can be processed in P-bodies or storedin stress granules.

The zipcode sequences contain the necessary in-formation for the successful transport of the corre-sponding mRNAs. They are usually located in theuntranslated regions (UTRs), mainly in the 39 UTR, butmay also be present in the coding region (Chabanonet al., 2004; Tian and Okita, 2014). Although zipcodesmay be sequence dependent, the secondary/tertiarystructure of zipcode RNA sequences may be the signalrecognized by trans-acting factors. For example, theyeast ASH1 RNA contains four zipcodes that shareweak sequence homology (Niedner et al., 2014). Threeof the zipcodes are located in the coding region, a lo-cation that may constrain their evolution. Although onezipcode is sufficient forASH1 localization, all four worksynergistically to ensure efficient transport on micro-filaments (Niedner et al., 2014). These zipcodes alsoserve to arrest translation, as the hairpin loop structureimpedes ribosome movement.

The zipcodes are recognized by specific RBPs. Typi-cal RBPs contain one or more conserved RNA-bindingmotifs. These include the RNA recognition motif(RRM), K homology motif, zinc finger, pentatricopep-tide repeat, cold shock domain, RGG (Arg-Gly-Gly)box, Puf RNA-binding repeats, and RNA helicaseDEAD/DEAH box (Git and Standart, 2002; Lundeet al., 2007; Glisovic et al., 2008; Bailey-Serres et al.,2009; Lorkovic, 2009; Lee and Kang, 2016). Many pro-teins that have RNA-binding activity, however, lacka conserved RNA-binding motif. A notable exampleis the yeast ASH1 RNA and its two RBPs, She2Pand She3P. Although She2P and She3P lack any rec-ognizable canonical RNA-binding domain, they spe-cifically recognize and directly bind to the zipcodeelements ofASH1 RNA (Böhl et al., 2000; Niedner et al.,2014). Much more atypical is the binding of VPS36,an Endosomal Sorting Complexes Required for Trans-port protein II (ESCRT-II) subunit, via its N-terminalGRAM-like ubiquitin-binding in EAP45 (GLUE)

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domain, to Drosophila bicoid mRNA (Irion and StJohnston, 2007).In addition to RNA-binding motifs, RBPs usually

contain a variety of auxiliary sequences or domains.Examples include peptide domains enriched in Gly,Arg, Pro, andArg-Ser repeats (Biamonti and Riva, 1994;Fedoroff, 2002; Maris et al., 2005; Lunde et al., 2007;Ambrosone et al., 2012; Lee and Kang, 2016). Theseadditional modules afford RBPs diverse binding abili-ties to other proteins, which can recruit other relevantproteins or factors to further specify RNA recognitionand mediate mRNA transport.While there are examples where mRNAs are trans-

ported passively by diffusion to an anchoring site(Forrest and Gavis, 2003; Chang et al., 2004; Palacios,2007), most are actively transported by molecular mo-tors along the cytoskeleton.Molecularmotors canmovebidirectionally along the cytoskeleton and are widelyused to transport organelles and other cargos, includingmRNP complexes (Bullock, 2007; Palacios, 2007). Theactive transport of mRNAs requires the core mRNPmachinery, specifically RBPs and other adaptor pro-teins, that recruits the corresponding motor proteins totransport mRNAs toward their final destination. Allthree families of molecular motors (myosins, dyneins,and kinesin) have been shown to transport variousmRNAs (Bullock, 2007; Palacios, 2007; Ryder and Lerit,2018). Inmany cases, however, howmRNPparticles areattached tomolecular motors has yet to be resolved andrequires further investigation.

MRNA LOCALIZATION IN PLANT CELLS

Localization of mRNAs has been poorly investigatedin vascular plants simply because most plant cells arenot amenable for study. While most plant cells are largeenough to be studied by light microscopy, they in-variably contain a large vacuole compartment thatsqueezes the cytoplasm to the periphery of the cell(Chou et al., 2019). Meristematic cells are cytoplasmi-cally dense, but their small sizes preclude them frombeing a system to study by light microscopy. An idealplant cell type is the cereal endosperm cell, which islarge and cytoplasmically dense. In this section,we mainly focus on storage protein mRNA localiza-tion in rice developing endosperm cells, a well-definedmodel for mRNA localization study in vascular plants.We briefly summarize studies of mRNA targeting tochloroplast and mitochondria and targeting to theplasmodesmata, the entry point for cell-to-cell andlong-distance movement.

mRNA Localization to the Cortical ER in CerealEndosperm Cells

An advantage of studying endosperm cells from riceis that, unlike other cereals that predominantly accu-mulate one type of storage protein in developinggrains, this cereal synthesizes two major types of

storage proteins, glutelin and prolamine, andmoreoverdeposits them in separate intracellular compartments.Although both storage protein types are synthesized onthe rough ER, prolamines are retained as intracisternalinclusions in the ER lumen, forming an ER-derivedprotein body I (PB-I), whereas glutelins are exportedto the Golgi and are subsequently transported to theprotein storage vacuoles (PSVs) to form PB-II. The bi-ogenesis of a prolamine intracisternal granule gives riseto two distinct morphological domains of the corticalER membrane complex: protein body ER (PB-ER) andthe interconnecting cisternal ER (cis-ER) network con-sisting of cisternal and tubular membranes (Li et al.,1993).These unique traits make rice endosperm cells an

ideal system to study mRNA targeting to the ER. Bio-chemical evidence obtained in the 1980s and 1990srevealed the asymmetric distribution of storage proteinmRNAs on the ER (Kim, 1988; Li et al., 1993): prolaminemRNAs are concentrated on the PB-ER, whereas glu-telin mRNAs are localized on the adjoining cis-ER. AsmRNA localization was not dependent on the synthesisof storage protein, a series of transgenic rice linesexpressing hybrid storage protein mRNA sequenceswere constructed. Confocal microscopic analysis ofseed sections subjected to in situ reverse transcription-PCR hybridization showed that the distinct distributionof prolamine and glutelin mRNAs to the PB-ER and cis-ER, respectively, is dependent on specific prolamineand glutelin cis-RNA sequences or zipcodes (Hamadaet al., 2003b; Washida et al., 2009, 2012). Based on thesefindings, a model of mRNA localization featuringthree mRNA transport and localization pathways tosubdomains of the cortical ER in rice endosperm cellwas proposed (Fig. 1; Crofts et al., 2005; Doroshenket al., 2012; Tian and Okita, 2014; Chou et al., 2019).Prolamine (as well as a-globulin) and glutelin mRNAsare transported to the PB-ER and cis-ER, respectively,using regulated pathways requiring zipcodes. A thirdzipcode-independent default pathway was also evi-dent. Genetic-biochemical studies showed that thethree mRNA transport pathways are not independentbut are instead arranged in a hierarchal interrela-tionship (Fig. 2). The glutelin mRNA localizationpathway is dominant over the prolamine mRNAtransport pathway that, in turn, is dominant over thedefault pathway (Crofts et al., 2005; Doroshenk et al.,2012; Tian and Okita, 2014; Chou et al., 2019). Themutant rice lines glup4 and glup6 exhibit mis-localization of glutelin mRNAs from the cis-ER to PB-ER, while glup2 mislocalizes prolamine mRNAs fromthe PB-ER to cis-ER. The interrelatedness of the threeRNA transport pathways suggests that they sharecommon trans-factors for targeting to the ER mem-brane, while glutelin and prolamine pathways requireadditional factors for selective transport (Chou et al.,2019). Indeed, our studies showed that prolamine andglutelin zipcode sequences bind identical trans-factors, Tudor-SN (Chou et al., 2017; Tian et al.,2018, 2019), RBP-P (Chou et al., 2017; Tian et al., 2018,

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2019), and RBP-L (Chou et al., 2017; Tian et al., 2018,2019).

Both prolamine and glutelin RNAs contain multiplecis-acting zipcode elements (Fig. 3). The two cis-actingsignals of prolamine are found in the coding region and

39UTR. Both contain a conserved 27-nucleotidemotif ineach zipcode element (Hamada et al., 2003b; Washidaet al., 2009, 2012). By contrast, glutelin RNA has threezipcode regions containing two sequencemotifs, 23 and27 nucleotides in length. Zipcode 1, located at the end of

Figure 1. Working model of mRNA transport pathways to the cortical ER in developing rice endosperm cells (adapted from TianandOkita, 2014). In the nucleus, storage proteinmRNAs are recognized and bound by various sets of RBPs, forming heterogenousnuclear ribonucleoprotein (hnRNP) complexeswhere they are exported from the nucleus. In the cytoplasm, themRNP complexesundergo dynamic remodeling and are actively targeted to the distinct ER subdomain via the cytoskeleton by three pathways.Prolamine (blue) and globulin (orange) mRNAs are targeted to the PB-ER, where the mRNAs are translated and their codedproteins are translocated into the ER lumen. Prolamine polypeptides assemble as an intracisternal granule to form PB-I, whileglobulins are rapidly exported to the Golgi for subsequent transport to the irregularly shaped PSVs by dense vesicles. GlutelinmRNAs (red) are localized to the cis-ER, and the coded glutelin proteins are transported to the PSV via the Golgi complex. Anadditional default pathway mediates general mRNA (green) transport to the cis-ER.

Figure 2. The interrelationship among the three mRNA transport pathways in developing rice endosperm (adapted from Chouet al., 2019). The glutelin mRNA localization pathway (red) is dominant over the prolamine mRNA transport pathway (blue),which, in turn, is dominant over the default pathway (gray). Analyses of mutant rice lines expressing defective Rab5 (glup4), Rab5-GEF (glup6), and Got1 (glup2) suggest the involvement of membrane trafficking in the transport of mRNAs to the cis-ER or PB-ER.The dominant mutation, glup5, misdirects only globulinmRNAs to the cis-ERwithout affecting the targeting of prolaminemRNAsto the PB-ER. The hierarchal interrelationship of the three RNA-transport pathways suggests that they may share common trans-factors for targeting to the ER membrane, while additional factors are required for selectively transporting glutelin and prolaminemRNAs.

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exon 4, is composed of three copies ofmotif 1. Zipcode 2contains a single motif 2 located within the 39 UTR,while zipcode 3 at exon 1 contains both motifs 1 and 2(Washida et al., 2009). Deletion analysis showed thatremoval of these zipcode elements resulted in the mis-localization of the corresponding mRNAs (Hamadaet al., 2003b; Washida et al., 2009, 2012). Individually,these zipcode elements functioned weakly on theirown, as two zipcodes are required for restricted local-ization of prolamine and glutelin mRNAs to the PB-ERand cis-ER, respectively (Hamada et al., 2003b;Washida et al., 2009, 2012).Asymmetric distribution of storage mRNAs is also

observed in other cereal endosperm cells. Similar to thepattern seen in rice endosperm, zein (a prolaminefamily protein) mRNAs are targeted to PB-ER whilelegumin-1 (an 11S globulin family protein) mRNAs arelocalized to the cis-ER in maize (Zea mays; Washidaet al., 2004). The spatial distribution of storage proteinmRNAs in the distinct ER domain may directly con-tribute to the deposition of zein in the ER-derivedprotein body and legumin-1 in PSV (Washida et al.,2006). The findings in maize and rice endospermssuggest thatmRNA localizationmay be a prevalent andconserved mechanism to sort storage proteins in cerealendosperm cells.

IDENTIFICATION OF TRANS-FACTORS THATRECOGNIZE STORAGE PROTEIN ZIPCODES

Using the prolamine zipcode sequences as bait, 15RBPs were selectively captured under stringentbinding-elution conditions (Crofts et al., 2010). Follow-up functional studies of five of these captured proteins,RBPs A, I, J, K, and Q, exhibited direct binding to pro-lamine zipcode RNA and assembled into at least threezipcode RNA-protein complexes (Yang et al., 2014).RBPs A-J-K and I-J-K formed two complexes, whileQ formed a third complex with other unidentifiedproteins (Yang et al., 2014). The finding of multiple

complexes is consistent with the dynamic remodelingof mRNP complexes in the multistep process of mRNAlocalization.Although similar affinity chromatography studies

failed to capture any specific RBPs using the glutelinzipcode RNA as bait, a northwestern-blotting approachidentified a key glutelin RNA-binding protein, RBP-P,which was also captured by prolamine zipcode se-quences (Crofts et al., 2010). RBP-P contains two RRMsflanked by Ala/Glu-rich N-terminal and Gly-richC-terminal domains. The two RRMs in RBP-P directlycontribute to its strong binding to glutelin and prola-mine mRNAs (Tian et al., 2018). The auxiliary domainsflanking the RRMs, especially the Gly-rich region,mediated both RNA binding and protein-protein in-teraction (Tian et al., 2018). Point mutations in theinterdomain (A252T) that separated the two RRMmotifs and the C-terminal Gly-rich region (G373E andG401S) reduced the association of RBP-P with pro-lamine and glutelin mRNAs in vivo and causedmislocalization of these mRNAs (Tian et al., 2018),suggesting an essential role of RBP-P in mRNAlocalization.The role of RBP-P in glutelin and prolamine mRNA

localization also requires its association with otherRBPs to form one or more multiprotein complexes. Ayeast two-hybrid screening analysis of a rice seedcDNA library identified twomajor interacting partners,RBP-L and RBP208 (Tian et al., 2018). These RBPs,which contain three RRM motifs, coassembled withRBP-P into multiprotein complexes that specificallybind to prolamine and glutelin zipcode RNAs, asrevealed by in vivo RNA-immunoprecipitation studies(Tian et al., 2018). The following lines of evidence fur-ther substantiated the involvement of RBP-L in glutelinand prolamine mRNA localization. First, RBP-L exhibi-ted strong binding to storage protein zipcode RNAs.Second, a microscopic analysis combining in situ reversetranscription-PCR and immunofluorescence labelingshowed that RBP-L colocalized with both glutelin andprolamine mRNAs in developing endosperm cells

Figure 3. Zipcode elements of prolamine (A) andglutelin (B) mRNAs (adapted from Tian et al.,2019). A, Prolamine zipcode elements, whichconsist of only a single zipcodemotif (*). At left, thelocations of the two zipcode elements within thecoding region and 39 UTR of prolamine mRNA areshown; at right, the consensus sequence of theprolamine zipcode motif is shown. B, GlutelinRNA zipcode elements, which consist of two typesof motifs, zipcode motif 1 (orange triangles) andzipcode motif 2 (magenta triangles). At top, thelocations and components of three glutelin zip-code elements are shown; at bottom, consensuszipcode motif sequences are shown. 59 and 39, 59UTR and 39 UTR.

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(Tian et al., 2019). Third, knockdown of RBP-L expres-sion induced by a T-DNA insertion in its 39 UTR dis-rupted the efficient assembly of the required mRNPcomplexes, which in turn caused partial mislocalizationof glutelin and prolamine mRNAs in rice endosperms(Tian et al., 2019). The RBP-P/RBP-L protein complexmay serve as a scaffold that recognizes and binds toprolamine and glutelin zipcodes for stringent selectionof prolamine and glutelin mRNAs from other unlocal-ized mRNAs.

Although the relationship of RBP-P and RBP-L toRBPs A, I, J, and K (A-J-K and I-J-K) for selectivetransport of prolamine mRNA localization remainsunresolved, RBP208 may serve as a link between thesetwo RBP groups. RBP208 interacts with both RBP-P andRBP-L, forming distinct complexes: the RBP-P/RBP208complex mainly locates in the nucleus, while RBP208only interacts with RBP-L in the cytoplasm (Tian et al.,2018). Additionally, RBP208 interacts with RBP-Q inboth the nucleus and cytoplasm (L. Tian and T.W.Okita, unpublished data). As mentioned earlier, RBP-Qforms a third multicomplex on the prolamine zipcodesequences. Hence, its interaction with RBP208 mayserve as a link between the prolamine and glutelintransport pathways. These findings reflect the multiplefunctions of these RBPs in RNA metabolism and reso-nate with the aforementioned interrelationship of pro-lamine and glutelin mRNA transport pathways.

NONZIPCODE TRANS-FACTORS

In addition to the zipcode-binding RBPs, other fac-tors are also involved in RNA localization. One suchprotein is OsTudor-SN. This RBP was initially identi-fied as a cytoplasm-localized, cytoskeleton-associatedprotein in developing rice grains (Sami-Subbu et al.,2001; Wang et al., 2008). It contains four tandemStaphylococcus nuclease domains (4SN module) and aTudor domain followed by an additional truncated SNdomain in the C terminus (Tsn module; Chou et al.,2017). The N-terminal 4SN module directly binds toglutelin and prolamine mRNAs, while the Tsn modulepossesses the capacity to interact with eight other RBPs(Chou et al., 2017). These binding activities collectivelycontribute to the involvement of Tudor-SN in glutelinand prolamine mRNA localization (Wang et al., 2008;Chou et al., 2017), as reflected by the partial mis-localization of storage protein mRNAs in rice linesexpressing Tudor-SN possessing mutations in thefourth SN and Tudor domains (Chou et al., 2017).

DOES RNA LOCALIZATION COOPTMEMBRANE TRAFFICKING?

The involvement of cytoskeleton-associated OsTudor-SN protein in mRNA localization raised the possibilityof active transport of prolamine and glutelin mRNAs.Indeed, employment of the GFP-based RNAmovement

system (Okita and Choi, 2002; Hamada et al., 2003a)showed that glutelin and prolamine RNAs weredetected asmoving particles. Movement of the particleswas overall unidirectional in a stop-and-go manner,although bidirectional, random, and oscillatory move-ment patterns were also observed. These movementpatterns and the velocity of the RNA transport particlesuggest the role of a cytoskeletal motor protein (Okitaand Choi, 2002; Hamada et al., 2003a). Indeed, drugsthat disrupt the integrity of actin filaments suppressedparticle movement, suggesting the involvement ofmyosin motor protein (Hamada et al., 2003a).

The shape of the RNA-transport particles duringmovement was not uniform but pleomorphic, sug-gesting that the particle’s structure was not a rigid ri-bonucleoprotein but rather a membrane vesicle. Such aview is consistent with the observations that storageprotein RNA localization requires several membrane-trafficking factors. In glup4 and glup6 mutant lines,glutelin mRNAs are mislocalized from the cis-ER to PB-ER (Doroshenk et al., 2010; Tian and Okita, 2014; Chouet al., 2019). Moreover, in both mutant rice lines, anextramural paramural body formed by aborted endo-cytosis is conspicuous. In addition to containing se-creted glutelin and a-globulin storage proteins, theparamural body contains marker proteins for variousendomembranes and mRNAs, the latter suggestingmRNA transport via membrane trafficking. Glup4codes for the small GTPase Rab5 while Glup6 codes forthe guanine nucleotide exchange factor of Rab5 (Rab5-GEF). Given the functions of Rab5 in endosomal traf-ficking and the finding that large amounts of mRNAswere trapped into extracellular paramural bodies (Yanget al., 2018), glutelin mRNA transport may involveendosomal trafficking. A third mutant line, glup2,which contains a defective Golgi Transport1 (Got1),mislocalizes prolamine and a-globulin mRNAs fromthe PB-ER to cis-ER (Fig. 2). Got1 is a membrane-relatedprotein found on COPII vesicles that mediates cargotransport from the ER to the Golgi (Lorente-Rodríguezet al., 2009; Fukuda et al., 2016). Apparently, prolamineand glutelin mRNA transport are assigned to differentmembrane-trafficking pathways.

The detailed molecular mechanism of how storageprotein mRNA localization coopts membrane traffick-ing requires further study. In budding yeast and fila-mentous fungi, mRNA transport has been directlylinked to membrane trafficking (Kraut-Cohen andGerst, 2010; Jansen et al., 2014; Haag et al., 2015;Niessing et al., 2018). A subset of yeast mRNAs arecotransported with the ER (Aronov et al., 2007;Fundakowski et al., 2012), while in the smut fungusUstilago maydis, mRNAs are cotransported on microtubule-powered endosomes (Niessing et al., 2018). There isalso evidence that mRNA localization is associatedwith the ER in metazoans. Xenopus Vg1 RBP (Vera), thetrans-factor of the Vg1 zipcode, colocalizes with the ER(Deshler et al., 1997; King et al., 2005). In mammaliancells, a substantial number of mRNAs that code forcytoplasmic proteins are closely associated with the ER,

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suggesting their direct transport from the nucleus(Chen et al., 2011). Indeed, the RBP p180 may serve asan ER receptor that anchors mRNAs independent ofribosomes and translation (Cui et al., 2012).

mRNA Targeting to Chloroplasts and Mitochondria

In addition to mRNA localization on the ER mem-brane, nucleus-encoded mRNAs are also targeted to ornear the surface of chloroplasts andmitochondria (Weiset al., 2013; Tian and Okita, 2014). Although chloro-plasts and mitochondria contain their own genomes,nearly all of the proteins that constitute these functionalorganelles are nucleus encoded. These proteins aresynthesized in the cytosol and then imported into theorganelles posttranslationally (Martin et al., 2002;Timmis et al., 2004). Emerging evidence over the years,however, indicates that directed mRNA localizationcoupled with cotranslation is also involved in proteintargeting to these endosymbiont organelles.Viruses use various intracellular membranes for

replication. The single-stranded (1) plant RNA virusesreplicate on the chloroplast envelope (Budziszewskaand Obrepalska-Steplowska, 2018). Although themechanism accounting for the localization of the rep-lication complex on the chloroplast membranes has yetto be elucidated, the targeting signal to chloroplast hasbeen suggested to be a 41-residue sequence located inthe two amphipathic helices located in the 140K/98Kprotein domains of Turnip yellow mosaic virus (Moriceauet al., 2017).Several plant viruses and viroids exploit the chloro-

plast as the site of replication. Replication of RNA vi-roids of the Avsunviroidae family occurs in thechloroplasts, although the import mechanism has yet tobe determined (Gómez and Pallás, 2010a, 2010b;Budziszewska and Obrepalska-Steplowska, 2018). A110-bp chloroplast-specific RNA-targeting signal wasidentified at the central region of the viroid RNA se-quence, while the remaining 59-end and 39-end se-quence contributed to RNA folding into a specificsecondary structure for enhancing mRNA targetingand the subsequent protein translation in the chloro-plast (Gómez and Pallás, 2010b). The trans-factors thatrecognize this targeting signal have yet to be identified.The minus-strand RNA of the Bamboo mosaic virus

(BaMV) is also imported into chloroplasts. The nucleus-encoded, plastid phosphoglycerate kinase interactswith the 39 UTR of BaMV RNA and together with heatshock proteins translocate the viral nucleoproteincomplex across the chloroplast membranes (Chenget al., 2013). In this instance, transport of the viralRNA may occur by hitchhiking along with the importof the plastid phosphoglycerate kinase into the stroma.While the overall contribution of RNA targeting to

the chloroplast surface for its biogenesis and functionremains to be determined, this process is an essentialmechanism for mitochondria. The mRNAs of manynucleus-encoded mitochondrial proteins are enrichedon themitochondrial surface (Ahmed and Fisher, 2009).

In general, mRNA targeting to the mitochondria isregarded as an essential process of gene expression andregulation closely linked to cellular functions and theformation of protein complexes that are involved inmetabolic pathways (Margeot et al., 2005). In vascularplants, this view is exemplified by the voltage-dependent anion channel (VDAC), an abundant mito-chondrial outer membrane protein. In Arabidopsis(Arabidopsis thaliana), VDACs are synthesized from twomRNA types that differ in the length of their 39 UTR.While the expression of the shorter mRNA species in aVDAC mutant line restores a normal mitochondriaphenotype, expression of the longer variant alters mi-tochondria size and number (Michaud et al., 2014).Recently, a genome-wide study of potato (Solanumtuberosum) mitochondria-targeted mRNAs via RNAsequencing demonstrated that targeted mRNAs spe-cifically encoded mitochondrial proteins involved inmetabolic pathways, suggesting a coordinated controlof mRNA localization within particular cellular func-tions (Vincent et al., 2017).Based on extensive studies in yeast, several parame-

ters of mitochondria targeting are well established.mRNA targeting to the mitochondrial surface is almostinvariably dependent on translation, as an importantmRNA localization signal is the mitochondrial target-ing sequence (MTS). The synthesis of this peptide signalsuggests its interaction with the translocase of the outermembrane (TOM) complex (e.g. Tom20 and Tom70),which is also required for mRNA localization to mito-chondria (Eliyahu et al., 2010; Gadir et al., 2011). Asecond independent targeting signal is located in the 39UTR. About half of mitochondria-enriched mRNAscontain recognition sites for Puf3 RBP, whichmay serveto anchor the localized mRNA to the mitochondriasurface (Gerber et al., 2004; García-Rodríguez et al.,2007; Saint-Georges et al., 2008). A prime example ofthese two localization signals is observed for the yeastATM1 mRNA, whose localization is dependent on ei-ther its MTS sequence or its 39 UTR (Corral-Debrinskiet al., 2000).In addition to MTS and 39 UTR signals, required for

binding TOM components and Puf3, respectively, thereare likely other factors yet to be identified. The Gerstlaboratory (Zabezhinsky et al., 2016) demonstrated thatCOPI, the complex that transports membrane vesiclesfrom the Golgi to the ER, has an essential role formRNA localization to the mitochondria. COPI inacti-vation was found to decrease the amount of OXA1mRNA associated with the mitochondria while redi-recting these mRNAs to the ER, suggesting a closeconnection betweenmRNA localization andmembranetrafficking.Although mRNA targeting to plant mitochondria is

not as well studied as that in yeast, studies of the Ara-bidopsis VDAC3 mRNA and potato malate dehydro-genase mRNA suggest that the cis-elements located inthe 39UTR are a key signal formRNA localization to themitochondria surface (Michaud et al., 2014; Vincentet al., 2017). Except for the 39 UTR, the upstream

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AUGs have also been proposed as potential regulatorsfor targeting the associated mRNAs to the potato mi-tochondrial surface (Vincent et al., 2017).

Symplastic Transport and Long-Distance mRNAMovement via the Phloem: Targeting tothe Plasmodesmata

In addition to the transport and localization ofmRNAs at specific intracellular locations, RNAs arealso transported between cells or long distances todifferent tissues via a plasmodesmata-mediated sym-plastic route in plants. These mobile RNAs includemRNAs, small RNAs, small interfering RNAs, micro-RNAs, and tRNAs. As long-distance movement hasbeen extensively reviewed in several recent articles(Saplaoura and Kragler, 2016; Kehr and Kragler, 2018;Liu and Chen, 2018; Morris, 2018), we instead focus onwhy and how mRNAs are transported.

In plants, cell-to-cell communication plays an essen-tial role in regulating plant development, growth,disease resistance, and responses to various environ-mental stresses. Intercellular communication involvesthe transport and movement of a large number ofmolecules, such as metabolites, proteins, and RNAs,and is operated by a plant-specific symplasmic path-way mediated by plasmodesmata. Cell-to-cell traffick-ing ofmRNAswas first observed formaizeKNOTTED1(Lucas et al., 1995) and later for the potato Suc trans-porter SUC1 (Kühn et al., 1997). It is now wellestablished that regulators of gene expression (i.e.transcriptional factors and small RNAs) are transportedbetween cells via the plasmodesmata to control organdevelopment and tissue patterning (Otero et al., 2016).

Considerable effort has been directed at under-standing long-distance transport via the phloem.Whileplasmodesmata mainly mediate the cell-to-cell move-ment of molecules, the plant vascular phloem systemconducts long-distance trafficking of photoassimilatesand macromolecules from source to sink tissues (Oteroet al., 2016). Since the first discovery of mRNA speciesin rice phloem sap (Sasaki et al., 1998), increasing evi-dence reveals that a considerable number of mRNAsmove non-cell-autonomously through the phloem (Liuand Chen, 2018; Winter and Kragler, 2018).

The composition of phloem-mobile mRNAs is ex-tensive, covering possibly one-third to one-half of atranscriptome (Morris, 2018). Hence, many phloem-mobile mRNAs may not serve any specific function,as they are constitutively expressed in many tissuesor encode organelle-localized proteins. Therefore,the phenomenon of phloem-mediated long-distancemovement of some mRNAs could be considered asnonspecific bulk flow. Alternatively, many of themRNAs may originate from young sieve cells, whichempty their contents when they mature within thephloem (Knoblauch et al., 2018). However, recentevidence from high-throughput sequencing analysesindicates that mRNA transport may be mediated by a

sequence motif recognition pathway (Thieme et al.,2015; Zhang et al., 2016a; Yang et al., 2019). One suchelement is the tRNA-like structures (TLSs) embedded inthe UTR. Transcripts harboring TLS motifs were foundto be enriched in the phloem-mobile mRNA population(Zhang et al., 2016a), suggesting that the TLS with pre-dicted stem-bulge-stem-loop structure is sufficient tomediate mRNA transport through graft junctions

Figure 4. RNA-labeling approaches to track RNA movement. A, RNAlabeling based on the MS2-GFP system. A GFP (green)-fused MS2 coatprotein (yellow) recognizes and binds to anMS2 hairpin sequence (blueline) that is appended to the target RNA (black line). Analogous strate-gies are the boxB RNA stem-loop, bacteriophage PP7 aptamer tagging,and spinach tracking systems. GFP can also be replaced by other flu-orescent proteins, such as RFP (red). B, Advanced double labeling basedon the fluorescence resonance energy transfer (FRET) system. Two dif-ferent loops, such as MS2 and PP7, are introduced into one (top) or two(bottom) different RNA molecules, which are recognized and bound bytheir corresponding coat proteins to track the movement of specificRNA molecules or simultaneously track two different RNA molecules.C, The split-fluorescent protein approach can also be introduced into anRNA-labeling system to further target specific RNAmolecules. The split-fluorescent protein method can also be applied in combination withdouble labeling of two RNA molecules, as shown in B.

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(Zhang et al., 2016a). Another known motif is the poly-pyrimidine track protein (PTB)-binding sequencedetected in multiple or distributed copies within,generally, ribosome entry sites or intron sequences.This motif composed of UUCUCUCUCUU (Hamet al., 2009), which is recognized and bound by PTBfamily proteins, is present in several known phloem-mobile mRNAs such as Arabidopsis GIBBERELLICACID INSENSITIVE (GAI) and potato StBEL5(Haywood et al., 2005; Huang and Yu, 2009; Choet al., 2015). However, due to the high abundance ofPTB motifs throughout the genome, PTB was questionedto be a specific motif enriched in graft-mobile transcripts(Zhang et al., 2016b; Yang et al., 2019). A more recentstudy from Arabidopsis seedlings suggested that al-though PTB and TLS motifs were enriched in small sub-sets of mobile transcripts, both motifs were not sufficientto predict the mobility of an mRNA (Yang et al., 2019).The study also discovered that m5C methylation mightguide systemic mRNA mobility over graft junctions, asthe modification was found to be common in the mRNAspreviously described as mobile (Yang et al., 2019). Col-lectively, these findings indicate that the mechanism re-sponsible for directing selective transport of mobilemRNAs is much more complex.Cooccurrence of mRNAs and RBPs (Kehr and

Kragler, 2018) revealed that mRNAs are transportedas mRNP complexes in phloem flow. RBPs are the mainbinding proteins to mRNAs through recognition ofRNA sequence motifs and thereby mediate theirtransport and/or protect them from degradation. Oneexample of the link between the transported mRNAand its RBPs is the potato StBEL5 mRNA. Over-expression of its binding proteins, StPTB1 and StPTB6,stabilized and enhanced the long-distance movement ofStBEL5 mRNA, while both its stability and phloemtransportwere decreased in suppression lines (Cho et al.,2015). Proteomics analysis on phloem exudates fromArabidopsis (Tetyuk et al., 2013), rice (Aki et al., 2008),pumpkin (Cucurbita maxima; Haebel and Kehr, 2001; Linet al., 2009), cucumber (Cucumis sativus; Walz et al.,2004), castor bean (Ricinus communis; Barnes et al.,2004), and Brassica napus (Giavalisco et al., 2006) showthat a significant protein class consists of RBPs, includingthe previously characterized pumpkin Phloem SmallRNA-binding Protein1 (PSRP1; Yoo et al., 2004), the16-kD pumpkin phloem protein (CmPP16; Xoconostle-Cázares et al., 1999), the eukaryotic translation initiationfactor 5A (eIF-5A; Pallas and Gómez, 2013), and the 50-kD RNA-binding protein RBP50 (Ham et al., 2009). Ininstances where the binding sequences recognized bythese RBPs have been established, such as for eIF-5A,which specifically recognizes CCUAACCA and AAU-GUCACAC (Xu and Chen, 2001), it provides a facileapproach to identify potential mRNA partners.

OUTLOOK

The advancement of high-resolution microscopy,high-throughput sequencing, and high-resolution mass

spectrometry in recent years will provide powerfulstrategies to investigate mRNA localization in vascularplants. Fluorescence-based mRNA-labeling systemscombined with high-resolution confocal microscopyallows for visualizing the real-time trafficking of targetmRNAs in live cells. Methods such as the GFP-MS2RNA-tracking system (Hamada et al., 2003a; Luo et al.,2018) and its alternative approaches, including theboxB RNA stem-loop (Urbinati and Long, 2011), bac-teriophage PP7 aptamer tagging (Wu et al., 2012), andspinach (Spinacia oleracea) tracking systems (Paige et al.,2011; Bann and Parent, 2012; Dean and Palmer, 2014;Tian and Okita, 2014), in combination with fluores-cence resonance energy transfer and split-fluorescenceprotein systems (Ibragimov et al., 2014; Rath andRentmeister, 2015; Mannack et al., 2016), can be uti-lized to simultaneously monitor the movement of dif-ferent species of mRNAs along the cytoskeleton (Fig. 4).While these systems will require optimization in plants,the employment of fast and sensitive microscopytechniques, such as multiphoton microscopy, totalinternal refection microscopy, and 3D structured illu-mination microscopy (Lange et al., 2008), will pro-vide information that was unprecedented before. Thecombination of biochemical and high-throughputmethods will enable large-scale profiling approachesto investigate mRNA localization. For example, througha combination of cross-linking immunoprecipitationwith high-throughput sequencing, transcriptome-wideRNA-binding sites of RBPs can be elucidated (Lee and

throughput approaches to

OUTSTANDING QUESTIONS

• Except for a few examples established in plant cells, how fundamental is mRNA localization in plant growth and development?

• Do mRNAs co-opt specific membrane trafficking processes to transport mRNAs to distinct subdomains of the endoplasmic reticulum, chloroplast, mitochondria, and plasmodesmata?

• What are the cis-determinants and trans-factors responsible for RNA localization to the plasmodesmata?

• Are mRNAs that code for proteins that share function or intracellular location co-transported as regulons?

• How could we further combine biochemical methods and high-decipher mRNP architecture and dissect the machinery involved in mRNA localization in plant cells?

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Ule, 2018). RNA-protein immunoprecipitation in tan-dem followed by high-throughput sequencing canfootprint the occupancy sites of RBPs and mRNPcomplexes (Singh et al., 2014). Ribosome profiling canglobally monitor the translation status of mRNA dur-ing the localization process to allow investigationof dynamic mRNP complexes (Brar and Weissman,2015). Proteomic profiling can reveal the global pro-tein occupancy on mRNAs to characterize the mRNA-protein interactome and, in turn, elucidate the bindingsites and targets of RBPs during mRNA localization(Baltz et al., 2012).

Further employment of computational approacheswill assist in quantifying the localized mRNAs, deci-phering mRNP architecture, and constructing feasi-ble models for the mRNA localization process (seeOutstanding Questions). Another point that needsconsideration is the modification of mRNAs. Cytosinemethylation is involved in the regulation of mRNAmobility across graft junctions via the phloem andis worthy of investigation for its possible role inmRNA localization. The above-mentioned biochemicalmethods, in combination with high-throughput se-quencing, will advance efforts to dissect the machineryinvolved in mRNA localization in plant cells.Received August 13, 2019; accepted October 1, 2019; published October 14,2019.

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