sorting of messenger rnas in the cytoplasm: mrna localization and the cytoskeleton

EXPERIMENTAL CELL RESEARCH 225, 219–236 (1996)ARTICLE NO. 0172

REVIEW

Sorting of Messenger RNAs in the Cytoplasm:mRNA Localization and the Cytoskeleton

JOHN E. HESKETH1

Intracellular Targeting Group, Rowett Research Institute, Greenburn Road, Bucksburn, Aberdeen, AB2 9SB United Kingdom

The targeting of newly synthesized proteins to their is susceptible to artifacts of fixation and can only dem-site of function plays a major role in cell organization; onstrate the apposition of two components; it cannotfor example, correct localization of proteins is critical provide evidence for a direct association of mRNAs orduring development, for the organization of macromo- polysomes with specific cell components unless com-lecular assemblies such as myofibrils and in cell polar- bined with the use of cytoskeleton-disrupting com-ity. Although a variety of protein motifs are involved pounds, the use of which introduces further caveats; inin the channeling and sorting of proteins themselves, addition, this approach can tell us little about the na-there is an increasing body of evidence to suggest that ture of polysome–cytoskeleton interactions. Usingthere is also a compartmentalization of the protein syn- light microscopy, descriptions of mRNA localization arethetic apparatus within cells. It has been known for relatively imprecise (e.g., peripheral or perinuclear cy-some time that the synthesis of membrane and secreted toplasm, or proximal axon) and at present in situ hy-proteins is carried out on polyribosomes associated bridization combined with electron microscopy, whichwith the endoplasmic reticulum [1] but more recently is required for precise localization, is not sufficientlyit has been found that mRNAs for certain other pro- sensitive to detect many individual mRNA species. Inteins show a distinct subcellular localization [reviewed contrast, a cell fractionation/biochemistry approachin 2–5]. In addition, there is now strong evidence that can lead to the production of fractions which are en-some polyribosomes and mRNAs are associated with riched in cytoskeletal components and the study ofthe cytoskeleton: such an interaction could provide a these and their interaction with polysomes has the po-basis for mRNA transport and localization. tential to produce evidence for direct cytoskeleton–

The aims of this article are twofold: to present and mRNA interactions for specific mRNA species. Eventu-discuss the evidence for the association of mRNAs and ally, this approach is essential to allow a full character-polysomes with the cytoskeleton, and to explore both ization of the cytoskeletal and other components in-the links between mRNA localization and association volved. Importantly, polysome analysis gives informa-of mRNAs with the cytoskeleton and the mechanisms tion on whether or not the association of a particularwhich underlie the ability of cells to sort mRNAs so that mRNA with the cytoskeleton is dependent on thatthey are translated in different parts of the cytoplasm. mRNA being translated and this approach has the po-Such capabilities may produce a spatial organization tential to allow study of the response of polysome–cy-of protein synthesis; this, in addition to known protein toskeleton interactions to physiological stimuli; in con-targeting mechanisms, is likely to be important in the trast localization per se gives no information about theefficient delivery of newly synthesized proteins. translation status of the mRNA. However, to date, the

To date, studies of the intracellular distribution of biochemical approach has given little informationmRNAs have largely involved either a microscopical about ‘‘geographical’’ localization of mRNAs and it suf-approach to study the subcellular location of mRNAs fers from possible artifacts produced during cell disrup-in intact cells or a biochemical approach to investigate tion and fractionation. This approach has producedthe association of mRNA–polyribosome complexes fractions enriched in cytoskeletal-bound polysomes butwith the cytoskeleton following subcellular fraction- improvements in fractionation and characterizationation. The two approaches are complementary, and techniques are required in order to derive maximumboth have their strengths and weaknesses. Microscopy information from this type of experiment. In this article

I discuss data which have been obtained using both1 Fax: (44)-1224-716622. E-mail: [email protected]. approaches since it is only by considering both sets of

219 0014-4827/96 $18.00Copyright q 1996 by Academic Press, Inc.

All rights of reproduction in any form reserved.

AID ECR 3177 / 6i0f$$$361 05-21-96 18:39:29 ecl AP: Exp Cell

220 JOHN E. HESKETH

information and by, in the future, the use of improve- in the case of actin, mRNA localization may provide amechanism to synthesize the various isoforms at differ-ments in both techniques that a full understanding of

mRNA sorting, transport, and localization will emerge. ent subcellular sites. Comparable mechanisms may op-erate with other proteins which exist in isoforms: cre-atine kinase isoform mRNAs exhibit different localiza-CERTAIN mRNAs ARE LOCALIZED IN THE CYTOPLASMtions in transfected myoblasts [22] while the mRNA forone isoform of the protein metallothionein (MT-1) isEarly evidence to indicate that specific mRNAs could

be localized in particular regions of the cytoplasm came found enriched in cytoskeletal-bound polysomes (CBP)and in the perinuclear cytoplasm whereas the mRNAfrom in situ hybridization studies of germ cells of Xeno-

pus and Drosophila [6, 7]. Such localized mRNAs repre- for a second isoform appears to be more weakly associ-ated with the cytoskeleton [23].sent a relatively small proportion of all mRNAs but

appear to code for proteins which are developmentally A number of studies suggest that the peripheral lo-calization of b-actin mRNA is required for the localimportant. Thus, the Vg1 and X-cat mRNAs are local-

ized in the vegetal half of the Xenopus oocyte [8, 9] synthesis of protein to provide a pool of actin duringlamellipodia extension and cell spreading. For exam-while, in Drosophila, bicoid and nanos mRNAs are

found localized to the anterior and posterior poles, re- ple, in wounded endothelial cells relocalization of b-actin mRNA to the cell periphery is associated withspectively [10–12], and cyclin B mRNA is found at the

posterior pole [13]. In the blastoderm embryo bicoid, increased amounts of b-actin protein in this area [24].In myoblasts the peripheral distribution of b-actineven-skipped, hairy, and fushi tarazu mRNAs all ex-

hibit localization in the apical compartment [14]. mRNA is paralleled by that of the protein [18]. Further-more, delocalization of b-actin mRNA by antisense oli-mRNA localization in oocytes and embryos is poten-

tially of considerable physiological significance. The lo- gonucleotides alters lamellipodia structure, stress fiberorganization and cell morphology [25]. In contrast, ad-calization of a restricted range of mRNAs may be the

basis for correct determination of embryonic polarity dition of serum to serum-starved 3T3 fibroblasts wasfound to alter b-actin mRNA localization without anyand subsequent development [12, 15] by providing a

mechanism for the synthesis of these specific proteins apparent effect on protein distribution [21] but thismay be accounted for by a failure to distinguish newlyin a precise part of the embryo.

The accumulation of RNA transcripts at particular synthesized actin from a large pool of preexisting pro-tein.cytoplasmic sites now appears to be a general phenome-

non which occurs not only in germ cells and early em- mRNAs have also been found to be localized in avariety of highly polarized cells: for example, in intesti-bryos but also in a wide variety of somatic cell types.

For example, in fibroblasts transfected with exons 2 nal epithelium cells actin mRNA is localized in the api-cal region of the cytoplasm [26] and in oligodendrogliaand 3 of the c-myc gene the mRNA is found to be pres-

ent only in the perinuclear cytoplasm (Fig. 1 and [16]). the mRNA for myelin basic protein has been found tobe localized in the extremities of the long cell processes,This distribution contrasts dramatically with that of

b-actin mRNA, which, in fibroblasts that are actively both in cultured cells [27] and in vivo [28, 29]. Fluores-cently tagged myelin basic protein mRNA is localizedspreading on the substratum, is found in the cell pe-

riphery in the cytoplasm underlying the lamellipodia after microinjection and time-course studies suggestthat the mRNA is transported along the cell processes[17]. In myoblasts b-actin mRNA is found to be in the

peripheral cytoplasm but the a- and g-isoform mRNAs [27]. In neurons, specific mRNAs are present in den-drites but not in axons [30–34] and the mRNA for theare largely in the perinuclear cytoplasm [18, 19]. Fur-

thermore, localization of b-actin mRNA responds to microtubule-associated protein, tau, is localized in theproximal portion of the axon in neurons grown in cellphysiological stimuli such as alterations in growth con-

ditions or addition of growth factors [20, 21]. In two culture [34]: in the case of MAP2, mRNA distributionhas been found to reflect the protein localization [30].similar studies it was found that in cells growing in

normal serum concentrations the mRNA was found Recently, two tropomyosin isoform mRNAs have beenfound to be differentially localized in developing neu-both in peripheral cytoplasm underlying the lamelli-

podia and in the perinuclear cytoplasm, but when the rons and the mRNA localization correlates with thetargeting of one isoform protein to growing axons [35].cells were deprived of serum the mRNA in the periph-

eral cytoplasm disappeared and was found only in the In fibroblasts vimentin mRNA is found in the peri-nuclear cytoplasm [36]. However, in muscle, whereperinuclear cytoplasm. Readdition of serum caused a

rapid (within 10 min) reappearance of b-actin mRNA desmin and vimentin are components of the costam-ere, both desmin and vimentin mRNAs exhibit a band-in the peripheral cytoplasm: this was unaffected by

inhibition of RNA synthesis and thus appeared to rep- ing pattern and their localization alters during forma-tion and development of the costameres and myofibrilsresent relocalization of existing mRNA. It appears that


FIG. 1. Localization of c-myc mRNA. In situ hybridization of fibroblasts transfected with a construct containing exons 2 and 3 of thec-myc gene which includes most of the coding sequence and the 3*UTR. Note the distinct perinuclear distribution of the staining and thelack of staining in the peripheral cytoplasm. For details see Ref. [16].

FIG. 3. Identification of an 86-nt region of the c-myc 3 *UTR which is required for perinuclear targeting of b-globin coding sequences.In situ hybridization showing the distribution of transcripts containing b-globin coding sequences. Transcripts were detected using digoxi-genin-labeled riboprobes, anti-digoxigenin linked to alkaline phosphatase and Vector red substrate to yield fluorescent product. Results areshown from untransfected rat embryo fibroblasts and from cells transfected with three different constructs in which globin coding sequencesare linked to either the whole c-myc 3*UTR (globin-myc) or to truncated c-myc 3*UTR sequences containing bases 194–440 (deletion 3) or280–440 (deletion 4). Note the perinuclear distribution of transcripts in the globin-myc and deletion 3 lines and the presence of transcriptthroughout the cytoplasm in deletion 4. Results are unpublished data from Hesketh, Veyrune, Campbell, and Blanchard; additional datain [103].

05-21-96 18:39:29 ecl AP: Exp Cell

222 JOHN E. HESKETH

[37, 38]; the timing of appearance of mRNA localiza- THE EVIDENCE FOR CYTOSKELETAL-BOUNDPOLYSOMES AND mRNAstion correlates with the incorporation of the proteins

into the costameres. In intact muscle both myosinTreatment of eukaryotic cells with low concentra-heavy chain mRNA and ribosomes have been reported

tions of nonionic detergents releases soluble cellularto be present in the myofibrillar cytoplasm [39, 40].components and plasma membrane components butDespite occasional vague banding reported in someleaves an insoluble cell matrix containing cytoskeletalstudies, most data show no clear banding pattern infilaments and other cellular material. Electron micros-the distribution of the myosin heavy chain mRNA incopy and sucrose gradient analysis have shown thatthe myofibrillar cytoplasm [40–43] except after elec-such cell matrix material from a variety of cell linestrical stimulation [44]. Thus, although the exact dis-contains polysomes, translation initiation factors, andtribution of myosin heavy chain mRNA in muscle isapproximately 70–85% of the cellular mRNA [45–47]:still the subject of debate it is clear that it is presentrecently, in situ hybridization using a poly(T) oligonu-in the cytoplasm between the myofibrils. The localiza-cleotide to detect most mRNAs has confirmed that ap-tion of the mRNAs coding for myosin, vimentin, andproximately 75% of cellular mRNAs are retained in thedesmin is compatible with their being translated incell matrix [48]. It is observations such as these thatclose proximity to the site of assembly of the proteinshave led to the suggestion that the majority of cell poly-into the myofibril; this could provide a mechanism forsomes are associated with the cytoskeleton.precise targeting of the proteins during myofibrillar

It is clear from such studies that mRNAs and poly-turnover or muscle growth.somes are present in the cell matrix, as illustratedThe increasing number of studies of mRNA localiza-by the positive immunostaining of the cell matrix bytion support the hypothesis that in a variety of cellsantibodies that recognize ribosomal components [49,and tissues there is subcellular localization and trans-50]. However, it is often unclear in these studiesport of specific mRNAs. In several cases, such as vimen-whether these mRNAs and polysomes are associatedtin, b-actin, tropomyosin, and MAP2, localization of thewith the cytoskeleton or with other cell componentsmRNA often corresponds closely to the protein localiza-also retained in the cell matrix. Many of the originaltion, suggesting that such mRNA sorting/targeting pro-studies ignored the existence of membrane-boundvides a mechanism for the synthesis of certain proteinspolysomes associated with the endoplasmic reticu-close to their site of function, i.e., for a targeting of thelum and it was assumed that all the polysomes in

protein synthetic apparatus. It remains to be seen to the cell matrix were associated with the cytoskeleton.how great a proportion of mRNAs are localized within However, there is strong evidence both that frag-the cytoplasm. However, the local synthesis of proteins ments of the endoplasmic reticulum and membrane-which such mRNA localization can provide is poten- bound polysomes remain (trapped?) in the cell matrixtially an energetically efficient mechanism for the tar- and that at least some of the polysomes released fromgeting of proteins: it may be important in the case of the cell matrix by deoxycholate are derived from theunstable proteins which otherwise might be largely de- endoplasmic reticulum (see Table 1). Electron mi-graded, it appears to allow the segregation of protein croscopy and studies of biochemical markers showisoforms, and it could prevent proteins from getting to that the cell matrix contains membranous fragmentsthe wrong part of the cell. Furthermore, it appears that of the unsolubilized endoplasmic reticulum [51, 52];mRNA localization can respond to growth factor stimu- furthermore, the cell matrix has been shown to con-lation; thus it potentially provides a mechanism for tain mRNAs for membrane proteins [53]—theseregulating the site of mRNA translation and protein mRNAs would be expected to be translated on thesynthesis within cells. endoplasmic reticulum. Breakdown of actin filaments

A number of different mechanisms have been pro- in the cell matrix, either by fragmentation of actinposed to account for the observed localization of filaments with cytochalasin D or by extraction of themRNAs: differential control of mRNA stability in dif- cell matrix with 130 mM KCl causes release fromferent cell compartments, vectorial nuclear export of the cell matrix of some mRNAs but not of those formRNA, targeted transport of mRNAs, and the anchor- membrane proteins whereas the mRNAs for mem-ing of mRNAs to localized cell components. The evi- brane-proteins such as b2-microglobulin and glucosedence for stability and nuclear export models is limited transporter 1 are present in polysomes released fromto a few examples in Drosophila oocytes [5] but there the microfilament-depleted cell matrix by deoxycho-is increasing evidence for the targeting and/or anchor- late [23, 54 –57]. These data emphasize that the pres-ing of mRNAs, and an increasing body of evidence indi- ence of translational components within the cell ma-cates that the cytoskeleton is involved in these pro- trix is not sufficient evidence for an association with

the cytoskeleton: furthermore, polysomes released bycesses.


223mRNA LOCALIZATION AND THE CYTOSKELETON

TABLE 1

Agents Which Induce either Solubilization of the Endoplasmic Reticulum or MicrofilamentDepolymerization Release Different Components from the Cell Matrix

Presence in cell Enriched in fractions released Released byComponent matrix by microfilament collapse deoxycholate

Membrane markersMembrane fragments [51, 67] / n.d. n.d.[14C]Choline [52] n.d. 0 /b-2 Microglobulin mRNA [55] n.d. 0 /Glucose transporter mRNA [23, 56] n.d. 0 /HLAB7 mRNA [53, 54] / 0 /

Cytoskeleton markersActin [23, 52, 56] / / 0Vimentin [23, 52, 56] / / /Cytokeratin [52] / / 0

Note. n.d., not determined.

double-detergent treatment following nonionic deter- fraction [48, 52, 65–67], thus indicating that actin de-polymerization induces polysome redistribution. Simi-gent treatment at low salt do not originate solely

from the cytoskeleton but also from the rough ER lar effects have been observed after actin depolymeriza-tion induced by DNase 1 [68] and after treatment ofand should not therefore be described as cytoskeletal-

bound polysomes [57]. the cell matrix with 130 mM KCl which causes actindepolymerization and also redistribution of polysomesStronger evidence for association of polysomes and

mRNAs with the cytoskeleton has come from two broad so that there is loss from the cell matrix and increasedrecovery in the soluble fraction [52, 69]. These threetypes of experiment: from a microscopical approach in-

vestigating the colocalization of components of the treatments release cytoskeletal proteins from the cellmatrix whereas deoxycholate treatment releases ERtranslational apparatus with filament structures and

from biochemical studies in which the release of poly- components (Table 1).Elongation factor 1 has sequence characteristicssomes/mRNA from the cell matrix is studied after

treatments causing disruption of the cytoskeleton. Im- which suggest that it is an actin-binding protein [70].This highly abundant protein (1–2% of cell protein,munohistochemical and histochemical studies have

shown that ribosomal material [49, 50], 5* mRNA bind- the second most abundant after actin) is found to becolocalized with actin filaments in Dictyostelium amoe-ing protein [58], and both initiation and elongation fac-

tors [59–61] are localized within cells in a pattern bae [71] and in fibroblasts [63] and has actin cross-linking activity [70–72]; in addition, the protein haswhich closely resembles that of the cytoskeletal net-

work. In situ hybridization techniques have also shown been reported to have microtubule severing activity[73]. Other translation factors, namely initiation factorthat poly(A)-containing mRNA colocalizes with cy-

toskeletal elements [48]. The combination of in situ 2 and elongation factor 2, have also been reported tobe colocalized with microfilaments and intermediatehybridization and electron microscopy has shown

mRNA species coding for actin, tubulin, and vimentin filaments [60, 61]. There is thus growing evidence, par-ticularily in the case of elongation factor 1 [72], thatto be clustered around filaments in the cell matrix [62]:

poly(T) probes, which detect all mRNAs with poly(A) regulatory translation components of polysome factorare associated with, and can interact with, the cytoskel-tails, have shown mRNAs to be associated with fila-

ments, particularly at the intersections or cross-over eton; these studies provide support for the concept thatsome polysomes are associated with the cytoskeleton.points where filaments interact [63]. These observa-

tions are consistent with the earlier high-voltage and The association of polysomes with the cytoskeletonhas also been reported to occur in plant cells. Extrac-deep-etch electron microscopy studies that showed

polysomes to be close to, or surrounding, filamentous tion of plant tissues has shown that cytoskeletal frac-tions contain polysomes [74, 75]. Recently it has beenstructures and at filament intersection points [64].

Pretreatment of cells with cytochalasins causes de- found that in the presence of neutral detergent KClconcentrations between 100 and 200 mM cause re-polymerization of actin and collapse of the microfila-

ment network. This is accompanied by loss of mRNAs lease of both cytoskeletal proteins and polysomes [76,77]. At present the precise conditions required to sep-and polysomes from the cell matrix and an increase in

the proportion of polysomes recovered in the soluble arate free, cytoskeletal, and membrane-bound poly-


224 JOHN E. HESKETH

somes, and the cell compartments from which such tural studies, in vitro studies and the use of depolymer-izing agents in combination with in situ hybridization,polysomes are derived, remain somewhat unclear; in-

deed cytoskeletal proteins have been reported to be do suggest that microtubules both interact with thetranslational apparatus and play a role in mRNA local-present in membrane-bound polysome fractions [76,

77]. Further characterization of the various polysome ization. There is evidence from electron microscopy andin vitro observations for an association of ribosomesfractions, particularily in terms of their mRNA com-

plement, will allow further dissection of polysome and RNA with microtubules, particularly with the mi-totic spindle and microtubule organizing centers [re-compartmentalization in plants. It appears that, as

in animal cells, some polysomes are associated with viewed in 78]. Furthermore the microtubule-associatedprotein tau has been reported to be present in brainthe cytoskeleton in plants.polysomes [79] and, more recently, it has been shownby electron microscopy that microtubule preparationsROLES OF DIFFERENT CYTOSKELETAL COMPONENTSfrom oocytes and embryos contain ribosomes or poly-IN CYTOSKELETON–RNA INTERACTIONSsomes [80]. The significance of these in vitro observa-tions is unclear since the specificity of the interactionsThe overall picture that emerges from the variety

of biochemical, immunohistochemical, and morpho- between polysomes and microtubule proteins remainsto be established [78]. An indication that such interac-logical studies indicates an association of polysomes

and mRNAs with the cytoskeleton. There is strong tions may be specific is provided by the observationthat in microtubule preparations generated from seaevidence to indicate that this association involves the

actin-containing microfilaments. Electron microscopy urchin eggs only ribosomes were found associated withmicrotubules whereas in preparations from embryos,of lens cells shows polysomes associated with micro-

filaments [66] and recently, the combination of elec- in which translation is much more active, polysomeswere also observed [80]; furthermore in the prepara-tron microscopy and in situ hybridization has shown

that, in fibroblasts, 72% of polyadenylated mRNA is tions from embryos there was evidence that the poly-somes contained specific mRNAs. However, in thisfound in juxtaposition to actin filaments [63]. Immu-

nohistochemistry has shown that in fibroblasts ribo- case, even if the interaction is of physiological signifi-cance, since the recovered ribosomes/polysomes thatsomes [49, 50] and translation factors [60, 61] colocal-

ize with actin filaments. These studies show close co- were associated with the microtubles accounted foronly 1–3% of the total cell complement, it is quantita-localization with part of the actin network —not all

filaments have polysomes associated with them [61] tively only a minor component of the mRNA/polysome–cytoskeleton interaction: these polysomes might repre-and this is also evident in in situ hybridization studies

of total mRNA distribution where digital imaging mi- sent a specific compartment of the protein syntheticapparatus.croscopy shows close association of mRNAs with some

but not all actin filaments [48]; it appears that it is Electron microscopy has shown that insect nutritivetubes contain large numbers of ribosomes associatedthe finer actin filaments, not the stress fibers, that

are associated with mRNA. with microtubules [81]. These cells are carrying out ahighly specialized transport function and it may be thatFurther evidence for microfilament–polysome/

mRNA association has also come from the use of agents the ribosome– microtubule interaction is due to a trans-port role of the microtubules. Interestingly, there iswhich induce filament depolymerization or stabilize

microfilaments: thus cytochalasins B or D, which in- evidence for a role of microtubules in mRNA transportfrom a number of inhibitor studies in oocytes. In Xeno-duce depolymerization, release polysomes and mRNAs

from the cell matrix; DNase I, which also induces de- pus oocytes the different effects of colchicine and cyto-chalasin indicate that microtubules are required forpolymerization, also causes release of polysomes [68]

while phalloidin, which stabilizes actin filaments, pre- translocation of the Vg1 mRNA to the cortical cyto-plasm of the vegetal half of the egg but microfilamentsvents the loss of actin and polysomes [52, 68]. Finally,

the fact that extraction of the cell matrix with 130 mM are involved in anchoring the mRNA in some way sothat its distribution was restricted to this specific partsalt (known to affect stability of microfilaments but not

that of intermediate filaments) leads to concomitant of the cytoplasm [82]. In Drosophilia oocytes there is agradient of microtubules from anterior to posterior poleloss of actin and polysomes from the cell matrix [50,

52, 69] also indicates the association of polysomes with [83] and the localization of specific mRNAs (e.g., bicoid,oskar) is sensitive to colchicine and thus probably de-the microfilament network. The importance of micro-

filament–polysome interactions is emphasized by the pendent upon microtubules [13, 84]; in addition mu-tants with a symmetric microtubule network exhibitincreasing number of observations which imply that

elongation factor 1 is an actin-binding protein [72]. localization of bicoid mRNA at both poles [85]. Thedistribution of staufen, a protein which binds to theIncreasingly, other approaches, namely ultrastruc-



localized bicoid mRNA, is also altered by colchicine protein vimentin, initiation factor eiF2 is still associ-ated with filaments [59]. On the other hand there aretreatment [86]. Furthermore, a fusion protein in which

b-galactosidase is linked to the microtubule motor pro- a number of observations indicating that there is anassociation of mRNA with such filaments; for example,tein kinesin is normally localized in Drosophila oocytes

but this localization is destroyed by colchicine and lost the 50-kDa cap-binding protein appears to colocalizewith IF [58] and, in oocytes the Vg1 mRNA is recoveredin mutants in which both staufen and oskar mRNA are

mislocalized [87]. Microinjection of myelin basic pro- in a cytokeratin/vimentin-enriched fraction [9, 90, 91].In addition, ultrastructural in situ hybridization sug-tein mRNA into oligodendroglia results in a transport

of the mRNA down the long cell processes [27] and gests that some 29% of mRNAs are present in closeproximity to intermediate filaments [63]. The discrep-confocal microscopy suggests a close apposition of the

mRNA ‘‘particles’’ and microtubules; the speed of trans- ancy in results for polysomes and for some individualmRNAs suggests that some specific mRNAs, possiblyport (0.2 mm/s) is compatible with a microtubule- and

kinesin-based mechanism [2]. The transported mRNA those that are in an untranslated form as opposed topolysomes, may be associated with IFs. Recently, it hasis not extracted by nonionic detergent and on this basis

has been described as associated with the cytoskeleton. been found that although in rapidly growing fibroblaststhe translation factor eEF-2 codistributes with micro-Recently, dual channel confocal microscopy has been

used to study the distribution of myelin basic protein filaments, in growth-arrested cells it appears to be dis-tributed in a pattern which resembles that of interme-mRNA and various components of the protein synthetic

apparatus in oligodendrocytes [88], and the results sug- diate filaments or microtubules [92]. This suggests thatthe interactions of the translational machinery withgest that the mRNA colocalized in granules with ribo-

somal components, elongation factors, and aminoacyl- the cytoskeleton are altered by growth conditions: fur-thermore, if eEF-2 remained with untranslatedtRNA synthetases; such granules may represent a su-

pramolecular complex which allows transport and lo- mRNAs during growth arrest this observation wouldalso be compatible with the hypothesis that while ac-calization of the mRNA.

The combination of electron microscopy and in situ tively translated mRNAs in polysomes are primarilyassociated with microfilaments, untranslated mRNAshybridization has recently been used to dissect the na-

ture of the cytoskeleton –mRNA interaction using are associated with intermediate filaments.In conclusion there is both strong evidence thatprobes to detect total polyadenylated mRNAs [62, 89].

Although in fibroblasts most mRNAs are found in close mRNAs and polysomes are associated with microfila-ments and also increasingly significant indications thatproximity to actin filaments and less than 10% are close

to microtubules, in cultured neurons over 55% of the some mRNAs and polysomes can be associated withmicrotubules and that microtubules may be involvedmRNA is closely apposed to microtubules, suggesting

a close association of mRNAs with microtubules [89]. in ribosome/mRNA transport. It is possible, althoughmore speculative, however, that intermediate fila-These data suggest that the extent of the mRNA–mi-

crotubule interaction varies between cell types, with ments interact with untranslated mRNAs. Thus theremay be a series of different interactions betweenthe greater interaction in cells which require transport

of RNAs over large distances. mRNAs, polysomes, and the cytoskeleton which formthe basis of mechanisms for compartmentalizing,Overall, there is an increasing body of evidence to

indicate that microtubules are involved in mRNA, ribo- transporting, and localizing mRNAs (Fig. 2).some, and perhaps polysome transport. This functionwould be compatible with the observation that microtu- CYTOSKELETAL-BOUND POLYSOMES CONTAINbule-associated polysomes represent a small proportion SPECIFIC mRNAsof total cell polysomes [80] since the proportion ofmRNAs and polysomes being transported on microtu- Detailed study of the nature and function of the poly-

some–cytoskeleton interactions requires the isolationbles could be small at one moment in time. Such aproportion would increase in cells which transport of specific cytoskeletal-bound polysomes (CBP). Some

success at releasing cytoskeletal-bound mRNAs andmRNAs over longer distances since the interactionwould be maintained for a considerably longer time. polysomes from the cell matrix has been achieved using

cytochalasins, DNase1, and 100 –200 mM KCl: theseThe evidence for interactions between intermediatefilaments and the protein synthetic apparatus is con- methods indicate 35–45% of polysomes are associated

with the cytoskeleton (microfilaments). Salt concentra-tradictory. On the one hand some data suggest thatthere is no interaction between polysomes and interme- tions above 100 mM have been shown to cause both

cytoskeletal disorganization [45], actin depolymeriza-diate filaments: polysomes are found in areas of thecytoplasm which are free of intermediate filaments [45, tion [69], progressive loss of RNA [67], and release of

polysomes [69, 76]. In myoblasts the salt treatment57] and in cells which lack the intermediate filament


226 JOHN E. HESKETH

FIG. 2. The role of the cytoskeleton in sorting of mRNA. A scheme showing how various cytoskeletal components may be involved inthe sorting, transport, and localization of mRNAs. The role of intermediate filaments is unclear and the suggested interaction with untrans-lated mRNAs speculative.

produced a fraction which contained specific mRNAs mRNA is released by the microtubule-disrupting agentcolchicine [93] and 70% of the Vg1 by cytochalasins[67]. It was shown that treatment of the cell matrix

with 130 mM KCl releases a fraction which differed in [82]. Cytoskeletal fractions from mammalian somaticcells are also enriched in certain mRNAs: the cytoskele-sedimentation profile and which was enriched in actin

and thus appeared to be a cytoskeletal fraction [52, 59]; tal fraction released from the cell matrix by salt treat-ment is enriched in c-myc mRNA [55] and histonefurthermore the polysome content of this fraction was

depleted by pretreatment of the cells with cytochalasin. mRNAs have been reported to be present in the cellmatrix and to be released by cytochalasins [54, 59, 67,Thus salt treatment of the cell matrix appears to pro-

duce a fraction enriched in microfilament-associated 94] and by increased hydrostatic pressure which alsodisrupts the microfilament network [95]. In analysis ofpolysomes; nonionic detergent treatment followed by

130 mM KCl treatment of the cell matrix and the ex- MPC-11 cells actin mRNA was recovered in the cyto-solic fraction in starved cells but in both the cytosolictraction with deoxycholate produces fractions of free,

cytoskeletal-bound, and membrane-bound polysomes. and cytoskeletal fractions after serum stimulation [96].This may reflect the altered b-actin mRNA localizationAlthough the methods used to release CBP from the

cell matrix cause breakdown of cytoskeletal compo- known to occur during stimulation [20, 21]: however,polysomes were not isolated from fractions in these ex-nents and are unlikely to retain intact cytoskeleton–

polysome complexes, it is possible to use this fraction- periments and it is not clear which isoform(s) was de-tected under the hybridization conditions used.ation approach to investigate which mRNAs are associ-

ated with the cytoskeleton. Analysis of mRNAs using The data from the analysis of these fractions give noinformation as to whether the mRNA is associated withhybridization techniques indicates that cytoskeletal

fractions or polysomes isolated from such fractions are the cytoskeleton in polysomes (i.e., while being trans-lated) or in some untranslated form. Isolation of poly-enriched in certain specific mRNAs (Table 2), as would

be expected if CBP are a distinct polysome compart- somes from such fractions, and their subsequent analy-sis by hybridization assays, is necessary to gain infor-ment involved in the synthesis of specific proteins.

A cytokeratin-enriched fraction from Xenopus oo- mation on those mRNAs being translated in CBP. In avariety of mammalian cell lines (fibroblasts, hepatomacytes has been shown to be enriched in Vg1 and Xcat

mRNAs [9, 90] and similar fractions from Drosophila cells, ascites cells) CBP have been found consistentlyto be enriched in c-myc mRNA [16, 23, 55, 56]: in addi-oocytes are enriched in bicoid mRNA [84]; bicoid



TABLE 2

Examples of mRNAs Recovered in Cytoskeletal Fractions, CBP and FP

mRNA Nature of fraction Reference

c-myc CBP released by salt [16, 23, 55, 56, 99]Cyclin A CBP released by salt [56]Ribosomal protein L4 CBP released by salt [56]Ribosomal protein S6 CBP released by salt [56]Metallothionein 1 CBP released by salt [23]Histones Released by cytochalasin from cell matrix [53, 54, 67, 94, 95]c-fos Released by cytochalasin from cell matrix [54]b-actin CBP released by salt, released by cytochalasin [55, 67, 94, 95, 99]Vg1 Cytokeratin-enriched fraction from oocytes [9, 90]Xcat Cytokeratin-enriched fraction from oocytes [9]Bicoid Cytoskeletal fraction from oocytes (released by colchicine) [84, 93]

b-globin FP released by Nonidet P40 [16]Cytosolic glutathione peroxidase FP released by Nonidet P40 [100]Phospholipid hydroperoxide FP released by Nonidet P40 [100]Glutathione peroxidase

tion, CBP are enriched in the mRNAs for ribosomal published observations), are also present in CBP, indi-cating that other classes of mRNAs are also associatedproteins L4 and S6, cyclin A [56], and metallothionein

1 [23]. Treatment of the cell matrix with cytochalasins with the cytoskeleton. In contrast, mRNAs for b-globin[16] and two glutathione peroxidases [100] have beenreleases CBPs which are enriched in c-fos and histone

mRNAs [54, 67]. In fibroblasts and myoblasts actin found not to be enriched in CBP but in the FP.Redistribution of c-myc mRNA from CBP to FP, withmRNA is recovered largely on CBP [55, 57, 94, 97] and

in several of these studies the CBP show an enrichment a loss of perinuclear localization, does not cause anarrest of translation, as judged by the presence ofin actin mRNA. In 3T3 fibroblasts [55] this was not the

case and the actin mRNA appeared to be present in mRNA in polysomes and the immunohistochemicaldemonstration of protein product [16, 101]. Thus,both FP and CBP. Further analysis of actin isoform

mRNAs in FP and CBP is important as it may produce mRNA localization and association with the cytoskele-ton are not obligatory for translation. However, prelim-some insight into the relationship between recovery of

an mRNA in CBP and its spatial localization. inary data suggest that these changes do affect subse-quent targeting of the protein product since cells inThe bulk of evidence suggests that CBP are enriched

in certain specific mRNAs and thus that they are in- which both association of c-myc mRNA with CBP andlocalization are lost have a smaller proportion of c-mycvolved in the translation of specific mRNAs. In order

to assess the full functional significance of CBP and protein in the nucleus than cells in which the c-mycmRNA is perinuclear [101]. This is compatible withtheir relationship to mRNA localization and transport

it is essential to identify those mRNAs present in CBP. the hypothesis that c-myc mRNA is retained on thecytoskeleton in the perinuclear cytoplasm to maintainThe mRNAs for histones, cyclin A, ribosomal proteins,

myc, and fos are all found in CBP and these proteins synthesis close to where the protein is needed and topromote the efficiency of subsequent protein importare all transported back into the nucleus after synthe-

sis. In addition, metallothionein 1 mRNA has been into the nucleus.Methods based on the KCl- or DNase-1-induced cy-found in CBP [23] and there is evidence that metallo-

thionein 1 can be found in the nucleus after stimulation toskeleton disorganization provide the best approachto isolate CBP from cultured cells at present. It shouldof hepatocytes with growth factors [98]. It may be that

mRNAs for proteins which are targeted to the nucleus be noted, however, that such procedures have their lim-itations. For example, these agents primarly affect theare one class of mRNAs translated on CBP [55, 56];

association with the cytoskeleton may permit the re- microfilament system and so may produce a subpopula-tion of CBP bound to microfilaments; the precise com-tention of these mRNAs in the perinuclear cytoplasm

as suggested by the perinuclear localization of c-myc partment from which CBP are released has not beenwell defined and it is not clear if such fractions repre-mRNA [16]. However, other mRNAs, such as those en-

coding actin [55, 67, 94, 97, 99, 100], vimentin, and sent polysomes from a distinct microfilament-associ-ated compartment, from several such compartments,glyceraldehyde-3-phosphate dehydrogenase (Hesketh,

Jodar, Johanessen, Partridge, Pryme, and Tauler, un- or from a number of heterogeneous cytoskeletal-bound


228 JOHN E. HESKETH

polysome populations. In addition, the optimal salt and association of mRNAs with the cytoskeleton appears tobe correlated with that mRNA being localized withindetergent concentrations for separation of the poly-

some fractions vary between cell lines and it is there- the cytoplasm, this association does not determine asingle location and can result in different localizationsfore vital to fully characterize a putative CBP fraction.

Improvements in these fractionation techniques are re- of different mRNAs. Second, the polysomes andmRNAs present on the cytoskeleton do not representquired in order to define with precision which mRNAs

are translated in which compartment. one subcellular compartment but two or more compart-ments. However, it does appear that in many casesan association of an mRNA with the cytoskeleton isLOCALIZED mRNAs ARE ASSOCIATED WITH THEaccompanied by its sorting and localization.CYTOSKELETON

A proportion of polysomes and mRNAs are releasedfrom cells by nonionic detergent treatment [57]. On theIn oocytes, localized mRNAs such as bicoid are recov-

ered in a nonionic detergent insoluble fraction which basis that they are released along with lactic dehydro-genase these polysomes have been considered as ‘‘free’’has been characterized as being derived from the cy-

toskeleton by identification of specific cytoskeletal pro- polysomes (FP) not associated with the cytoskeletonand therefore released during the initial extraction. Inteins [9, 84, 90]. Both Vg1 and Xcat-2 mRNAs are only

localized at certain stages of oocyte development and studies of transfected cells both b-globin mRNA and c-myc coding sequences linked to the b-globin 3*UTRit is only at these stages that the mRNAs are recovered

in the detergent-insoluble fraction [9]. Similarly, in fi- have been found to be present largely in FP and, by insitu hybridization, to be exhibit no particular cyto-broblasts transfected with a control c-myc construct the

c-myc mRNA is associated with CBP [16], as in normal plasmic localization but to be present throughout thecytoplasm [16, 103, 104]. Thus, at least for thesefibroblasts [55], and also shows a distinct perinuclear

localization [16]; however, in cells transfected with a mRNAs, presence in FP is accompanied by a lack ofmRNA localization: this is consistent with FP beingchimeric construct the mRNA is no longer localized and

no longer associated with the cytoskeleton [16]. Thus, free polysomes derived from the cytosol and withmRNAs present in FP not being localized. However,studies, in parallel, of mRNA localization by in situ

hybridization and association with the cytoskeleton by although the mRNAs encoding both cytosolic and phos-pholipid hydroperoxide glutathione peroxidases havecell fractionation suggest that localized mRNAs are in-

deed associated with the cytoskeleton. been found at highest abundance in FP [100] there isa significant difference between the relative enrich-This view is supported by experiments showing that

b-actin mRNA localization is destroyed by disruption of ment of the two mRNAs in FP. This indicates that FPmay not be a homogenous population but may includemicrofilaments [102]. However, although b-actin and c-

myc mRNAs are both found associated with the cytoskel- mRNAs from different compartments, possibly even inpolysomes loosely associated with some cell structureeton, and both mRNAs are localized, the two mRNAs

are localized to different parts of the cell, namely the such as the cytoskeleton. In contrast, it appears thatmRNAs in CBP are those that are localized for transla-peripheral cytoplasm and perinuclear cytoplasm. Under

extraction conditions (nonionic detergent and 100 mM tion at specific sites.KCl) which would be expected to partly destabilize thecytoskeleton approximately 50% of b-actin mRNA is re- mRNA SORTING: ROLE OF 3*UTR ANDtained in the cell matrix both under control conditions CYTOSKELETONand after induction of mRNA relocalization by forskolin[20], suggesting that b-actin mRNA is associated with The presence of specific mRNAs in CBP and the local-

ization of mRNAs require a mechanism to segregatethe cytoskeleton whether the mRNA is localized to thecell periphery or present in the perinuclear cytoplasm. such mRNAs from those that are not localized or which

are translated on the ER or FP. Localization of b-actinIn MPC-11 cells growth conditions were found to alteractin mRNA association with the cytoskeleton [96] but mRNA in spreading fibroblasts is independent of na-

scent polypeptide chains or intact ribosomes [17] andin this case the two isoform mRNAs were not analyzedseparately and the response of serum was examined after thus appears to depend upon signals within the specific

mRNA; it is also dependent on intact microfilamentsa longer delay and involved RNA synthesis. In oocytesand early embryos association with the cytoskeleton is [102]. Furthermore, microinjection of exogenous

mRNA, either into oocytes [105, 106] or oligodendrogliaalso not correlated with any one particular localizationof mRNAs [9, 84]: thus, mRNAs which exhibit localiza- [27], results in correct localization of the mRNA. It thus

appears that both mRNA localization and interactiontion to different poles or to the mitochondrial cloud areall associated with the cytoskeleton. with the cytoskeleton depend on signals which reside

within the RNA itself.These data have several implications. First, although



The introduction of modified or chimeric genes in the different 3*UTRs [19, 25]. More recently, the3*UTR of tau mRNA has been shown to be requiredwhich either the whole or parts of the 3*UTR are de-

leted or in which 3*UTR regions are linked to a reporter for localization of this mRNA in transfected primaryneurons [110] and in situ hybridization studies havesequence has produced considerable evidence not only

that localization depends upon information within the shown that in myoblasts the myosin heavy chain andvimentin 3*UTRs can localize b-globin coding se-mRNA but also that localization and association with

the cytoskeleton depend upon the 3*UTR and that the quences [104] and that localization of creatine kinasemRNAs is dependent on 3*UTR sequences [22].3*UTR contains directional information which can di-

rect a reporter mRNA to a particular subcellular site. The use of in situ hybridization for analysis of mRNAlocalization in many of these studies gives no informa-Thus, microinjection of RNA into oocytes showed that

the 3*UTR of the Vg1 mRNA was essential for correct tion as to whether the localized mRNA is translated orif it is present in an untranslated ribonucleoproteinlocalization and that a chimeric construct in which the

Vg1 3*UTR was linked to the coding sequence of Xeno- particle. Indeed it is likely that many of the localizedmRNAs in oocytes, for example Vg1 [82, 105], are pres-pus b-globin was localized as per the native Vg1 [105].

In Drosophila it has proved possible to produce ent in a stored, untranslated form. In contrast, poly-some fractionation allows study of mRNAs which aretransgenic flies in which the gene for a localized mRNA

such as bicoid has been modified within the 3*UTR or translated and in the case of c-myc mRNA the localizedmRNA is apparently associated with the CBP and lossexpressing chimeric globin transcripts with different

3*UTRs [14, 107–109]. These studies indicate that for of localization is associated with a change in the associ-ation of polysomal mRNA (i.e., translated) with thea number of localized mRNAs such as bicoid, even-

skipped, fushi tarazu, and hairy the 3*UTR either is cytoskeleton [16]: this suggests that even the mislocal-ized mRNA is being translated. Furthermore, as shownrequired for localization or is capable of localizing a

reporter sequence [14, 109]. by immunocytochemistry correct c-myc mRNA localiza-tion is not essential for synthesis of the protein [101].Similarily, in mammalian cells transfected with a c-

myc gene containing the normal 3*UTR the mRNA is Overall, these observations suggest that, depending onthe message and the cell type, localization of both un-found in the perinuclear cytoplasm and in association

with the cytoskeleton but removal of the 3*UTR from translated mRNA and translated mRNA can occur.In summary, it is emerging that the localization ofc-myc and its replacement by that of b-globin resulted

in a loss of the perinuclear localization of the mRNA mRNAs and their association with the cytoskeleton,not only in oocytes of amphibia and insects, but also in[16]; in addition, whereas the normal c-myc mRNA is

found in CBP and normal b-globin in FP the chimeric differentiated mammalian cells, are dependent on the3*UTR region of the mRNAs concerned (Tables 3a andtranscripts in which the c-myc coding sequences are

linked to the b-globin UTR are found in FP and those 3b). It appears that mRNA targeting by 3*UTR se-quences and the cytoskeleton is a general mechanismin which the c-myc coding sequences are linked to the

b-globin 3*UTR are found in FP [16]. Thus, in the case utilized by a variety of eukaryotic cells [3, 4, 16] fromXenopus oocytes to mammalian neurons, fibroblasts,of c-myc mRNA the 3*UTR appears essential for associ-

ation of c-myc mRNA with the cytoskeleton and for and myoblasts. At present it is not clear at what stageafter entering the cytoplasm the mRNAs are sorted;perinuclear localization. Further studies have shown

that whereas both the normal b-globin mRNA and the this may depend on the mRNA and the cell with somebeing localized in ribonucleoprotein particles and oth-b-globin coding sequence without the 3*UTR are found

throughout the cytoplasm [103, 104], chimeric tran- ers either before or after they become part of anmRNA–ribosome or polysome complex.scripts in which the b-globin coding sequences are

linked to the whole, or parts, of the c-myc 3*UTR arelocalized to the perinuclear cytoplasm [103]. The simi- mRNA LOCALIZATION SIGNALSlar distribution of globin transcripts with and withoutthe 3*UTR indicate that there is no localization signal Although a variety of mRNAs are localized in oocytes

and embryos of Xenopus and Drosophila, there is as yetwithin the globin 3*UTR: the effects of the c-myc3*UTR indicate therefore that an active signal is re- no clear concensus as to the nature of the localization

signals involved. In the case of the bicoid mRNA, 3*UTRquired for perinuclear localization and association withCBP. Similar experiments using transfection of con- deletion analysis using chimeric mRNAs showed that

deletion of 120–150 nt from each end of the 3*UTRstructs in which actin 3*UTR sequences are linked tothe b-galactosidase coding region have shown that the destroyed mRNA localization [108]. Microinjection

studies have suggested that deletion of a short 9-ntb-actin 3*UTR is able to localize the reporter sequencesto the peripheral cytoplasm and that the differential sequence present in the 5* end of the 3*UTR of bicoid

causes partial loss of localization and this sequencelocalization of a- and b-actin mRNAs is dependent on


230 JOHN E. HESKETH

TABLE 3a

3*UTR Sequences Target mRNAs to the Cytoskeleton and to More Than One Cytoplasmic Location in Mammalian Cells

Association with Role of 3*UTR inmRNA cytoskeleton Localization localization

c-myc CBP Perinuclear /b-Globin 0 None 0b-Globin-c myc 3*UTR / Perinuclear /b-Globin-c-myc 3*UTR (bases 194 –440) / Perinuclear /b-Globin-c-myc 3*UTR (bases 280 –440) 0 None 0b-Globin-c-myc 3*UTR (bases 194 –280) / Perinuclear /b-Actin CBP Cell periphery /a-actin ? Perinuclear /LacZ-b-actin 3*UTR ? Peripheral /LacZ-a-actin 3*UTR ? Perinuclear /Metallothionein-1 CBP Perinuclear, but less so than c-myc ?Vimentin CBPa Perinuclear/costameres ?b-Globin-vimentin 3*UTR ? Perinuclear /Myosin heavy chain / Intermyofibrillary cytoplasm/myofibrils ?b-Globin-myosin heavy chain 3*UTR ? Perinuclear /Creatine kinase M ? Perinuclear /Creatine kinase B ? Peripheral /tau ? Proximal axon /

a Hesketh et al., unpublished observations.

shows conservation among the mRNAs known to be 3*UTR is required for bicoid mRNA localization. Inter-estingly, the BLE1 element appears necessary for thelocalized in Drosophila [111]. Studies using the bicoid

mRNA itself have given different results: the 120 nt at early stages of localization whereas the conserved 9-ntsequence in the 5* end of the 3*UTR was required tothe 5* end of the 3* UTR were not required for localiza-

tion but a 50-nt sequence (BLE1) in the middle of the retain injected RNA in the anterior cytoplasm, an assaywhich may detect regions of the 3*UTR required for3*UTR was found to be essential for the early stages

of localization [108]; in addition a number of deletions the later anchoring stage of mRNA localization ratherthan translocation. Localization of cyclin B mRNA,impaired localization but did not abolish it. The reasons

for the contradictory data are unknown but it may be which has been reported to occur via a two-step mecha-nism, also requires two separate regions of the 3*UTRthat either the generation of different 3*UTR second-

ary structures depends on the influence of the attached (one 94 nt and one 87 nt) for localization [112].Transfection of fibroblasts with chimeric constructscoding region or multiple signals exist and these have

different levels of effectiveness depending on the in which b-actin sequences with a series of deletionsare linked to the b-galactosidase coding region has alsomethod used to assess localization function. However,

both studies suggest that more than one region of the identified two regions of the b-actin 3*UTR as being

TABLE 3b

Comparison of 3*UTR Sequences and Structure of Some mRNAs Found on CBPand/or in Perinuclear Cytoplasm: Similarity in One Stem-Loop Region

mRNA Localization/association with cytoskeleton Homology with 3*UTR of c-myc mRNAa

c-myc Perinuclear/CBP [Bases 194–280 required for localization]Cyclin A CBP Bases 238–317 (70%)g-Actin Perinuclear Bases 265–321 (70%)Metallothionein-1 CBP Predicted to have stem-loop structure

similar to that of bases 200–360

a 3*UTR sequences were compared with the c-myc 3*UTR using the Bestfit programme; region of c-myc 3*UTR showing homology is shownwith percentage similarity in parentheses. Secondary structure was predicted using RNAFOLD programme. Note the regions of homologyand area of secondary structure similarity overlap with the region implicated in c-myc localization (see above and text).



FIG. 4. The sorting of mRNA. A hypothetical scheme illustrating how different signals in the 3*UTR could be involved in sorting mRNAsso they are either retained on the cytoskeleton in the perinuclear cytoplasm (e.g., c-myc) or transported to the cell periphery (e.g., b-actin).

involved in localization; a 54-nt region (close to the for localization, is retained in the perinuclear cytoplasm.If, as proposed in the case of localized mRNAs in oocytescoding region) which is necessary for full localization

activity and a 43-nt homologous segment which was [13, 82], the localization of bicoid, cyclin B, and b-actinconsists of at least two steps involving first translocationalso able to target mRNA to the cell periphery but was

less active [19, 25]; mutational and deletion analyses and then anchoring of the mRNA it is possible to envisagea need for more than one localization signal, one to linksuggest that several motifs may contribute to localiza-

tion. Antisense oligonucleotides to the identified b-ac- the mRNA to microtubules during translocation and onefor anchoring the mRNA at the appropriate site (see thetin localization signals caused a delocalization of the

mRNA but did not destabilize the mRNA [25]. model proposed in Fig. 4). On the other hand retentionof c-myc on the perinuclear cytoskeleton may only requireIn contrast to bicoid and b-actin mRNAs, localization

of tau and c-myc mRNAs appears to depend on a single an anchoring signal to bind to microfilaments. Presum-ably no single motif is responsible for mRNA localizationregion within the 3*UTR. The region of the c-myc

3*UTR responsible for perinuclear localization in fi- or interaction with the cytoskeleton but at least two:whatever interactions determine localization there mustbroblasts has been investigated by creating a series

of chimeric globin constructs with the b-globin coding be a selective mechanism to sort those mRNAs which areretained on the cytoskeleton in the perinuclear cytoplasmsequences linked to increasingly small segments of the

c-myc 3*UTR [103]. Deletion of an 86-nt sequence from from those that are translocated to the cell periphery(Table 3, Fig. 4).the 3*UTR caused loss of perinuclear localization and

loss of association with the cytoskeleton (Fig. 3). Simi- The relative importance of secondary structure andsequence in the localization signals is largely unknown.larly, localization of tau mRNA depends on a singe 91-

nt region of the 3*UTR [110]. Although sequence comparisons have implicated a 9-nt sequence in oocyte mRNA localization [111], in mostThe requirement of a single or multiple regions of the

3*UTR for localization may depend upon the subcellular cases relatively large 3*UTR regions (ú40 nt) are gen-erally required for localization and this suggests that,destination of the mRNA. b-actin, cyclin B, and bicoid

mRNAs, which contain at least two 3*UTR localization whether single or multiple regions of the 3*UTR havebeen implicated in localization, the signals dependregions, are translocated, either to the anterior pole or

to the peripheral cytoplasm underlying the lamellipodia, upon secondary structure rather than short sequencemotifs. In the case of the c-myc mRNA 3*UTR an 86-while c-myc mRNA, which has a single region required


232 JOHN E. HESKETH

FIG. 5. The localization signal in the c-myc 3 *UTR. The secondary structure for the c-myc 3*UTR predicted using the RNAFOLDprogram. The region between bases 194 and 280 is that required for perinuclear localization of reporter sequences and their associationwith the cytoskeleton [103]. The conserved AUUUA implicated in localization is also marked. Note that both the 86-nt localization regionand the AUUUA are within a predicted stem-loop structure. Since mutation of the AUUUA is predicted to destroy this stem-loop and alsodestroys c-myc localization [103, 113], our present hypothesis is that features of the stem-loop region form a critical part of the localizationsignal.

nt segment has been found capable of targeting globin in localization is predicted to alter the secondary struc-ture dramatically with loss of the stem-loop [113].sequences to the cytoskeleton and perinuclear cyto-

plasm [103]. Mutation of an AUUUA sequence within Overall, despite the speculative nature of secondarystructure predictions, the data suggest that features ofthis 86-nt region destroys the localization of c-myc

mRNA as well as the ability of the 86-nt to localize this long stem-loop region are part of the localizationsignal in c-myc mRNA.chimeric b-globin transcripts to the perinuclear cyto-

plasm and CBP [103]. Neither the mutation nor the The details of the mRNA– protein interactionswhich result in mRNA localization and association86-nt segment of the 3*UTR confer mRNA instability

and thus they appear to be part of a localization signal. with the cytoskeleton are essentially unknown but itis likely that it will involve specific proteins whichSeveral observations from computer predictions of sec-

ondary structure and sequence comparisons suggest bind to a range of localization signals in the 3*UTR.Until very recently, the nature of the binding pro-both that secondary structure is important in the local-

ization signal and that some features of this signal may teins and their interactions with 3 *UTR signals werelargely unknown. However, a number of RNA-bind-be shared with other mRNAs that are targeted to CBP

or the perinuclear cytoplasm: first, computer prediction ing proteins which may be involved in localizationhave now been found, and interestingly there are sev-of the secondary structure of the c-myc 3*UTR suggests

that this 86-nt sequence forms part of a long stem- eral examples of proteins which appear to mediatemRNA interactions with microtubules. The bestloop structure (Fig. 5); second, comparison of the c-myc

3*UTR sequence with that of cyclin A (enriched in CBP) studied is the protein staufen which is required forbicoid and oskar mRNA localization, colocalizes withand that of g-actin (perinuclear localization) reveals

homology within the identified 86-nt region and within bicoid mRNA during translocation of the mRNA inoocytes [114], and contains a RNA-binding motif; athe sequence corresponding to that of the predicted

stem-loop structure (Tables 3a and 3b; Fig. 5); third, peptide containing this motif binds double-strandedRNA in vitro [115]. Localization of the protein de-computer prediction of the secondary structure of the

3*UTR of metallothionein-1 mRNA (enriched in CBP) pends upon an interaction with bicoid mRNA, spe-cifically the 3*UTR [86]. The binding appears to in-suggests that it can form a single long stem-loop struc-

ture similar in length to the one implicated in c-myc volve three regions, each of which may potentiallyform a long stem-loop structure. Staufen forms com-localization; fourth, mutation of the AUUUA motif

within the 86-nt segment of c-myc 3*UTR implicated plexes with injected bicoid 3 *UTR and the localiza-



tion of these is destroyed by colchicine, thus sug- are translated on the ER, possibly to different com-partments.gesting that it is dependent on intact microtubules

[86]. It appears therefore that, at least in the caseof bicoid mRNA, the cytoskeleton does not interact FUTURE PERSPECTIVESdirectly with the mRNA but that a ‘‘linker’’ protein

Our concept of the organization of the protein syn-such as staufen is needed to interact with the mRNA.thetic apparatus has changed markedly over the pastStudies with galactosidase– kinesin fusion proteins20 years. It is now clear that mRNAs are not simplyimplicate kinesin as the motor driving mRNA trans-translated either on the endoplasmic reticulum or inport in this system [87]. Similarly, in the case of Vg1the cytosolic ‘‘soup’’ in a spatially random fashion butmRNA an mRNA binding protein has been identifiedthat some of those mRNAs which code for intracellu-which appears to mediate an interaction with micro-lar proteins are found localized in specific areas oftubules [116]. Such proteins appear not to be re-the cytoplasm and some mRNAs are associated withstricted to oocytes since a protein from testis andcytoskeletal structures (Fig. 4). There appears to bebrain has been found to bind to specific mRNAs anda general mechanism for the localization of mRNAsto microtubules [117]. It remains to be seen whetherand this involves the cytoskeleton and localizationSpnr, a mRNA-binding protein which localizes withsignals within 3*UTRs of specific mRNAs [3, 4, 57].microtubules [118], is involved in mRNA localization.This mechanism sorts mRNAs to be targeted to differ-Finally, cross-linking studies have shown that theent sites, either for translocation to the cell periphery91-bp region of the 3*UTR responsible for localizationor for retention in the periuclear cytoplasm; this sort-of tau mRNA binds two proteins of 43 and 38 kDa,ing appears to ensure that, at least for the small num-and these proteins are also present in microtubuleber of mRNAs thus far studied, the proteins are syn-preparations [110]. A protein which binds to thethesized in specific compartments close to the site ofBLE1 motif has been detected [119] but its interac-function. There is evidence from permeabilized celltions with the cytoskeleton remain to be studied. Thesystems that the protein synthetic machinery isseveral studies showing interactions between mRNAhighly organized [123] and the localization of mRNAsbinding proteins and microtubules support the hy-and association of polysomes with the cytoskeletonpothesis that microtubules may be important inmay play an important part in such spatial organiza-mRNA transport.tion of the translational apparatus.

The cell matrix of fibroblasts contains proteins The cytoskeleton appears to play a key part in thiswhich bind to the b-actin 3 *UTR [120] but these pro- organization but the nature and extent of this roleteins have not been shown to bind specifically to the are still unclear. It is important to determine howregions identified as containing localization signals; great a proportion of mRNAs are either localized,however, their presence in the cell matrix suggests translated on CBP, or targeted by 3*UTR-basedthat they play a role in the association of b-actin mechanisms so as to assess the extent to which themRNA with the cytoskeleton. As the 3 *UTR regions these mechanisms are used by cells. CBP are en-involved in localization become known it will be pos- riched in specific mRNAs and so represent a compart-sible to use band-shift, North-Western, and cross- ment which is distinct from FP, but it is still unclearlinking assays to investigate the proteins which bind whether FP and CBP represent single compartmentsto such localization signals and how they are linked with defined subcellular localizations or whetherto the cytoskeleton. Recent analysis of CBP has indi- they each represent a number of different compart-cated the presence of annexin II (Vedeler, personal ments with different localizations. This needs to becommunication), indicating that this protein may be defined and will require improvements in fraction-involved in the anchoring or transport of mRNAs. ation techniques and greater sensitivity of in situLocalization of Vg1 mRNA requires not only 3 *UTR hybridization methods. These improvements are alsosequences but also the correct localization of the non- needed to elucidate the roles of different cytoskeletalcoding Xsirt rRNA [121], suggesting that other RNA components, particularly whether microtubules andspecies as well as proteins may have a role in mRNA microfilaments do indeed have distinct transport andtargeting. anchoring roles (Fig. 4). Future work should also fo-

In neurons there is compartmentalization of cus on defining the localization signals in sufficientmRNA for the secreted arginine vasopressin and this mRNAs to allow the search for consensus sequencescan be altered by a single nucleotide change [122]. or structures, for stem-loop structures, and for bind-Such data suggest that, in addition to the known sig- ing proteins. The identification of the binding pro-nal sequence mechanism, some mRNA localization teins will then open the way for studies of how the

mRNAs are linked to the cytoskeleton, for studies ofsignals play a role in localization of mRNAs which


234 JOHN E. HESKETH

25. Kislauskis, E. H., Xiaochun, Z., and Singer, R. H. (1994) J.transport and mRNA anchoring, and for studies ofCell Biol. 127, 441–451.how localization and mRNA–cytoskeleton interac-

26. Cheng, H., and Bjerknes, M. (1989) J. Mol. Biol. 210, 541–tions can be regulated.549.

Knowledge of which mRNAs are associated with27. Ainger, K. D., Avossa, F., Morgan, S. J., Hill, C., Barry, E.,CBP, which are localized, and what is the effect of Barbarese, and Carson, J. H. (1993) J. Cell Biol. 123, 431–

mRNA relocalization in terms of protein synthesis and 441.subsequent protein targeting should illuminate the 28. Verity, N. A., and Campagnoni, A. T. (1988) J. Neurosci. Res.functional significance of mRNA–cytoskeleton interac- 21, 238–248.tions. Ultimately, this should allow us to assess 29. Kritennsson, K., Holmes, K. V., Duchala, L. S., Zeller, N. K.,

Lazzarini, R. A., and Dubois-Dulcq, M. (1986) Nature 322,whether CBP and mRNA localization, through cy-544–547.toskeletal reorganization and/or mRNA binding pro-

30. Garner, C. C., Tucker, R. P., and Matus, A. (1988) Nature 336,teins, is a mechanism by which the cell, in response to674–677.extracellular signals, can modulate the subcellular site

31. Bruckenstein, D. A., Lein, P. J., Higgins, D., and Fremeau,of gene expression. R. T., Jr. (1990) Neuron 5, 809–819.

32. Kleiman, R., Banker, G., and Steward, O. (1990) Neuron 5,The author’s work is supported by the Scottish Office Agriculture, 821–830.

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Received November 27, 1995Revised version received March 19, 1996


sorting of messenger rnas in the cytoplasm: mrna localization and the cytoskeleton

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