Conserved 3′-Untranslated Region Sequences Direct Subcellular Localization of Chaperone Protein mRNAs in Neurons

Abstract

mRNA localization provides polarized cells with a locally renewable source of proteins. In neurons, mRNA translation can occur at millimeters to centimeters from the cell body, giving the dendritic and axonal processes a means to autonomously respond to their environment. Despite that hundreds of mRNAs have been detected in neuronal processes, there are no reliable means to predict mRNA localization elements. Here, we have asked what RNA elements are needed for localization of transcripts encoding endoplasmic reticulum chaperone proteins in neurons. The 3′-untranslated regions (UTRs) of calreticulin and Grp78/BiP mRNAs show no homology to one another, but each shows extensive regions of high sequence identity to their 3′UTRs in mammalian orthologs. These conserved regions are sufficient for subcellular localization of reporter mRNAs in neurons. The 3′UTR of calreticulin has two conserved regions, and either of these is sufficient for axonal and dendritic targeting. However, only nucleotides 1315–1412 show ligand responsiveness to neurotrophin 3 (NT3) and myelin-associated glycoprotein (MAG). This NT3- and MAG-dependent axonal mRNA transport requires activation of JNK, both for calreticulin mRNA and for other mRNAs whose axonal levels are commonly regulated by NT3 and MAG. mRNA localization provides polarized cells with a locally renewable source of proteins. In neurons, mRNA translation can occur at millimeters to centimeters from the cell body, giving the dendritic and axonal processes a means to autonomously respond to their environment. Despite that hundreds of mRNAs have been detected in neuronal processes, there are no reliable means to predict mRNA localization elements. Here, we have asked what RNA elements are needed for localization of transcripts encoding endoplasmic reticulum chaperone proteins in neurons. The 3′-untranslated regions (UTRs) of calreticulin and Grp78/BiP mRNAs show no homology to one another, but each shows extensive regions of high sequence identity to their 3′UTRs in mammalian orthologs. These conserved regions are sufficient for subcellular localization of reporter mRNAs in neurons. The 3′UTR of calreticulin has two conserved regions, and either of these is sufficient for axonal and dendritic targeting. However, only nucleotides 1315–1412 show ligand responsiveness to neurotrophin 3 (NT3) and myelin-associated glycoprotein (MAG). This NT3- and MAG-dependent axonal mRNA transport requires activation of JNK, both for calreticulin mRNA and for other mRNAs whose axonal levels are commonly regulated by NT3 and MAG. IntroductionTargeting of mRNAs to subcellular regions is a highly selective process that provides spatial control to gene expression (1Besse F. Ephrussi A. Nat. Rev. Mol. Cell Biol. 2008; 9: 971-980Crossref PubMed Scopus (256) Google Scholar). Localized mRNA translation has been linked to polarized growth in fibroblasts and migration of cancer cells (2Shestakova E.A. Singer R.H. Condeelis J. Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 7045-7050Crossref PubMed Scopus (165) Google Scholar, 3Adereth Y. Dammai V. Kose N. Li R. Hsu T. Nat. Cell Biol. 2005; 7: 1240-1247Crossref PubMed Scopus (105) Google Scholar). In neurons, protein products of dendritically localized mRNAs contribute to synaptic plasticity (4Bramham C.R. Wells D.G. Nat. Rev. Neurosci. 2007; 8: 776-789Crossref PubMed Scopus (466) Google Scholar). Protein products of mRNAs transported into axons have been linked to growth and injury responses (5Lin A.C. Holt C.E. Curr. Opin. Neurobiol. 2008; 18: 60-68Crossref PubMed Scopus (123) Google Scholar, 6Willis D.E. Twiss J.L. Curr. Opin. Neurobiol. 2006; 16: 111-118Crossref PubMed Scopus (104) Google Scholar). The mRNA population localizing into neuronal processes is quite complex, with several hundred mRNAs localizing into axonal and/or dendritic processes (7Willis D.E. van Niekerk E.A. Sasaki Y. Mesngon M. Merianda T.T. Williams G.G. Kendall M. Smith D.S. Bassell G.J. Twiss J.L. J. Cell Biol. 2007; 178: 965-980Crossref PubMed Scopus (229) Google Scholar, 8Taylor A.M. Berchtold N.C. Perreau V.M. Tu C.H. Li Jeon N. Cotman C.W. J. Neurosci. 2009; 29: 4697-4707Crossref PubMed Scopus (276) Google Scholar, 9Vogelaar C.F. Gervasi N.M. Gumy L.F. Story D.J. Raha-Chowdhury R. Leung K.M. Holt C.E. Fawcett J.W. Mol. Cell. Neurosci. 2009; 42: 102-115Crossref PubMed Scopus (76) Google Scholar, 10Poon M.M. Choi S.H. Jamieson C.A. Geschwind D.H. Martin K.C. J. Neurosci. 2006; 26: 13390-13399Crossref PubMed Scopus (171) Google Scholar, 11Matsumoto M. Setou M. Inokuchi K. Neurosci. Res. 2007; 57: 411-423Crossref PubMed Scopus (46) Google Scholar). However, it is clear that neurons do not send all mRNAs into their distal processes, and these cells must actively select which mRNAs to localize. For example, although β-actin and γ-actin mRNAs encode remarkably similar proteins, only β-actin mRNA is transported into neuronal processes (12Bassell G.J. Zhang H. Byrd A.L. Femino A.M. Singer R.H. Taneja K.L. Lifshitz L.M. Herman I.M. Kosik K.S. J. Neurosci. 1998; 18: 251-265Crossref PubMed Google Scholar, 13Tiruchinapalli D.M. Oleynikov Y. Kelic S. Shenoy S.M. Hartley A. Stanton P.K. Singer R.H. Bassell G.J. J. Neurosci. 2003; 23: 3251-3261Crossref PubMed Google Scholar, 14Zheng J.Q. Kelly T.K. Chang B. Ryazantsev S. Rajasekaran A.K. Martin K.C. Twiss J.L. J. Neurosci. 2001; 21: 9291-9303Crossref PubMed Google Scholar). Moreover, some mRNAs are selectively transported into dendrites or axons (15Garner C.C. Tucker R.P. Matus A. Nature. 1988; 336: 674-677Crossref PubMed Scopus (442) Google Scholar, 16Aronov S. Aranda G. Behar L. Ginzburg I. J. Neurosci. 2001; 21: 6577-6587Crossref PubMed Google Scholar), whereas other mRNAs are targeted to both axons and dendrites (13Tiruchinapalli D.M. Oleynikov Y. Kelic S. Shenoy S.M. Hartley A. Stanton P.K. Singer R.H. Bassell G.J. J. Neurosci. 2003; 23: 3251-3261Crossref PubMed Google Scholar, 17Lu R. Wang H. Liang Z. Ku L. O'donnell W.T. Li W. Warren S.T. Feng Y. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 15201-15206Crossref PubMed Scopus (252) Google Scholar, 18Antar L.N. Li C. Zhang H. Carroll R.C. Bassell G.J. Mol. Cell. Neurosci. 2006; 32: 37-48Crossref PubMed Scopus (204) Google Scholar).Untranslated regions (UTR) 4The abbreviations used are: UTRuntranslated regionNT3neurotrophin 3MAGmyelin-associated glycoproteinDRGdorsal root ganglionLVlentivirusFISHfluorescence in situ hybridizationGFPgreen fluorescent proteinGFPmyrGFP with myristoylation elementNFneurofilamentPBSphosphate-buffered salineROIregion of interestFRAPfluorescent recovery after photobleachingDIVdays in vitroRTtranscriptase-coupled PCRANOVAanalysis of varianceNGFnerve growth factorJNKc-Jun N-terminal kinaseBSAbovine serum albuminGFAPglial fibrillary acidic proteinNFneurofilament. of mRNAs, particularly the 3′UTRs (19Jambhekar A. Derisi J.L. RNA. 2007; 13: 625-642Crossref PubMed Scopus (114) Google Scholar, 20Jansen R.P. Nat. Rev. Mol. Cell Biol. 2001; 2: 247-256Crossref PubMed Scopus (292) Google Scholar), often include elements that confer subcellular localization. These cis-elements are thought to provide binding sites for proteins needed for subcellular localization of mRNAs (21Kiebler M.A. Bassell G.J. Neuron. 2006; 51: 685-690Abstract Full Text Full Text PDF PubMed Scopus (428) Google Scholar). There is little cumulative knowledge of what constitutes a cis-element responsible for targeting neuronal mRNAs to subcellular sites. RNA structures that drive localization along with the specific proteins that recognize these structures will undoubtedly play a key role in regulation of mRNA transport and localization. For example, the cis-element or “zipcode” of β-actin consists of a 54-nucleotide segment in its proximal 3′UTR that is bound by zipcode-binding protein 1 (ZBP1) (22Kislauskis E.H. Li Z. Singer R.H. Taneja K.L. J. Cell Biol. 1993; 123: 165-172Crossref PubMed Scopus (270) Google Scholar, 23Farina K.L. Huttelmaier S. Musunuru K. Darnell R. Singer R.H. J. Cell Biol. 2003; 160: 77-87Crossref PubMed Scopus (193) Google Scholar). Localization elements can also provide a means to regulate the translation of mRNAs by actively preventing translational initiation during their transport (24Wells D.G. J. Neurosci. 2006; 26: 7135-7138Crossref PubMed Scopus (60) Google Scholar). The 3′UTR of rat β-actin is sufficient for axonal localization of reporter mRNAs in adult sensory neurons, including ligand-dependent mRNA transport (7Willis D.E. van Niekerk E.A. Sasaki Y. Mesngon M. Merianda T.T. Williams G.G. Kendall M. Smith D.S. Bassell G.J. Twiss J.L. J. Cell Biol. 2007; 178: 965-980Crossref PubMed Scopus (229) Google Scholar). However, the β-actin zipcode shows no clear homology with the 3′UTRs of roughly 200 mRNAs that we have isolated from axons of cultured sensory neurons. Here, we have asked what cis-elements direct subcellular localization of mRNAs encoding the endoplasmic reticulum chaperone proteins calreticulin and Grp78/BiP. Despite the lack of homology between calreticulin and Grp78/BiP mRNA 3′UTRs, each shows high sequence identity with their mammalian orthologs. These conserved noncoding sequences are sufficient for subcellular localization of both mRNAs. Calreticulin mRNA further contains two distinct elements that confer subcellular localization, but these elements are functionally distinguished by their responsiveness to ligand stimulation.DISCUSSIONSeveral hundred different neuronal mRNAs have been shown to be transported into axons and dendrites (7Willis D.E. van Niekerk E.A. Sasaki Y. Mesngon M. Merianda T.T. Williams G.G. Kendall M. Smith D.S. Bassell G.J. Twiss J.L. J. Cell Biol. 2007; 178: 965-980Crossref PubMed Scopus (229) Google Scholar, 8Taylor A.M. Berchtold N.C. Perreau V.M. Tu C.H. Li Jeon N. Cotman C.W. J. Neurosci. 2009; 29: 4697-4707Crossref PubMed Scopus (276) Google Scholar, 9Vogelaar C.F. Gervasi N.M. Gumy L.F. Story D.J. Raha-Chowdhury R. Leung K.M. Holt C.E. Fawcett J.W. Mol. Cell. Neurosci. 2009; 42: 102-115Crossref PubMed Scopus (76) Google Scholar, 10Poon M.M. Choi S.H. Jamieson C.A. Geschwind D.H. Martin K.C. J. Neurosci. 2006; 26: 13390-13399Crossref PubMed Scopus (171) Google Scholar, 11Matsumoto M. Setou M. Inokuchi K. Neurosci. Res. 2007; 57: 411-423Crossref PubMed Scopus (46) Google Scholar). Despite increasing interest in RNA localization, there have been only a few RNA elements identified that distinguish localizing from nonlocalizing mRNAs. Moreover, only a handful of elements have been identified for neurons and even fewer for axonally localized mRNAs (43Vuppalanchi D. Willis D.E. Twiss J.L. Results Probl. Cell Differ. 2009; 48: 193-224PubMed Google Scholar). Here, we have used complementary methods to show that neuronal calreticulin and Grp78/BiP mRNAs are localized into neuronal processes, and this is driven through conserved 3′UTR elements.The 3′UTRs of localizing mRNAs frequently contain sequence elements responsible for their transport to subcellular domains. Farina et al. (23Farina K.L. Huttelmaier S. Musunuru K. Darnell R. Singer R.H. J. Cell Biol. 2003; 160: 77-87Crossref PubMed Scopus (193) Google Scholar) defined the minimal consensus mRNA-binding site for ZBP1 by SELEX analyses as RCACCC. Rat and mouse calreticulin 3′UTRs include the ACACCC motif (see supplemental Fig. S2), but this is not seen in the 3′UTR of Grp78/BiP or in the other mammalian orthologs of calreticulin mRNA. Moreover, neither calreticulin nor Grp78/BiP mRNAs were detected in a recent screen of mRNAs co-purifying with IMP1, the human ortholog of ZBP1 (44Jønson L. Vikesaa J. Krogh A. Nielsen L.K. Hansen T. Borup R. Johnsen A.H. Christiansen J. Nielsen F.C. Mol. Cell. Proteomics. 2007; 6: 798-811Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). These observations plus the studies here on ligand-dependent transport suggest that other neuronal RNA-binding proteins beyond ZBP1 contribute to subcellular localization of calreticulin and Grp78/BiP mRNAs.Secondary structure rather than primary sequence more typically defines RNA localization elements. Detecting commonalities in RNA secondary structure has proven difficult for more than just a few transcripts such that biologically testing for localization elements has been the most successful approach (19Jambhekar A. Derisi J.L. RNA. 2007; 13: 625-642Crossref PubMed Scopus (114) Google Scholar, 45Colegrove-Otero L.J. Minshall N. Standart N. Crit. Rev. Biochem. Mol. Biol. 2005; 40: 21-73Crossref PubMed Scopus (72) Google Scholar). Sequence comparisons of calreticulin and Grp78/BiP mRNA 3′UTRs to their mammalian orthologs show more than 95% primary sequence identity. For Grp78/BiP, this spans nearly its entire 3′UTR; for calreticulin mRNA, this consisted of two ∼100-nucleotide segments with >80% identity in adjacent regions (supplemental Fig. S2). Analogous queries among mammalian orthologs for the 3′UTR of the nonlocalizing γ-actin mRNA showed much lower conservation (≤50%). The conserved UTR segments exceed homology of mRNA sequence in the coding regions of these chaperone protein mRNAs, and both of the 3′UTRs are sufficient for subcellular localization in neurons. However, we did not see any homology comparing calreticulin and Grp78/BiP mRNA sequences available for amphibian, flies, and nematodes.Dendrite-specific localization elements have been described for the 3′UTRs of the mRNAs encoding microtubule-associated protein 2 (MAP2), calcium-calmodulin kinase IIα (CAMKIIα), and protein kinase Mζ (PKMζ) (46Mayford M. Baranes D. Podsypanina K. Kandel E.R. Proc. Natl. Acad. Sci. U.S.A. 1996; 93: 13250-13255Crossref PubMed Scopus (260) Google Scholar, 47Blichenberg A. Schwanke B. Rehbein M. Garner C.C. Richter D. Kindler S. J. Neurosci. 1999; 19: 8818-8829Crossref PubMed Google Scholar, 48Muslimov I.A. Nimmrich V. Hernandez A.I. Tcherepanov A. Sacktor T.C. Tiedge H. J. Biol. Chem. 2004; 279: 52613-52622Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Considering that the DRGs do not develop dendritic processes in culture, we anticipated that the two distinct localization elements in calreticulin mRNA would provide differential transport into axons versus dendrites. However, either localization element was sufficient for localization into axons and dendrites of polarized central nervous system neurons. To date, there has been no report of neuronal mRNA transcript that contains separate dendritic and axonal localization elements. The zipcode element of β-actin confers both axonal and dendritic localization (13Tiruchinapalli D.M. Oleynikov Y. Kelic S. Shenoy S.M. Hartley A. Stanton P.K. Singer R.H. Bassell G.J. J. Neurosci. 2003; 23: 3251-3261Crossref PubMed Google Scholar). Map1b mRNA similarly shows both axonal and dendritic localization, but the RNA elements responsible for these have not been fully determined (17Lu R. Wang H. Liang Z. Ku L. O'donnell W.T. Li W. Warren S.T. Feng Y. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 15201-15206Crossref PubMed Scopus (252) Google Scholar, 18Antar L.N. Li C. Zhang H. Carroll R.C. Bassell G.J. Mol. Cell. Neurosci. 2006; 32: 37-48Crossref PubMed Scopus (204) Google Scholar, 49Davidkova G. Carroll R.C. J. Neurosci. 2007; 27: 13273-13278Crossref PubMed Scopus (93) Google Scholar). Interestingly, early PCR-based studies of dendritic growth cones showed a number of mRNAs that are now known to localize to axons, including the mRNA encoding GAP-43, a classic axonal growth cone protein (50Crino P.B. Eberwine J. Neuron. 1996; 17: 1173-1187Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar). Thus, axons and dendrites clearly share some RNA transport mechanisms, with the same RNA elements and likely the same RNA-binding proteins used for both.The 3′UTR elements in calreticulin mRNA are distinguished by their ligand responsiveness. Other localizing mRNAs have been shown to contain more than one element used for subcellular localization. For example, nanos mRNA contains four elements in its 3′UTR that function cooperatively to confer localization in Drosophila oocytes (51Gavis E.R. Lehmann R. Cell. 1992; 71: 301-313Abstract Full Text PDF PubMed Scopus (288) Google Scholar, 52Gavis E.R. Lunsford L. Bergsten S.E. Lehmann R. Development. 1996; 122: 2791-2800Crossref PubMed Google Scholar). Localization of protein kinase Mζ mRNA into dendrites is specified by two cis-elements. One element in the 5′UTR, and the coding sequence is needed for export from the cell body, and a element in the 3′UTR element is for of the mRNA into distal dendrites I.A. Nimmrich V. Hernandez A.I. Tcherepanov A. Sacktor T.C. Tiedge H. J. Biol. Chem. 2004; 279: 52613-52622Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). The 3′UTR of protein mRNA contains two cis-elements for localization in One element is needed for transport of the mRNA along the processes, and a element is for localization of the mRNA to sites of the processes K. D. C. J. Cell Biol. PubMed Scopus (256) Google Scholar). For calreticulin mRNA, either cis-element is sufficient for transport into distal neuronal processes, and ligand both show axonal localization that with the the that or of NT3 in the or in the provides a for localization of through the proximal ligand-dependent segment of calreticulin distinct responses of calreticulin mRNA elements to NT3 versus are The neurons that were the only to show a localization of calreticulin mRNA, even calreticulin mRNA is also in the and neurons. Other mRNAs that show expression in neuronal are selectively regulated by For example, axonal levels of mRNA are by but not by or that is also by and neurons (7Willis D.E. van Niekerk E.A. Sasaki Y. Mesngon M. Merianda T.T. Williams G.G. Kendall M. Smith D.S. Bassell G.J. Twiss J.L. J. Cell Biol. 2007; 178: 965-980Crossref PubMed Scopus (229) Google Scholar). it is that expression of the RNA-binding proteins needed to these transcripts are to the and neurons, that nucleotides 1315–1412 are both and sufficient for the NT3- and MAG-dependent on axonal localization of calreticulin mRNA. the was not to neuronal activation of by NT3 and that on a transport for the proximal 3′UTR calreticulin localization element is a more likely for the shown with this activation of is for both the NT3- and MAG-dependent of axonal calreticulin mRNA has been shown to play a role in axonal and L. J. Neurosci. 2006; 26: PubMed Scopus Google Scholar, A.M. J. Neurosci. 1998; 18: PubMed Google Scholar, V. J. J. Cell Biol. 2005; PubMed Scopus Google Scholar). However, for the activation is typically to occur through activation of the rather than through and that selectively to and L.F. R. B. Biol. Sci. 2006; PubMed Scopus Google Scholar). the other is known to through K.C. R. R. Z. Nature. PubMed Scopus Google Scholar, S.T. K.C. M. M.M. Nat. Neurosci. PubMed Scopus Google Scholar), and MAG-dependent in transport of the was not to neuronal with expression of in all neuronal in the The of transport of calreticulin mRNA by the and the selective of neurons a for calreticulin activation of has been in cells J. Proc. Natl. Acad. Sci. U.S.A. 2003; PubMed Scopus Google Scholar), and that this can occur in neurons as it is not clear NT3 and share this for calreticulin mRNA other axonal mRNAs transport regulation by and NT3 also Thus, suggest the on RNA transport that is with other mRNAs to the localization of or of mRNAs in IntroductionTargeting of mRNAs to subcellular regions is a highly selective process that provides spatial control to gene expression (1Besse F. Ephrussi A. Nat. Rev. Mol. Cell Biol. 2008; 9: 971-980Crossref PubMed Scopus (256) Google Scholar). Localized mRNA translation has been linked to polarized growth in fibroblasts and migration of cancer cells (2Shestakova E.A. Singer R.H. Condeelis J. Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 7045-7050Crossref PubMed Scopus (165) Google Scholar, 3Adereth Y. Dammai V. Kose N. Li R. Hsu T. Nat. Cell Biol. 2005; 7: 1240-1247Crossref PubMed Scopus (105) Google Scholar). In neurons, protein products of dendritically localized mRNAs contribute to synaptic plasticity (4Bramham C.R. Wells D.G. Nat. Rev. Neurosci. 2007; 8: 776-789Crossref PubMed Scopus (466) Google Scholar). Protein products of mRNAs transported into axons have been linked to growth and injury responses (5Lin A.C. Holt C.E. Curr. Opin. Neurobiol. 2008; 18: 60-68Crossref PubMed Scopus (123) Google Scholar, 6Willis D.E. Twiss J.L. Curr. Opin. Neurobiol. 2006; 16: 111-118Crossref PubMed Scopus (104) Google Scholar). The mRNA population localizing into neuronal processes is quite complex, with several hundred mRNAs localizing into axonal and/or dendritic processes (7Willis D.E. van Niekerk E.A. Sasaki Y. Mesngon M. Merianda T.T. Williams G.G. Kendall M. Smith D.S. Bassell G.J. Twiss J.L. J. Cell Biol. 2007; 178: 965-980Crossref PubMed Scopus (229) Google Scholar, 8Taylor A.M. Berchtold N.C. Perreau V.M. Tu C.H. Li Jeon N. Cotman C.W. J. Neurosci. 2009; 29: 4697-4707Crossref PubMed Scopus (276) Google Scholar, 9Vogelaar C.F. Gervasi N.M. Gumy L.F. Story D.J. Raha-Chowdhury R. Leung K.M. Holt C.E. Fawcett J.W. Mol. Cell. Neurosci. 2009; 42: 102-115Crossref PubMed Scopus (76) Google Scholar, 10Poon M.M. Choi S.H. Jamieson C.A. Geschwind D.H. Martin K.C. J. Neurosci. 2006; 26: 13390-13399Crossref PubMed Scopus (171) Google Scholar, 11Matsumoto M. Setou M. Inokuchi K. Neurosci. Res. 2007; 57: 411-423Crossref PubMed Scopus (46) Google Scholar). However, it is clear that neurons do not send all mRNAs into their distal processes, and these cells must actively select which mRNAs to localize. For example, although β-actin and γ-actin mRNAs encode remarkably similar proteins, only β-actin mRNA is transported into neuronal processes (12Bassell G.J. Zhang H. Byrd A.L. Femino A.M. Singer R.H. Taneja K.L. Lifshitz L.M. Herman I.M. Kosik K.S. J. Neurosci. 1998; 18: 251-265Crossref PubMed Google Scholar, 13Tiruchinapalli D.M. Oleynikov Y. Kelic S. Shenoy S.M. Hartley A. Stanton P.K. Singer R.H. Bassell G.J. J. Neurosci. 2003; 23: 3251-3261Crossref PubMed Google Scholar, 14Zheng J.Q. Kelly T.K. Chang B. Ryazantsev S. Rajasekaran A.K. Martin K.C. Twiss J.L. J. Neurosci. 2001; 21: 9291-9303Crossref PubMed Google Scholar). Moreover, some mRNAs are selectively transported into dendrites or axons (15Garner C.C. Tucker R.P. Matus A. Nature. 1988; 336: 674-677Crossref PubMed Scopus (442) Google Scholar, 16Aronov S. Aranda G. Behar L. Ginzburg I. J. Neurosci. 2001; 21: 6577-6587Crossref PubMed Google Scholar), whereas other mRNAs are targeted to both axons and dendrites (13Tiruchinapalli D.M. Oleynikov Y. Kelic S. Shenoy S.M. Hartley A. Stanton P.K. Singer R.H. Bassell G.J. J. Neurosci. 2003; 23: 3251-3261Crossref PubMed Google Scholar, 17Lu R. Wang H. Liang Z. Ku L. O'donnell W.T. Li W. Warren S.T. Feng Y. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 15201-15206Crossref PubMed Scopus (252) Google Scholar, 18Antar L.N. Li C. Zhang H. Carroll R.C. Bassell G.J. Mol. Cell. Neurosci. 2006; 32: 37-48Crossref PubMed Scopus (204) Google Scholar).Untranslated regions (UTR) 4The abbreviations used are: UTRuntranslated regionNT3neurotrophin 3MAGmyelin-associated glycoproteinDRGdorsal root ganglionLVlentivirusFISHfluorescence in situ hybridizationGFPgreen fluorescent proteinGFPmyrGFP with myristoylation elementNFneurofilamentPBSphosphate-buffered salineROIregion of interestFRAPfluorescent recovery after photobleachingDIVdays in vitroRTtranscriptase-coupled PCRANOVAanalysis of varianceNGFnerve growth factorJNKc-Jun N-terminal kinaseBSAbovine serum albuminGFAPglial fibrillary acidic proteinNFneurofilament. of mRNAs, particularly the 3′UTRs (19Jambhekar A. Derisi J.L. RNA. 2007; 13: 625-642Crossref PubMed Scopus (114) Google Scholar, 20Jansen R.P. Nat. Rev. Mol. Cell Biol. 2001; 2: 247-256Crossref PubMed Scopus (292) Google Scholar), often include elements that confer subcellular localization. These cis-elements are thought to provide binding sites for proteins needed for subcellular localization of mRNAs (21Kiebler M.A. Bassell G.J. Neuron. 2006; 51: 685-690Abstract Full Text Full Text PDF PubMed Scopus (428) Google Scholar). There is little cumulative knowledge of what constitutes a cis-element responsible for targeting neuronal mRNAs to subcellular sites. RNA structures that drive localization along with the specific proteins that recognize these structures will undoubtedly play a key role in regulation of mRNA transport and localization. For example, the cis-element or “zipcode” of β-actin consists of a 54-nucleotide segment in its proximal 3′UTR that is bound by zipcode-binding protein 1 (ZBP1) (22Kislauskis E.H. Li Z. Singer R.H. Taneja K.L. J. Cell Biol. 1993; 123: 165-172Crossref PubMed Scopus (270) Google Scholar, 23Farina K.L. Huttelmaier S. Musunuru K. Darnell R. Singer R.H. J. Cell Biol. 2003; 160: 77-87Crossref PubMed Scopus (193) Google Scholar). Localization elements can also provide a means to regulate the translation of mRNAs by actively preventing translational initiation during their transport (24Wells D.G. J. Neurosci. 2006; 26: 7135-7138Crossref PubMed Scopus (60) Google Scholar). The 3′UTR of rat β-actin is sufficient for axonal localization of reporter mRNAs in adult sensory neurons, including ligand-dependent mRNA transport (7Willis D.E. van Niekerk E.A. Sasaki Y. Mesngon M. Merianda T.T. Williams G.G. Kendall M. Smith D.S. Bassell G.J. Twiss J.L. J. Cell Biol. 2007; 178: 965-980Crossref PubMed Scopus (229) Google Scholar). However, the β-actin zipcode shows no clear homology with the 3′UTRs of roughly 200 mRNAs that we have isolated from axons of cultured sensory neurons. Here, we have asked what cis-elements direct subcellular localization of mRNAs encoding the endoplasmic reticulum chaperone proteins calreticulin and Grp78/BiP. Despite the lack of homology between calreticulin and Grp78/BiP mRNA 3′UTRs, each shows high sequence identity with their mammalian orthologs. These conserved noncoding sequences are sufficient for subcellular localization of both mRNAs. Calreticulin mRNA further contains two distinct elements that confer subcellular localization, but these elements are functionally distinguished by their responsiveness to ligand